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Regulation of bone morphogenic protein 4 (BMP4) in age -related macular degeneration (AMD)

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Regulation of bone morphogenic protein 4 (BMP4) in age -related macular degeneration (AMD)

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Content NOTE TO USERS Page(s) not included in the original manuscript and are unavailable from the author or university. The manuscript was scanned as received. 37-38 This reproduction is the best copy available. ® UMI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REGULATION OF BONE MORPHOGENIC PROTEIN 4 (BMP4) IN AGE-RELATED MACULAR DEGENERATION (AMD) by Jian Wu A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) May 2005 Copyright 2005 Jian Wu Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3180361 Copyright 2005 by Wu, Jian All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3180361 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION W ith love and profound gratitude To m y parents whose enormous generosity, encouragement and support made this work possible. To Tao whose love, respect, understanding and support that lead me not ju st through the good tim es but also the bad times. To my little sister who has been the sunshine o f m y life. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS M y foremost acknowledgement and gratitude goes to my fellow researchers for their great input and cooperation. I am immensely indebted to my mentor Dr. David Hinton, who has made my graduate education thoroughly enjoyable and fulfilling. I thank him for continued encouragement, advice, and support which began when I first entered his laboratory. He has provided a rich learning environment, every opportunity to apply advanced technology, and encouragement to pursue new ideas. He helped me to develop personally, intellectually, and scientifically. Besides o f being an excellent supervisor, David was as close as a relative and a good friend to me. It has been a wonderful experience and unforgettable chapter o f my life. I thank Dr. Ram Kannan for his help in several aspects of my research. I am thankful for his ready willingness to assist with constructive comments on my papers and thesis writing. His scientific knowledge and keen guidance is very much appreciated. I would like to thank Dr. David W arburtou and Dr. Timothy Triche, for agreeing to serve as my thesis committee members and for their advice in preparation of my thesis. They have been critical, but at the same time, considerate and supportive. They provided me great inspiration by their expert comments and insight o f my project. I would like to express my deep appreciation to C hris Spec, the finest lab manager one can ever ask for. I am deeply grateful to all her constant unwavering support. Her continued encouragement and patience has facilitated m y work to proceed smoothly. Needless to say, in her, I have acquired a great professional and personal friend. I would like to thank Dr. Pradip Roy-Burmam for kindly providing guidance and giving me an opportunity to start my very first graduate research in his lab. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I thank several o f my fellow colleagues: Shekun He, M anila Jin, Jiehao Zhou, Ning Zhang, Cynthia Chinn, Audreea Gamulescu, Jennifer Yaung, Candy Chan, Klma Khankan, A runi D e S iv a, Yi Ding for being good friends and stickingg with me in good and bad times. Their true support and kind generosity have made my PhD study ail unforgettable experience. I thank Ernesto B arron and Tony Rodriguez for all the technical support in the crucial microscopic component o f my thesis work. L ast but not the least I thank Lisa Doumak, helpful secretary in pathology department, for taking tim e out o f her busy schedule to solve all my problems. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS D EDICA TION ..................................................................... ii ACKNOW LEDGEM ENTS ................... iii LIST OF FIG U RES .......... .......viii A B STR A C T.................................................................................. ix Chapter 1 Introduction: Bone Morphogenic Proteins (BMPs) and Growth Factor Regulation in Ocular D iseases.............................................. 1 1-1. Summary...........................................................................................................................................I 1-2. Bone Morphogenic Proteins (BM Ps)................................................................................................. 2 1-2-1. Bone Morphogenic Proteins (BM Ps)........................................................................................2 1-2-2. BMP Receptors and Signaling................................................................................................... 3 1-2-3. BM P4................................................................................................................................................4 1-3. Human Retinal Pigment Epithelium.................................................................................................. 5 1-3-1. RPE Anatomy and H istology................ 5 1-3-2. RPE cellular functions....................................................................... 6 1-4. Age-Related Macular Degeneration (A M D )....................................................................................8 1-4-1. D rusen..............................................................................................................................................9 3-4-2. Geographic A trophy..................................................................................................................... 9 1-4-3. W et AM D/Neovascular.............................................................................................................. 10 1-5. Regulation of the RPE cell functions and behaviors by growth factors/cytokines in Age- Related M acular Degeneration..............................................................................................................11 1-5-1. RPE Activation and Growth Factors and C ytokines............................................................1 1 1-5-2. Regulation of RPE by Growth Factors in A M D ...................................................................12 1-6. R eferences.........................................................................................................................................14 Chapter 2 Bone Morphogenic Protein 4 (BMP4) and Its Role in The Pathogenesis o f Dry Age- related M acular Degeneration (AM D)...........................................................................................................22 2-1. Summary..................................................................................................................................................22 2-2. Introduction..................................................................................................................... 24 2-3. M aterials and methods..........................................................................................................................26 2-3-1. A nim als.......................................................................................................................................... 26 2-3-2. Human Sam ples............................................................................... 26 2-3-3. Human RPE C u ltu re....................................................................................................................27 2-3-4. ARPE-19 Culture with BMP4 Treatm ent..............................................................................27 2-3-5. ARPE-19 Monolayer Grown on Permeable Transwell Filters and BMP4 E L IS A 27 2-3-6. P-galactosidase A ctivity............................................................................................................. 28 2-3-7. RT-PCR .................................................. 28 2-3-8. Immunohistochemistry.............................................................................................................. .29 2-3-9. Immunogold Electron M icroscopy............................... 29 2-3-10. Scanning Electron M icroscopy ....................................... 29 2-3-11. Senescence-associated p-galactosidase Staining........................................... 30 2-3-12. BrdU Incorporation............................. 30 2-4. R esults .......... 30 2-4-1. BMP4 is Preferentially Expressed in Retinal Pigment Epithelium and Ciliary Body. 30 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-4-2. Expression o f BMP4 and Its Three Receptors (BMPRIA, BMFRIB, and BMPRII) in RPE Cultures................................................................................................. 31 2-4-3. Polarized BMP4 Expression o f BMP4 in ARPE Cultures on Transwell F ilters 31 2-4-4. Expression o f BMP4 in Human Norma! and Macular Degeneration T issues.............. 33 2-4-5. Electron-Microscopic Immunohistochemistry o f BMP4 in Human Macular Degeneration Tissues ........................................................................................................... 36 2-4-6. Induction o f Senescence and Proliferation Arrest by BMP4 in ARPE-19 C ells.......... 36 2-5. Discussion.............................................................................................................................................. 41 2-6. References ...... 44 Chapter 3 Bone Morphogenic Protein 4 (BMP4) As A Negative Regulator in Choroidal N eovascularization..................................................................................... 48 3-1. Summary.................................................................................................................................................48 3-2. Introduction............................................................................................................................................49 3-3. Materials and methods......................................................................................................................... 51 3-3-1. A nim als..........................................................................................................................................51 3-3-2. Human Sam ples.................................................................. 51 3-3-3. Induction o f CNV........................................................................................................................ 52 3-3-4. Histopathological Study.................. 52 3-3-5. Fluorescein A ngiogram s............................................................................................................52 3-3-6. P-galactosidase Activity.............................................................................................................53 3-3-7. Human and Bovine Choroidal Endothelial Cell C ulture....................................................53 3-3-8. In Vitro Tube Form ation............................................................................................................54 3-3-9. RT-PCR..........................................................................................................................................54 3-3-10. Relative Quantitative Real-Time R T -PC R ..........................................................................54 3-3-11. Statistic A nalysis....................................................................................................................... 55 3-4. Results..................................................................................................................................................... 55 3-4-1. BMP4 Expression Is Absent in Surgically Removed Human Choroidal Neovascularization Membrane (CNVM )........................................................................................... 55 3-4-2. BMP4 Is Down-regulated During Laser-induced CNV Progression.............................. 56 3-4-3. BMP4 Haploinsufficiency Promoted Neovascularization in Laser-Induced CNV M odel..........................................................................................................................................................59 3-4-4. Preferential Expression of BMP4 Receptors in Choroidal Endothelium in vivo and Up-regulation o f BMPRII by ECM in fCEC in vitro.......................................................................62 3-4-5. BMP4 Inhibits In Vitro Choroidal Endothelial Cell Tube Formation............................. 62 3-4-6. BM P4 Is Regulated by VEGF and TN Fa in fRPE.............................................................. 65 3-5. Discussion................................................................................................................................................67 3-6. R eferences........................... 70 Chapter 4 Future Direction: Development o f a Cell-type-specific Cre Transgenic Model and Its Potential Application for Site-Specific Mutation o f BMP4 in the Retinal Pigment Epithelium (RPE).......................................................................................................................................................................73 4-1. Summary ...... 73 4-2. Introduction.............................................................................................................................................75 4-3. M aterials and m ethods............................................... ................................ ........................................77 4-3-1. Transgene Construction ...... 77 4-3-2. Generation of PB-Cre Transgenic M ice................................................................................. 78 4-3-3. Cross Breeding o f PB-Cre mice with R26R Reporter Strain and RXRct Floxed Strain .......................................................................................................................................................................78 4-3-4. RT-PCR................ 79 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-3-5. X-gal Staining and H istology ....................... 79 4-3-6. DNA Preparation and PCR Analysis ofRXRot A lleles..................................................... 80 4-3-7. Tissue Preparations.................................................................................................................... 80 4-4. Results.................................................................................................................................................... 80 4-4-1. Generation o f ARR2PB-Cre4 M ice..................................................................................... 80 4-4-2. Tissue-specific Cre recombination A ctivity ............................................................... 83 4-4-3. Onset and Extent o f Cre Activity' During Postnatal D evelopm ent ....... 90 4-4-4. Evidence for the Utility? o f The Model: Conditional Mutation o f RXRa alleles ......... 92 4-5. Discussion ....... 95 4-6. R eferences..................................................................... 98 Chapter 5 Conclusion.............................................................................................. 102 BIBLOGRAPHY..............................................................................................................................................105 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 2-1: Beta-gal Staining.. Error! Bookm ark not defined. Figure 2-2: R T -PC R Error! Bookm ark not defined. Figure 2-3: Immunofluorescent Staining ....Error! Bookm ark not defined. Figure 2-4: Immunofaistochemieal Staining E rror! Bookm ark not defined. Figure 2-5: Electron-Microscopic IH C E rror! Bookm ark not defined. Figure 2-6: Scanning EM and BrdU Incorporation E rror! Bookm ark not defined. Figure 3-1: Immunohistochemical Staining E rror! Bookm ark not defined. Figure 3-2: Quantitative Real-Time P C R E rror! Bookm ark not defined. Figure 3-3: Beta-gal Staining E rror! Bookm ark not defined. Figure 3-4: Fluorescein Angiogram o f CNV............................................ E rror! Bookm ark not defined. Figure 3-5: H-E Stainning............................................................................ E rror! Bookm ark not defined. Figure 3-6: R T -PC R .......................................................................................E rror! Bookm ark not defined. Figure 3-7: Tube Form ation......................................................................... Error! Bookm ark not defined. Figure 3-8: Quantitative Real-Time P C R ..................................................Error! Bookm ark not defined. Figure 4-1: ARR2 P B -C re.................................................................... ...Error! Bookm ark not defined. Figure 4-2 Beta-gal S taining....................................................................... E rror! Bookm ark not defined. Figure 4-3: Cre Activity in Seminal V esicles.......................................... Error! Bookm ark not defined. Figure 4-4: Cre Activities in Testes and Ovaries..................................... Error! Bookm ark not defined. Figure 4-5: Postnatal Prostatic Cre A ctivity..............................................E rror! Bookm ark not defined. Figure 4-6: PCR................................................................................................E rror! Bookm ark not defined. viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Identification of a gene that causes AMD or a condition that very strongly resembles AMD allows us to delineate the biochemical pathway in which causes o f the disease might be understood and various treatment strategies might be developed. In this current study, we investigated the regulation of BMP4 expression in AMD and examined the possible roles it may play in AMD pathogenesis. Hopefully, it will lead us to a better understanding of AMD pathogenesis and better disease prevention in the future. We systemicaily investigated BMP4 expression and its detailed location in different stages o f dry AMD. We established an in vitro RPE monolayer model to study the preferential secretion o f BMP4. Further, we created an in vitro RPE senescence model which enables us to demonstrate for the first time that BMP4 inhibits RPE proliferation and facilitates their progression into senescence. The pathogenesis o f wet form o f AMD is more complicated and involves more than one cell type. W e provided both in vivo and in vitro data suggesting BM P4 is a major inhibitor o f angiogenesis in wet AMD, including inhibition o f CEC in in vitro angiogenesis, more severe wet AMD phenotype in BMP4 haploinsufficient mouse model and regulation o f BMP4 by TNF and VEGF. We have successfully generated prostate epithilium-specific Cre mouse model and the model has been applied to generate various gene inactivation in the prostate. Despite the tissue difference, same concept and technology can be applied to the retinal tissue and BM P4 inactivation in RPE can be achieved using either virus-mediated Cre or transgenic Cre mouse model. The results will provide strong information to dissect mediating pathways o f RPE activation in AMD and a potential therapeutic target in AMD. ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In summary, identification o f genes whose function or expression is altered in AMD rem ains critical for understanding and preventing AMD. This project successfully demonstrated that BMP4 plays critical roles in the regulation o f RPE during different stages of AMD. An understanding o f the mechanisms involved and how they are regulated provides insight into the pathophysiology and potential therapeutic targets in AMD. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1 Introduction: Bone Morphogenic Proteins (BMPs) and Growth Factor Regulation in Ocular Diseases 1-1. Summary This PhD thesis is focused on the differential regulation o f Bone Morphogenic Protein 4 (BMP4) by retinal pigment epithelial cells (RPE), and how this regulation o f BMP4 alters RPE structure and function. Furthermore we evaluate the role o f dysregulated BMP4 as a potential mediator of age-related macular degeneration (AMD). This chapter gives an overview o f the histology and function o f RPE cells, an introduction to AMD pathogenesis, and a review o f the primary role of cytokines/ growth factors and RPE cells in this disorder. A focused review o f the structure and function o f BMP4 and its receptors is also provided. Chapter 2 describes how increased BMP4 m ay contribute to the pathogenesis o f dry form o f AMD. We show the vectorial secretion o f BMP4 by RPE in vitro, the up-regulation o f BM P4 in dry AMD tissues, and the induction o f RPE senescence by BMP4 in vitro. Chapter 3 demonstrates how dysregulated BM P4 may play a role in the pathogenesis of neovascular AMD, and the inter relatioship between BMP4 and other growth factors found in AMD such as vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF). In chapter 4 the creation o f a tissue-specific Cre model in the prostate and the potential application of this novel technology to ocular tissue to further study the role o f RPE expression o f BMP4 in mouse model of AMD are discussed. Chapter 5 presents conclusions and future directions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1-2. Bone M orphogenic Proteins (BMPs) 1-2-1. Bone Morphogenic Proteins (BMPs) Bone morphogenic proteins (BMPs) were initially purified from demineralized bone extracts by their ability to induce the formation o f ectopic cartilage and bone after transplantation into the connective tissue o f rodents. ’’ 2 M olecular cloning revealed that they belong to die TGF-j3 superfamily. 3' 4 There are more than 20 members in species ranging from C. elegans to humans that can be subgrouped according to the homology in their sequence.3’ 4 Based on the degree o f sequence identity or homology in the mature carboxyl domain, BMPs are grouped into several classes. 5 Among the classes, BMP2, BMP4, BMPS, BMP6 , BMP7, BMP8 A, and BMP8B are best characterized. BMPs were originally named for their ability to induce ectopic bone formation, ’ ’ 6 later they were found to have important roles in a wide range of cellular processes, including cellular proliferation, differentiation, motility, adhesion, cell death and the specification of developmental cell fate during embryogenesis. 5’ 7’ 8 T hey are involved in the formation and patterning of various tissues, including the central nervous system (CNS), skeleton, heart, kidney, gut, lung, liver, teeth and eyes. 5’ 9 ’ 10 These multifunctional proteins which are not only important during mouse development, but also in the adult function o f various organs. BMPs are synthesized and folded as large dimeric pro-proteins in the cytoplasm and cleaved by proteases during secretion. 1 1 , 5 Each monomer contains about 300 amino acid residues as the pro- protein. The functional carboxyl region (100-120 amino acid residues in each monomer) approximately 21-25 kd in size, is then released into the extracellular matrix to bind to membrane receptors on target cells. Dimerization relies on the disulfide bonds between the two subunits. u Although homodimers are considered the standard form, there are natural heterodimers with equal bioactivity. 1 3 , 1 4 Furthermore, the exact concentration o f active BMPs seems to be important for rendering a specific effect. Therefore, there are numerous proteins known as BMP antagonists binding to specific BMPs in the extracellular com partment to tightly regulate their functions. 13 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These antagonists directly interact w ith BMPs and prevent the binding o f BMPs to their receptors. The large number and still growing list o f BMP antagonists includes noggin, chordin, chardin-like, follistatin, FSRP, the DAN protein family, and sclerostin. 16’ i7, 18' 1 5 Therefore, the regulation o f BM P activities and functions is very complex at the molecular and biochemical levels. 1-2-2. BMP Receptors and Signaling Despite the growing number o f BMP family members, there are only three type-I (activin receptor like kinase-2 (ALK-2), BMP type IA receptor (BMPR1A) and BMPR1B) and three type-II receptors (BMPRII, ActR-II and ActR-IIB) are known. 1 9 , 5’ 2 0 Different BMPs bind with different affinity to the three type I receptors, for instance, BMP4 preferentially binds to BM PR1A and BMPR1B while BMP? binds with higher affinity to ALK-2 and BMPR1B. Binding o f the BMP to at least one type-I and one type II receptor is necessary for activation of the BMP signal. 2 1 - 1 9 BMPs bind with high affinity to the heteromeric type I/type II receptor complex. Upon ligand- receptor complex formation, type I receptors possessing constitutively active kinase activity phosphorylate type I receptors. The activated type I receptor initiates intracellular signaling by phosphorylating specific downstream SMAD proteins (R-SMADs). 2 2 , 23 There are three distinct classes o f SMAD proteins: the receptor-activated SMADs (R-SMADs), the common-mediator SMADs (Co-SMADs) and the inhibitory SMADs (I-SMADs). Upon phosphorylation by type I receptor R-SMADs are released rapidly to interact with Co-SM ADs to form hetero complexes which then translocate into the nucleus to regulate the transcription o f various target genes. In the nucleus, SMADs exert transcriptional activity through direct binding to DNA as well as through cooperation w ith co-activators and co-repressors. 24’ 25 BMP-induced activation o f R- and Co- SMADs is negatively regulated by inhibitory SMADs. I-SMADs can antagonize the BMP signaling pathway by interacting w ith activated type I receptors and thereby preventing access o f R-SMADs to the type I receptors. 2 6 , 27 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Although BMPs activate the SMAD pathway to modulate gene transcription, there is growing evidence that other pathways distinct from the SMAD pathway are initiated downstream of the receptor complex. 28 BMPs have been shown to activate the p38 mitogen activated protein kinase (MAPK) pathway to control apoptosis. RAS and ERK pathway can also been activated by BMP2 to stimulate osteoblasts. 1-2-3. BMP4 BMP4 is a 408 aa prepropeptide with a 19 aa signal sequence, a 273 aa pro-region, and a 116 aa mature segment. Comparison o f the mature regions o f human, mouse and rat reveals 98% aa identity. 29’30 BMP4 is closely related to BMP2 and to Drosophila decapentaplegic (DPP). BMP4 plays an important role in embryonic development. BMP4 is expressed in multiple embryonic tissues, including the heart, lung, kidney, brain and eye. 5 ’ 30 BM P4 helps to establish the dorsal-ventral axis in early Xenopus embryo 31 and it has a similar critical function in vertebrates. Mouse embryos with homozygous inactivation o f BMP4 die between 6.5 and 9.5 days p.c. with little or no mesodermal differentiation. 3 0 BM P4 also participates in later development processes, including outgrowth and patterning o f facial primordia, 3 2 patterning limb buds 33 and induction of cardiac myogenesis. j 4 BMP4 has crucial roles for optic development, especially for the lens induction process in mice. In the eye, BMP4 expression is first identified in the distal optic vesicle and overlying surface ectoderm at the 8-12 somite stage and later in the dorsal portion o f the developing optic cup. Lens induction is absent in the homozygous BM P4 mutants and can be rescued by exogenous BM P4. 35 Heterozygous deficiency o f BMP4 results in elevated IOP and anterior segment abnormalities, including malformed, absent or blocked trabecular meshwork and Schlemm’s canal drainage structures. 3 6 BMP4 and Msx genes are generally involved in morphogenesis, cell differentiation, and also induction o f apoptosis. 3 7 ' 3 j ' 38 BMP4 stimulates the phosphorylation and translocation to the nucleus of Sm adl, where it regulates the transcription o f target genes such as homeobox genes 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. encoding M sxl and Msx2 in various developing systems. l9’ 40 Overexpression of MSX2 perturbs BM P4 signaling in the developing eyes and results in optic nerve aplasia and microphthalmia in ail transgenic anim als.4 1 BMP4-mediated activation o f downstream Smads involves ligand binding to the liigh-affinity receptor BMPRI and subsequent recruitment o f BMPRII into the complex. This is followed by phosphorylation o f Smads 1, 5, and 8, association with Smad4, and translocation to the nucleus. M embers o f the BMP-Smad pathway can also physically interact w ith components of other signaling pathways to establish cross talk. 42 Thus BMP4 ligand may trigger multiple downstream pathways; p38 MARK, ERK1/2 and INK are all activated by BMP4 in lung fibroblasts to exert anti-proliferative and pro-differentiation effects. 4j In osteoblasts p38M APK kinase positively regulates BMP4-stimulated VEGF synthesis. 44 1-3. Human Retinal Pigment Epithelium 1-3-1. RPE Anatomy and Histology Retinal pigment epithelial (RPE) cells form a monolayer critically located between the neural retina and the vascular choroid. In flat preparations, the RPE cells are hexagonal and darkly pigmented with melanin. The RPE cells are bound together by junctional complexes at the lateral domains. The tight junction is the most apical component o f the junctional complex. The zonulae occludens between adjacent RPE cells form a tight intercellular junction as a result of interaction between extracellular domains o f adjacent occludin proteins, leading to the high transepithelial resistance of the RPE monolayer. 45, 46’ 4 7 The adherens junction is located near the apical region o f the plasma membrane, binds to a continuous belt o f actin filaments (the adhesion belt) and functions to hold neighboring cells together through a family o f C a2+-dependent cell-cell adhesion molecules known as cadherins 48 The RPE junctional complex is thought to be the main structure responsible for the outer blood-retina barrier derived from the leaky choroidal epithelium by preventing effusion of large molecules through the space between adjacent RPE cells. Polarity o f the epithelium also 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. allows unique cellular communication at apical surface o f the RPE with photoreceptor ceils and basally with the fenestrated choroidal vasculature. 43 At its apical surface, the RPE cell membrane has many microvilli, which interdigitate with the outer segments o f photoreceptor rods (ROS) and cones. However the plasma membranes of RPE and photoreceptors do not have any points o f contact, but rather are separated by a very thin layer o f extracellular matrix material called the interphotoreceptor matrix (IPM). The IPM, in conjunction with interdigitations between RPE microvilli and ROS and adhesion molecules expressed on the apical surface o f RPE cells are thought to be one o f the structures involved in maintaining adhesion and proximity o f the sensory retina to the RPE. 4 9 Close proximity of the large surface area o f the photoreceptor and RPE plasma membrane also facilitates exchange o f nutrients and wastes between these two cell types. 45 The subretinal space is a potential space between RPE and photoreceptors, that is manifest in the presence o f a retinal detachment. The basal plasma membrane domains of the RPE cell also contain many infoldings, which increase the surface area o f the membrane, thus facilitating exchange o f wastes and nutrients between the RPE cell and the choroidal blood supply. The RPE cell sits on a thick five-layered basement membrane known as Bruch’s membrane, which is composed of the basement membrane of RPE, inner collagenous zone, elastic fiber area, outer collagenous zone, and the basement membrane of the endothelium o f the choriocapillaris. Bruch’s membrane contains molecules typically found in basement membrane, including type IV collagen, heparan sulfate proteoglycan and laminin.49 The negatively charged proteoglycans in Bruch’s membrane may also contribute to blood-retina barrier by acting as a filter for macromolecules arriving via the choroidal circulation.30 Situated between the choroidal blood supply and the photoreceptor cell layer, this enzyme rich RPE cells mediate various critical functions o f the sensory retina. The barrier function of RPE cells allows the tight control the movement o f water and catabolites between retina and choriocapillaris, 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. which is critical for the neuro-retina attachment and nutrition. The RPE maintains retinal attachm ent by actively keeping the subretinal space dehydrated by moving water from the subretinal space with ionic transport systems including Na7K7-ATP pump and HCO3 ' transport system .51' 3 Z The breakdown of blood-retinal barrier has serious consequences for the health o f the eye and is present in many retinal diseases. RPE cells also participate in several activities that are critical in photorecptor function and viability. These activities include phagocytosis of shed photoreceptor rod outer segments (ROS), metabolism o f retinol, and interaction with light by its melanin granules. The internalization and subsequent degradation of shed ROS is one o f most important functions o f RPE ceils. The mechanism involved in ROS phagocytosis is a specific, receptor-mediated process. The receptors may include the mannose receptor, CD36, and OtvPs integrins.45 In the normal eye, RPE cells are thought to be highly selective for ROS phagocytosis. However, an in vitro study from our lab using subconfluent cells showed that activated RPE cells are able to phagocytose extracellular matrices (ECM), especially provisional ECM such as fibronectin and collagen type I. This may represent a novel mechanism for remodeling provisional ECM by RPE during the outer retinal wound healing process.53 In the normal eye, RPE cells secrete a wide spectrum o f cytokines and growth factors. Secreted cytokines and growth factors in the monolayer may act in an autocrine or paracrine way on the cell o f origin or adjacent RPE, or on adjacent photoreceptors or choroidal cell. In situ studies o f the RPE monolayer reveal expression o f transforming growth factor-beta (TGF-(3), fibroblast growth factor-1 and - 2 (FGF-i, FGF-2), and platelet-derived growth factor (PDGF). Local production of TGF-|3 by RPE results in an immunosuppressive microenvironment, while FGFs may be acting as survival-promoting factors.45 Chemokines and inflammatory cytokines are secreted in significant amounts only after RPE activation. 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Due to its critical anatomic location and its ability to interact with surrounding tissue elements, the RPE cell is often involved in retina! pathophysiology, specially in age-related diseases. The RPE shows specific features associated with aging. (M s become more irregular in size and shape, deposits from RPE accumulate in Bruch’s membrane, and lipofuscin appears in the cell’s cytoplasm. With increasing age, a gradual loss o f RPE cells occurs, and the remaining cells increase in size. 54 In culture, RPE cells exhibit decreased proliferative capacity with age. 5 3 All these aging modification inevitably makes changes in the RPE-Bruch’s membrane-choriocapillaris complex, w hich further may contribute to the pathogenesis o f AMD. 1-4. Age-Related Macular Degeneration (AMD) AMD is the leading cause of visual impairment and blindness in the United States and the developed world among people 65 years and older. 56 AMD prevalence was observed in 9% o f the population from ages 52-85 years old in the Framingham study and in 33% of autopsied eyes obtained after death from people older than 65 years. 5 7 ' 58 Roughly 30% o f the human population 75 years or older has some degree o f AMD. As the average life span o f humans continues to increase, particularly in the developed countries, the incidence o f AMD is expected to nearly double within the next 25 years. 59 AMD is a chronic and progressive degeneration o f photoreceptors, the RPE, Bruch's membrane and possibly the choriocapillaris in the macula. 60’ 61' 6 2 The macula is a highly specific region, 6 mm in diameter, located in the central retina temporally to the optic disc. A t the center o f the macula lies the fovea, 0.35 mm in diameter, which contains a population o f photoreceptor cells responsible for reading and color vision. The clinical presentation o f AMD includes drusen, hyperplasia o f RPE, geographic atrophy (dry AMD), and choroidal new vessels (CNVs) (wet AMD). 6 3 Both dry and w et forms are considered more advanced forms o f AMD. To date there is no proven treatment for advanced AMD. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1-4-1. Dmsen The earliest morphologic feature of AMD is the development o f dmsen. Dmsen are yellowish deposits of extracellular materials, localized between the basement membrane of the RPE and inner collagenous layer o f Bruch’s membrane. Clinically, dmsen are broadly classified as soft or hard according to morphology. Hard drusen are globular deposits o f hyalinized material with amorphous appearance on electron microscopy. They are less than 63 pm in diameter. 64 Soft drusen are generally larger (greater than 125 pm) and have a soft appearance. 65 They have an obvious thickness and tend to become confluent because o f indistinct margins. On fluorescein angiography, soft dmsen display weaker and delayed hyperfluorescence. With increasing age, drusen can become calcified or filled with cholesterol, appearing crystalline or polychromatic. Typically dmsen are clustered in the central macula. 66’61 D m sen constitute a major risk factor for visual deterioration with aging. Large or confluent dmsen are regarded as indicative of insufficient cellular function in the RPE due to various insults. Because its special location, drusen likely create a hydrophobic barrier that mpede the normal balance between the RPE and choriocapillaris, eventually resulting in detachment and loss o f overlying RPE. Dmsen may also perturb photoreceptor cell function by placing pressure on rods and cones or by distorting photoreceptor cell alignment. Dmsen has been shown to be a significant strong risk factor leading to development o f both advanced dry or w et AMD. 6 8 ,6 9 ,7 0 The presence o f soft, large or confluent dmsen is correlated with the subsequent development o f geographic atrophy or CNV. However, precise information about the origin and composition o f dmsen is still lacking. 1-4-2. Geographic Atrophy Geographic atrophy is the end result o f the atrophic form o f dry AMD. It is characterized by changes in the RPE including attenuation, atrophy, hypertrophy, hyperplasia, hypopigmentation or absence of the RPE. 6 9 , 71 Loss o f overlying photoreceptor layer, 7i’ 73 and occasionally absence o f 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. underlying choriocapillaris are also accompanied. Drusen o f all types accompany geographic atrophy. Atrophy o f the RPE and overlying photoreceptor layer is present in 37% o f AMD eyes at autopsy. 7‘ Affected areas of geographic atrophy gradually result in lost visual function, most likely because loss o f the RPE is associated with fallout of photoreceptors which are metabolically dependent on underlying RPE cells. Choriocapillaris at atrophic areas tend to have relatively less vessel density and narrowing vessel lumens. Attendant loss o f VEGF and other trophic factors following the loss of RPE cells may account for the atrophy o f the choriocapillaris endothelium. The presence o f drusen measuring 250 ,ttm or greater is a risk factor for the development o f geographic atrophy. Increased fundus autofluorescence precedes the development and enlargement o f geographic atrophy. 74 Thus, excessive RPE lipofuscin accumulation also may play a critical role in geographic atrophy pathogenesis in AMD. The RPE continuously discharge cytoplasmic material into Bruch’s membrane, which could lead to decreased nutrients and increased metabolic abnormalities. Atrophy of the RPE may be a response to such abnormalities in areas o f excessive accumulation o f extracellular debris. 1-4-3. W et AMD/Neovascular The major feature o f wet AMD is choroidal neovascularization (CNV) and its associated manifestations such as serious RPE detachment, RPE tears, pigment alteration, and vitreous hemorrhage. 75 The early stage o f CNV is observed in a small percentage o f eyes and may be promoted by cellular breakdown of Bruch’s membrane. 76 M ost eyes with CNV are clinically occult as defined by fluorescein angiographic features: fluorescein leakage o f undetermined origin 77 and fibrovascular RPE detachm ent.78 Histopathologically, CNV appears to begin in the choroid and extends into a plane between basal lamina of the RPE and the remainder of Bruch’s membrane. 71 Occasionally, the vessels extend through the RPE into the subretinal space. Following penetration through breaks in Bruch’s membrane, the new vessels proliferate laterally and develop a more organized vascular system with 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fibrous tissue. ' 1 Because the endothelial cells in the neovascular tufts lack the barrier function of m ature endothelial cells, they can leak fluid and fluorescein. D espite the theories and concepts that have been advanced, it still remains unknown what causes the vascular ingrowth o f choroidal vessels. Soft drusen, which some investigators believe suggest widespread RPE abnormality,69 have been histopathologically associated with CNV. Drusen may act as an indirect angiogenic factor by attracting macrophages from the choroid.79 However, w hether these macrophages act as mediators o f the degenerative changes seen in Bruch’s membrane or directly stimulate new vessel growth remains unknown. Additionally, angiogenic factors such as vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) may contribute to the newvascular formation. 8 0 Dysregulation o f these and other growth factors may result in an imbalance between stimulating and inhibiting modulators. 1-5. Regulation of the RPE cell functions and behaviors by growth factors/cytokines in Age- Related M acular Degeneration 1-5-1. RPE Activation and Growth Factors and Cytokines RPE plays a pivotal role in AMD pathogenesis. The functional status o f RPE cells in the disease is poorly understood. Histopathologic studies reveal pigment clumping, loss o f pigment, heterogeneity o f cell sizes and shapes, formation o f multiple layers, and presence o f pigmented cells in the subretinal space.71’ 73 A growing body o f evidence suggests that RPE cells are programmed to react to injury or environmental changes with alterations in cell phenotype, function, and gene expression.8 1 , 82 These morphologic and functional changes may be referred to as activation. Mediators of activation may include ECM components, growth factors, cytokines, accumulated products in Bruch’s membrane, Drusen, or hypoxia. These mediators, acting alone or in concert, result in alterations in gene expression o f RPE. Although the functional significance o f 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. these RPE changes in fibrocontractive diseases is apparent, their function in AMD is much less clear. Growth factors, chemokines and cytokines are cell-secreted mediators o f autocrine and paracrine functions, involved in cell maintenance, survival, growth and death, as well as in angiogenesis, vascular permeability, inflammation and other processes. A wide spectrum o f cytokines and growth factors are secreted by the RPE. Secreted cytokines and growth factors in the monolayer may act in an autocrine or paracrine way on the cell o f origin or adjacent RPE, or they may have paracrine effects on adjacent photoreceptors or choroidal cells. M any growth factors have been implied in AMD, mainly based on experimental data obtained from studies in cultured RPE and from histopathological studies in surgically removed human CNV or in laser-induced CNV in normal or transgenic rodent models 8j' 84. These studies have identified the RPE not only as an important source o f growth factors involved in tissue maintenance, homeostasis, inflammation and O f O o A CNV formation, but also as the central cell type acting in and regulating these processes . 1-5-2. Regulation ofR PE bv Growth Factors in AMD AMD involves aging changes plus additional pathological changes. Cellular senescence is observed in a wide variety o f human cell types, and has been accused of contributing to the development o f AMD. M arkers o f senescence, such as altered gene expression and shortening o f chromosomal telomeres, have been identified in cultured RPE cells exposed to advanced glycation endproducts (AGE), which form in Bruch’s membrane with age, 87 The presence o f senescence-related beta- galactosidase activity in RPE o f older m onkey eyes has been documented.88’ 89 Little is known about the m olecular changes involved in RPE senescence, but studies in other cells suggest that senescent cells show different patterns o f gene expression from non-senescent cells. Both the near ultraviolet-irradiated RPE cells and aged RPE cells express markedly less pigment epithelial derived factor (PEDF) and tissue inhibitor o f metalloproteinase-3 (TIMP-3) . 90 The former is a known neurotrophic and anti-angiogenic factor. Successive passage o f cultured RPE ceils results in 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. diminished PEDF production,9" suggesting an age-related decline, although this is not universally observed.9^ The retina is particularly susceptible to oxidative damage. RPE dysfuction mediated by reactive oxygen intermediates (ROI) has been suggested as a possible cause of AMD. Oxidative stress results in RPE injury which elicits an inflammatory response in the Bruch’s membrane and the choroid. RPE injury may also foster the production o f abnormal ECM which in turn, results in further damage to the RPE and the retina. Findings from in vitro and in vivo animal studies indicate that basal laminar deposit may form as a result of free radical-induced lipid peroxidation of RPE cell membranes with subsequent membrane blebbing and accumulation in the sub-RPE space. Advanced giycation endproducts (AGE) occur at sites of oxidant stress with hydroxyl radical formation. AGE occur in soft drusen, in basal laminar and basal linear deposits, and in the cell cytoplasm o f RPE associated with CNVs .9j Oxidative stress may also promote neovascularization. H2 O2 induces a dose-dependent reduction in the expression o f PEDF, an endogenous anti- angiogenic molecule.94 ROI also upregulate VEGF in RPE. 95 Oxidative stress leads to deposition of extracellular matrix along Bruch’s membrane and increased levels o f the angiogenic factor fibroblast growth factor 2 (FGF2) in RPE cells.96 The mechanisms of CNV and associated CNV membrane (CNVM) formation are being elucidated but are still not fully understood. 9 7 ' 9 8 CNV tissues have been found to contain high amounts of several growth factors including basic fibroblast growth factor (bFGF), transforming growth factor- 8 (TGF-B), insulin-like growth factor-1 (IGF-1), and VEGF. VEGF is the major angiogenic stimulant in the development of CNV. The source of VEGF is likely to be the RPE cells which are known to produce VEGF. It is, however, not clear how RPE cells are stimu lated to release VEGF in the chain o f events leading to CNV. In addition to VEGF, the two main members o f the angiopoietin family o f growth factors, Ang-1 and Ang-2, were also demonstrated at the protein level in human CNV tissue in migrating RPE cells, suggesting involvement o f these growth factors 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in vascular maturation, stabilization and remodeling in CNV 99. Recently, expression of connective tissue growth factor (CTGF) in human CNV was reported !0°. CTGF is a pro-fibrotic factor also capable o f inducing angiogenesis. In summary, one possible mechanism in the pathogenesis o f AMD is related to a dysfunction o f the RPE with production o f growth factors and cytokines which results in an impairment of normal homeostasis o f the retina. 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Matsunaga H, Handa JT, Gelftnan CM, Hjelmeland LM: The mRNA phenotype of a human RPE cell line at replicative senescence. Mol Vis 1999, 5:39 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93. Ishibashi T, Murata T, Hangai M, Nagai R, Horiuchi S, Lopez PF, Hinton DR, Ryan SJ: Advanced glycation end products in age-related macular degeneration. Arch Ophthalmol 1998,116:1629-1632 94. Ohno-Matsui K, Morita I, Tombran-Tink J, Mrazek D, Onodera M, Uetama T, Hayano M, Murota SI, Mochizuki M: Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF. J Cell Physiol 2001, 189:323-333 95. Kuroki M, Voest EE, Arnano S, Beerepoot LV, Takashima S, Tolentino M, Kim RY, Rohan RM, Colby KA, Yeo KT, Adamis AP: Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest 1996, 98:1667-1675 96. Mousa SA, Lorelli W, Campochiaro PA: Role o f hypoxia and extracellular matrix- integrin binding in the modulation o f angiogenic growth factors secretion by retinal pigmented epithelial cells. J Cell Biochem 1999,74:135-143 97. Campochiaro PA, Soloway P, Ryan SJ, Miller JW: The pathogenesis o f choroidal neovascularization in patients with age-related macular degeneration. Mol Vis 1999, 5:34 98. Zarbin MA: Age-related macular degeneration: review o f pathogenesis. Eur J Ophthalmol 1998,8:199-206 99. Hangai M, Murata T, Miyawaki N, Spee C, Lim JI, He S, Hinton DR, Ryan SJ: Angiopoietin-1 upregulation by vascular endothelial growth factor in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2001,42:1617-1625 100. He S, Jin ML, Worpel V, Hinton DR: A role for connective tissue growth factor in the pathogenesis o f choroidal neovascularization. Arch Ophthalmol 2003,121:1283-1288 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Bone Morphogenic Protein 4 (BMP4) and Its Role in The Pathogenesis of Dry Age-related M acular Degeneration (AMD) 2-1. Sum m ary To investigate the expression and pathogenic potential o f Bone M orphogenic Protein 4 (BMP4) in dry age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) expression of BM P4 was investigated in a BMP4 haploinsufficient mouse with P-galactosidase reporter activity. Reverse transcription-polymerase chain reaction (RT-PCR) was used to study the expression of BMP4 and its receptors in RPE culture. Vectorial secretion of BMP4 was assessed using transwell culture and enzyme-linked immunosorbent assay (ELISA). Expression and localization o f BMP4 in the retina and RPE under normal and pathologic conditions was investigated using immunohistochemistry and immunogold electron microscopy. The effects o f BMP4 on induction of replicative senescence in RPE was studied by serial passaging o f ARPE-19 cells. In BMP4 haploinsufficient mouse eyes BMP4 reporter expression is restricted to the RPE and the epithelial cells of the ciliary body. No expression o f BMP4 in retina or choroids was detected. RT -PCR demonstrated abundant expression o f BMP4 and BMP4 receptors (BMPR1A, BMPR1B and BMPRII) in human RPE cultures. ARPE-19 cells grown as monolayers on transwells revealed preferential secretion of BMP4 from the basolateral domain. Immunohistochemistry on human tissue samples from early AMD showed a marked increase in BMP4 expression in RPE and in the vicinity o f Bruch’s membrane adjacent to hard and soft drusen. In contrast, BMP4 expression was not seen in the RPE or vascular tissue from normal aging controls. Electron microscopy demonstrated the sub-RPE localization o f BMP4 in human AMD samples. Continuous treatment 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o f ARPE-19 cells with BMP4 induced a senescence phenotype and decreased proliferation. Our findings suggest that BMP4 may serve as a significant negative growth regulator that exhibits differential effects in the pathogenesis o f dry AMD. Modulation o f expression o f the BMP4 may provide a novel approach to control various processes involved in the progression o f age-related m acular diseases. 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-2, Introduction AM D is the leading cause o f blindness and visual disability in patients 60 years or older in the western hemisphere.1 0 1 Macular degenerative changes have typically been classified into two clinical forms, dry or wet, both o f which can lead to visual loss. The visual loss is usually gradual. One o f the earliest clinical abnormalities s the accumulation o f Drusen. Larger drusen may becom e confluent and progress to promote RPE atrophy or to exudative disease. Despite the fact that different concepts relevant to the cell biology o f AMD have been p u rsu ed /02 the pathogenesis o f AMD is still poorly understood. RPE is one o f the main cellular participants in the pathogenesis of dry A M D.1 0 3 The deposition o f lipids and lipofuscin granules in the RPE increase with age, thereby altering the physiological functions of the RPE . 1 0 4 The features o f RPE aging are present in AMD eyes and may contribute to the pathogenesis o f AMD. It has been widely accepted that senescent cells accumulate in vivo, where their altered phenotype contributes to age-related pathology . 1 0 5 ’ 1 0 7 The major characteristics o f senescence include irreversible replication exhaustion, enlarged and flattened cell morphology and altered gene expression . 1 0 8 1 0 9 RPE cells lose replicative capacity in culture after a specific number o f population doublings and have been largely studied as an in vitro model of aging. 1 1 0 ’ 113 Senescent RPE cells have been found both in vivo and in vitro . 1 14 Recently, a technique utilizing a histochemical staining procedure for beta galactosidase to study senescent cells in vivo has been developed 1 1 5 and applied to study RPE cells in vivo. Senescence-activated p-galactosidase (SA-P- gal; pH 6.0) has been used as a biomarker to identify senescence in human cells. In addition to RPE, AMD also affects the interaction between the RPE and Bruch’s membrane and choriocapillaris. Increased amount o f several growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been shown to be associated with the dysfunctional R PE.8 3 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Boris morphogenetic proteins (BMPs) are a large sub-family (more than 20 members) of the TGF- p superfamily, which includes TGF-ps, activins/inhibins, and M ullerian inhibiting substance (M IS).116 Along with their ability to induce bone formation as suggested by the name,6 BMPs are involved in many biological processes as diverse as cell proliferation, differentiation, migration 117 5 118 ’ 10 and apoptosis, cell-fate determination and morphogenesis. BMP4 plays an important role in embryonic development. Homozygous BMP4 mutants die early in embryonic development and show little or no mesodermal differentiation.30 BMP4 is also involved in later developmental processes, including outgrowth and patterning o f facial primordial/ 2 patterning o f limb bu d s120 33 and induction of cardiac myogenesis.3 4 During development, BMP4 is expressed in multiple tissues including the heart, lung, kidney, brain and 5 30 eye. In the eye, indirect suppression o f BM P4 expression resulted in optic nerve aplasia and microphthalmia in M sx2 transgenic m ice .41 Lens induction is absent in homozygous disruption o f BM P4oS while a variety o f ocular abnormalities including anterior segment dysgenesis (ASD), elevated intraocular pressure (IOP), and posterior segment abnormalities developed in heterozygous BMP4tacZneo m utants .3 6 As originally reported by Lawson et al,1 2 1 the first protein coding exon o f the BMP4 gene was replaced w ith a reporter cassette encoding beta-galactosidase with a N-termina! nuclear localization signal. BMP4lacZneo heterozygotes are appropriate to precisely monitor temporal and spatial levels o f BMP4 expression and thus were used in our study. Together, these mice provide strong evidence that BM P4 is an im portant candidate to study ocular development and diseases. There are at least thee types of BMP receptors; a 53 kDa type I receptor, a 70-85 kDa type II receptor and a 200-400 kDa type III receptor. 1 2 2 Only type I and type II receptors, which are transmembrane serine-theonine kinases receptor complexes, appear to play significant roles in BMP binding and signaling.1 2 3 U4 Type II receptors may be the primary ligand-binding subunit in 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the functional receptor-binding complex, however, the activity o f both o f the kinases is necessary for signal transduction in response to ligand binding. The physiologic significance o f this combinatorial system o f receptor usage is poorly understood. However, there is growing evidence to support the hypothesis that the BMP receptors are differentially regulated during development and that they have both unique and overlapping functions. M ost reported studies describe the role of BMP4 in vitro or in situ in development models, but little is known on the role of BMP4 in AMD. Only recently BMP4 was found to negatively regulate RPE cell proliferation in vitro. 1 2 5 The purpose o f our study was to localize the expression o f BMP4 and its receptors in normal human eyes and in eyes with dry AMD, and to elucidate its possible roles in the dry AMD pathogenesis. 2-3. M aterials and methods 2-3-1. Animals BMP4lac~ ” eo mice used for expression analysis were developed by Dr. Brigid LM Hogan3 6 and regenerated by Dr. Yihsin Liu at USC. All experiments were performed in accordance with the USC Animal Care and Use Committee and the ARVO Statement for the Use o f Animals in Ophthalmic and Vision Research. 2-3-2. Human Samples Cryostat sections ( 8 pm) o f macula areas o f two donors with early AMD, one with GA, and one with drusen and three aged normal donor eyes were obtained from Lyon’s Eye Bank o f Oregan (Portland, OR). The study was conducted in accordance with the guidelines in the Declaration of Helsinki for research involving human tissue. M acular tissue sections from donors with early AMD and from aged control and drusen-containing eyes were identified by histopathology, clinical records, or both. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-3-3. Human RPE Culture RPE cells were isolated from human eyes obtained from the Advanced Bioscience Resources, Inc., (Alameda, CA) and cultured in Dulbecco’s minimal Eagle’s medium (DMEM; Fisher Scientific, Pittsburgh, PA) with 2 mM L-giutamine, 100 U/snl penicillin, 100 p. g/ml streptomycin (Sigma, St. Louis, MO), and 10% heat inactivated fetal bovine serum (FBS; Irvine Scientific, Santa Ana, CA) as previously described.126 Second- to fourth-passage cells grown to confluence for 48 to 72 fa were used for these experiments. APRE-19 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham ’s F12, containing 3 mM L-glutamine, 100 U/ml penicillin, 100 p g/ml streptomycin and 10% fetal bovine serum, as previously described . 1 2 7 2-3-4. ARPEH9 Culture with BMP4 Treatment ARPE-19 cells (ATCC, M anassas, VA) were grown in medium with 1% FCS and 100 ng/ml of human recombinant BMP4 fl&D Systems, Minneapolis, MN) continuously present. Control ARPE-19 cells were grown in parallel in 1% FCS. The medium ± BM P4 was replaced every other day, and the cells were passaged before confluence. At various time during the BMP4 treatment, flasks o f ARPE-19 cells ± BM P4 were grown to near confluence, trypsinized, and counted by hemocytometer to compare growth rates. 2-3-5. ARPE-19 Monolayer Grown on Permeable Transwell Filters and BMP4 ELISA Trans well filters (0.4pm pore, Coming Incorporated, Coming, MA) were coated with laminin (BD Biosciences, San Jose, CA). ARPE-19 cells were seeded at a seeding density o f 1.66 x IQ5 cells/cm2 in DMEM/F-12 medium with 1% FBS. A corresponding amount of culture medium was added to the basolateral compartment, leveling the height of the liquid to prevent hydrostatic pressure. The medium was changed twice a week. Transepithelial electrical resistance (TEER) was measured once a week using an EVOM™ Epithelial Voltohmmeter (World Precision Instruments, 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sarasota, FL). The cell monolayers were cultured for at least a month before use. At this time the 1 r ) 1 ! ? 8 cultures are highly polarized. Experiments were performed with 12 transwell filters with confluent ARPE-19 cells. Medium was removed from the filters and replaced with fresh medium before the experiments. The filters were incubated in n ormal growth conditions for 24 or 48 h. After 24 h or 48 h medium from the upper and lower compartments was collected, snap-frozen, and stored at-80°C until farther analysis. M edium was concentrated though centrifugal filter devices (Millipore, Bedford, MA). Each sample o f medium was tested in triplicate. Levels of BMP4 production of the ARPE cells toward the basal or the apical side were assayed by enzyme-linked immunoabsorbent assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. 2-3-6. B-galactosidase Activity M ice were euthanized and eyes were immediately enucleated and fixed in 4% paraformaldehyde in PBS (pH 7.3) overnight at 4 °C. After fixation, samples were washed in PBS and incubated in 30% sucrose overnight at 4 °C. Thin sections (10 micron) were cut and stained in X-Gal staining solution for 1 h at 37 °C as previously described (Manipulating Mouse Embryo, second edition, Cold Spring Harbor Laboratory Press). 2-3-7. RT-PCR Total RNA was isolated from cultured RPE cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and the contaminating genomic DNA was removed with a kit (DNA- free; Ambion, Austin, TX). Reverse transcription was performed using 1 jig total RNA, oligo(dT)i5 primer and AM V leverse transcriptase (Promega, Madison, WI). PCR were performed as previously described . 1 2 9 The oligonucleotide primer sequences for human BMP4, BMPR-1A, BMPR-1B and BMPRII were also used as previously described 1 2 9 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-3-8. Immunohistochemistry RPE/choroidal tissues were dissected, snap frozen and cryostat sectioned at 8 tim. Thawed tissue sections were air dried, rehydrated with PBS (pH 7.4) and blocked with 1% BSA for 15 min. Sections were incubated with the primary anti-BMP4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min, followed by biotinylated secondary antibody for 30 min, and then streptavidin peroxidase for another 30 minutes. The AEC chomogen was developed; sections were counterstained with hematoxylin. 2-3-9. Immunoaold Electron Microscopy Paraformaldehyde fixed tissue specimens were processed and embedded in LR White acrylic resin (London Resin Company; 60°C). Blocks were sectioned (75 mn) and sections placed on pailadian coated nickel grids. Sections were sequentially etched (0-5% sodium metaperiodate), blocked (5% BSA), incubated with primary BMP4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), incubated with secondary antibody conjugated to 10 nm colloidal gold (Ted Pella), and stained with uranyl acetate/lead citrate. Sections were visualized using the Zeiss EM10 TEM and digital images evaluated. 2-3-10. Scanning Electron Microscopy APRE-19 cells (both control and BMP4-treated) were grown on 10mm coverslips placed in a 24 well plate. Cells on coverslips were immersion fixed overnight at 4°C in X A strength K am ovsky’s fixative and then rinsed in 0.1M cacodylate buffer (pH 7.4). Cells were next post-fixed with 1% osmium tetroxide for 2 hours at room temperature. After a cacodylate buffer rinse, coverslips were dehydrated through an alcohol series to 1 0 0 % ethanol and then transferred from 1 0 0 % ethanol to 100% hexamethyldisilasane (HMDS). After two changes in HMDS, coverslips were allowed to air dry for 24 hours. They were next mounted on to stubs and coated with gold and palladium on a sputter-coater. The cells were imaged with a Hitachi S 570 Scanning Electron Microscope (filament voltage at 15 or 20 KV). 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-3-11. Senescence-associated ft-galactosidase Staining A modified procedure of Ditnri et al and Matsunaga et al 1,3 114 was used to stain cultures for senescence-associated p-galactosidase activity. Cultures of APRE-19 cells (both control and BMP4-treated) were trypsinized and replated in four-well chamber slides. Celts v«re fixed for 4 m in at room temperature in 3% paraformaldehye followed by three 5-min washings in PBS. Wells were filled with 1 mg/ml of a solution of 2.45mM X-ga! in 40mM citric acid-sodium phosphate buffer (pH 6) for total 4 fa. After staining, cell monolayers were again washed thee times in PBS and fixed for 20 min in acidic alcohol fixative at -20°C. Observation o f stained cells was made using bright-field microscopy. 2-3-12. BrdU Incorporation A proliferation ELISA kit based on the DNA incorporation o f 5-bromo -20-deoxyuridine (BrdU) (Roche, Indianapolis, IN) was used. Briefly, 5000 ARPE-19 ± BMP4 cells was cultured in 96 well plates for 24 h followed by 12 h incubation with BrdU. After removing the culture medium, cells were fixed and DNA denatured. An anti-BrdU-POD antibody was added, and after 90 min incubation at room temperature, the cells were washed and the peroxidase substrate 2,2’-azino-di- (3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) was added. The substrate reaction was terminated by adding 1 M H 2 SO4 , and the absorbance was measured spectrophotomefrically at 450 nm. 2-4. Results 2-4-1. BMP4 is Preferentially Expressed in Retinal Pigment Epithelium and Ciliary Body Eyes o f BMP4IacZ n eo heterozygotes w ere evaluated for P-galactosidase expression at postnatal ages P10, P60, and PI 80. Expression was found selectively in RPE and the epithelial cells o f the ciliary body (Fig. 1A, IB, arrows). Expression levels remain unchanged throughout P I80. There was no apparent BMP4 expression in retina or choroid. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-4-2. Expression of BMP4 and Its Three Receptors (BMPRIA. BTviPRIB. and BMPRIH ir. RPE Cultures The type I (1A and IB) and type II receptors are required for BMP4 signaling. Using RT-PCR with primers specific for BMP4, BMPR-1A, BMPR-1B and BMPRII, products o f appropriate sizes were amplified from RNA from several primary RPE cultures derived from various donors (Fig. 2). 2-4-3. Polarized BMP4 Expression of BMP4 in ARPE Cultures on Transwell Filters The RPE is positioned at the interface between the vascularized choroid and the avascular retina, forming part o f the blood-retina barrier. We have shown that BMP4 is produced by RPE cells in vivo. We further investigated whether there is a polarized secretion of BMP4 by the RPE cells. After at least four weeks in vitro i ■ f t ' ■Vi t | | s | l | A . \ \ \ v Ciliary body C jV ::'. r a tetma Figure 2-1: Beta-gal Staining p-gal staining of frozen section o f eyes from BMP4,acZneo heterozygotes to detect BMP4 expression in situ (A. 20X; B. 40X). Insets are magnification o f areas pointed by arrows. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BM P4 BMPR1A BMPR1B BMPRH N F igure 2-2: RT-PCR N N N RT-PCR for BMP4 and its thee receptors (BMPR1A, BMPR1B, BMPRII) mRNA expression in four different fetal RPE primary cultures. All four RPE cultures showed prominent BMP4 and its receptor expression and results from two donors are presented in this figure. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. culture, human ARPE-19 cells formed distinct polarized monolayers on transwell filters. Transepithelial electrical resistance (TEER) on the monolayers increased gradually and maximum net TEER reached above 30 Q cm2 after approximately 2 weeks and piateaued thereafter. The polarity o f the RPE monolayers was verified by immunoflurescence staining o f the cell layers for the tight junction-associated protein ZD-1 (Fig. 3A). The cells were uniform in appearance and form ed confluent monolayers o f cuboidal to columnar epithelium. Taken together, these results indicated that cultured ARPE cells represent a model for differentiated resting RPE in vivo. Differentiated ARPE monolayers were grown for more than 30 days. Media from the apical and basal sides were assayed for BMP4 secretion by ELISA after 24 or 48 h o f fresh media change. BM P4 secretion increased over time and a preferential BMP4 secretion from the basal surface of the monolayers was observed (Fig. 3B). 2-4-4. Expression of BMP4 in Human Normal and Macular Degeneration Tissues The expression o f BMP4 was weak and sparsely detected in the control (non AMD) RPE/choroid tissue sections by immunohistochemistry (Fig. 4A). In contrast, RPE/choroid tissues from patients with pathologically diagnosed early dry AMD showed prominent increased expression o f BMP4 in RPE (Fig. 4B) and in the vicinity o f Bruch’s membrane adjacent to hard (Fig. 4C) and soft (Fig. 4D) drusen. In some samples o f geographic atrophy, BMP4 expression was prominent in the RPE and thickened Bruch’s membrane adjacent to areas o f RPE loss, and in association with the choroidal vasculature (Fig. 4E, arrow head). 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apical Basal Figure 2-3: Immunofluorescent Staining Irnmunofluorescent staining of ZO-1 o f AEPE-19 monolayer grown on transwell inserts (A). The cells have formed confluent monolayers o f cuboidal to columnar epithelium. ELISA of BMP4 to detect secreted BMP4 protein from the ARPE-19 monolayer (B). Both apical and basal medium were collected and concentrated and final BMP4 level was determined 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F igure 2-4: Immunohistocheinical Staining hmnmohistDchemical staining of BMP4 in normal aging eye and eyes with AMD. IHC staining with BMP4 showed weak staining in non-dry AMD tissue section (A) but marked increase in RPE in early dry AMD (B; arrow) and in the vicinity of Bruch’s membrane adjacent to hard (C; arrows) and soft (D; arrow) 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. drusen. In some geographic atrophy areas, the RPE and thickened Bruch’s membrane adjacent to areas of RPE loss also showed prominent BMP4 expression (E, arrows). 2-4-5. Electron-Microscopic Immunohistochemistry of BMP4 in Human Macular Degeneration Tissues Soluble morphogens are kept in the solid state by extracellular matrix and bone morphogenic proteins (BMPs) are intimately bound to heparan sulphate, heparin, and type IV collagen.130 To further localize the distribution o f BMP4 in macular degeneration tissues with higher spatial resolution, electron-microscopic immunocytochemistry (immuno-EM) was performed. By immuno-EM, we found large amount o f BMP4 deposited in the elastic layer o f the Bruch’s membrane (Fig. 5A), along with some deposited in the inner collagenous layer and inside the RPE basal infoldings (Fig. 5 A; arrow and inset). Some BMP4 was also found in the basal cytoplasm o f the RPE cells further supporting the contention that RPE produce BMP4 and secrete it from the basal surface. Additional BMP4 immuno reactivity was found in the vicinity areas o f hard drusen (Fig. 5B). Binding BMP4 to extracellular matrix converts it into an insoluble morphogen to act locally and may protect it from proteolysis and prolong its half life1 30. 2-4-6. Induction o f Senescence and Proliferation Arrest bv BMP4 in ARPE-19 Cells Cultures of ARPE-19 cells treated continuously with BMP4 showed reduced growth compared with untreated cells. A change in morphology was apparent after 3 wk o f treatment. The slower- growing cells appeared elongated, larger, flattened, and more granular than untreated cells under regular bright field microscope. Scanning electron microscopy (SEM) was applied to visualize finer detail o f the surface morphology. Compared to normal dome-shape o f control cells, the slower-growing BMP4-treated cells had a larger surface area and more flattened fried-egg shape (Fig. 6A, B). BMP4 treatment induced ARPE-19 cells to assume an enlarged, flattened appearance 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission E • 1 2 Figure 2-6: Scanning EM and BrdU Incorporation Scanning electron microscopy o f serial passaged ARPE-19 and control ARPE-19 culture. BMP4 treatment induced a significantly enlarged and much more flattened morphology (B) compared to control cells (A). Senescence-associated beta-galactosidase staining o f sequential passaged ARPE- 19 cells grown on chamber slides showed that continuous treatment o f BMP4 dramatically increased the number of RPE cells positive for senescence-associated beta-galactosidase (D) compared to control (C). BrdU incorporation demonstrated a significant decrease from growth factor induced stimulation in BMP4 treated cell (E). 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-5. Discussion A M D is a degenerative disease that affects the outer neural retina, RPE, Bruch’s membrane, and the choroid. Almost 90% of this disease is nonneovascular (dry) form while 10% accounts for the neovascular form (CNV). The absence o f an animal model that faithfully recapitulates the nonneovascular AMD hampers study o f its pathogenesis. A further understanding o f the factors triggering RPE injury, senescence, or accumulation o f drusen is another major challenge. Discovering and investigating these new factors will definitely help to elucidate the molecular mechanisms o f AMD pathogenesis. In this study, we have shown that the posterior ocular segments from patients with dry AMD express BMP4 protein. A prominent increase in BMP4 expression in RPE was detected in both RPE and the vicinity o f Bruch’s membrane adjacent to hard and soft drusen. In addition, BMP4 expression was prominent in the RPE and thickened Bruch’s membrane adjacent to areas o f RPE loss in geographic atrophy. An in vitro model representing differentiated RPE cells revealed a preferential scretion o f BMP4 protein from the basallateral surface. Immuno EM detection of BM P4 deposited in the inner collagenous layer o f Bruch’s membrane and basal laminar o f RPE cells close to drusen further suppored the finding from the in vitro RPE model. Furthermore, in vitro continuous treatment o f ARPB-19 cells with BM P4 induced strong senescent phenotype. The presence o f possible growth factors in the dry form AMD has not yet been estalished. The presence o f prominent BMP4 expressions prominent in the RPE and thickened Bruch’s membrane adjacent to drusen and areas o f RPE loss in geographic atrophy implicates that BMP4 may play a role in the early and late stages o f dry AMD. The finding o f the localization of BMP4 in dry AMD samples is of considerable interest. W hether BM P4 secretion is selective to a polarized RPE d om ain has not been studied so far. W e hypothesize there is a preferential basal BMP4 protein secretion from RPE. This was tested with two independent procedures. We were able to culture 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ARPE-19 cells into polarized monolayer in vitro. The monolayer has structural and functional properties characteristic of RPE cells in vivo. Preferential basal secretion of BMP4 protein was detected in the ARPE-19 monolayers. In addition, immunogold EM was performed on dry AMD samples embedded in plastic. BMP4 protein predominantly was localized to the Bruch’s membrane and the basal infoldings of RPE. The rationale for the presence of large amount of BMP4 inside Bruch’s membrane may be that BMP family members have strong affinity for extracellular matrix and that deposition in ECM protects them from degradation from endogenous proteases. Accumulation o f BMP4 in the collagen-rich extracellular matrix may strongly provide prolonged effect and sustain the cascade o f the events that lead to progressive RPE senescent change. Based on the specificity and vicinity o f BMP4 expression, it is conceivable that BMP4 is secreted by RPE and exerted its effect on RPE in both autocrine and paracrine manners in the pathologic dry AMD condition. BM P4 has been found to be a negative proliferation regulator of RPE cells in vitro. 1 2 5 It is by far the only study that had been performed to illustrate the BMP4 functions in RPE. BMP4 induced senescence in lung carcinoma cells. 1 3 1 In our study, BMP4 accelerated the process of RPE senescence both morphologically and functionally. ARPE cells continuously treated with BMP4 showed dramatically decreased proliferation rate and increased senescence-associated beta galactosidase activity and acquired similar morphology change o f senescence. Besides oxidative stress and photo-oxidative stress, replicative senescence is an additional paradigm for understanding the development o f aged R P E .1 ' 2 The senescence is not equivalent to cellular quiescence because senescence cells cannot reenter the cell cycle upon stimulation with growth factors. Senescent RPE cells have been found both in vivo and in vitro.88'1 RPE cells at replicative senescence not only stain for SA-j3-gal but also have shortened chomosomal telomeres.’1 4 Morphologically, they appear flattened, either hyper- or hypopigmented and accumulate autofluorescent granules o f lipofuscin. It has been reported that cellular senescence of 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RPE cells altered several candidate genes.92 Although it is still unclear whether senescent RPE cells contribute to the development o f AMD, the increased amount o f BM P4 found in AMD and the acceleration o f the process o f RPE senescence by BM P4 in our study strongly support the concept o f senescent RPE involvement during the AMD development. The development o f dry AMD is a multi-gene and multi-factor involved event. Damage of any sort to RPE cells causes either substantial de novo expression of growth factors, or at feast, substantial up-regulation in an effort to facilitate repair of the damaged tissues. It is crucial to maintain the balance o f inhibitory and stimulating factors. Sfeveral autocrine loops involving positive growth regulators in RPE cells have been discovered.80 BM P4 appears to be negatively regulating RPE growth; it is reasonable to conceive that BMP4 may act antagonistically with other positive growth factors in the regulation o f RPE proliferation, differentiation and growth. An excess production o f the BMP4 in dry AMD might indicate an imbalance between genes conferring suscecptibility to and protecting against AMD. The evolution o f novel technologies such as microarray analysis may help identify such gene framework. BMP4 and its receptors participate in development of lens, retina, and ciliary body and angiogenesis during embryonic development.35 134 1 3 5 36 There is only limited information available on the role o f BMP4 and its receptors in the human postnatal eye. BMP4 binds first to the type II receptor and subsequently phosphorylates the type I receptors to trigger the Smad protein-signaling pathways.136 The specific pathway activated by BMPR-II may depend on w hether preformed type I/type II heterodimers are stimulated by ligand (Smad-dependent) or whether ligand leads to recruitment o f type I and II receptors to the signaling complex (p38M APK- dependent).!j7 Elucidation of ligand-receptor interactions may provide fundamental information to our understanding o f disease pathology in patients with AMD. In conclusion, we localized the expression o f BMP4 in both normal and AMD eyes. The localization o f protein indicates both autocrine and paracrine functions in the complex o f RPE, 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B ruch’s membrane and choriocapiilaris in the pathological conditions. Because BMP4 is a pleiotrophic factor with multiple functions on varied cell types, its exact role in dry AMD and possible therapeutic possibilities are yet to be established. 2-6. References 1. Evans I, Wormald R: Is the incidence of registrable age-related macular degeneration increasing? Br J Ophthalmol 1996, 80:9-14 2. Zarbin MA: Current concepts in the pathogenesis o f age-related macular degeneration. Arch Ophthalmol 2004,122:598-614 3. Young RW: Pathophysiology of age-related macular degeneration. Surv Ophthalmol 1987,31:291-306 4. Sheraidah G, Steinmetz R, Maguire J, Pauleikhoff D, Marshall J, Bird AC: Correlation between lipids extracted from Bruch's membrane and age. Ophthalmology 1993, 100:47- 51 5. Martin GM, Sprague CA, Epstein CJ: Replicative life-span of cultivated human cells. Effects o f donor's age, tissue, and genotype. Lab Invest 1970, 23:86-92 6. Schneider EL, Mitsui Y: The relationship between in vitro cellular aging and in vivo human age. Proc Natl Acad Sci U S A 1976, 73:3584-3588 7. West MD: The cellular and molecular biology of skin aging. Arch Dermatol 1994, 130:87-95 8. Rubin H: Cell aging in vivo and in vitro. Mech Ageing Dev 1997,98:1-35 9. Campisi J: The biology o f replicative senescence. Eur J Cancer 5997,33:703-709 10. Burke JM: Cytochrome oxidase activity in bovine and human retinal pigment epithelium: topographical and age-related differences. Curr Eye Res 1993,12:1073-1079 11. Burke JM, McKay BS: In vitro aging o f bovine and human retinal pigment epithelium: number and activity o f the Na/K ATPase pump. Exp Eye Res 1993, 57:51-57 12. Flood MT, Gouras P, Kjeldbye H: Growth characteristics and ultrastructure o f human retinal pigment epithelium in vitro. Invest Ophthalmol Vis Sci 1980, 19:1309-1320 13. Song MK, Lui GM: Propagation o f fetal human RPE cells: preservation of original culture morphology after serial passage. J Cell Physiol 1990,143:196-203 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14. M atsanaga H, Handa JT, Aotaki-Keen A, Sherwood SW, West MD, Hjelmeland LM: Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999,40:197-202 15. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al.: A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 1995,92:9363-9367 16. Frank RN: Growth factors in age-related macular degeneration: pathogenic and therapeutic implications. Ophthalmic Res 1997, 29:341-353 17. Kingsley DM: The TGF-beta superfamily: new members, new receptors, and new genetic tests o f function in different organisms. Genes Dev 1994, 8:133-146 18. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA: Novel regulators of bone formation: molecular clones and activities. Science 1988,242:1528-1534 19. Mabie PC, Mehler MF, Kessler JA: Multiple roles of bone morphogenetic protein signaling in the regulation o f cortical cell number and phenotype. J Neurosci 1999, 19:7077-7088 20. Hogan BL: Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 1996,10:1580-1594 21. Graham A, Francis -West P, Brickell P, Lumsden A: The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 1994, 372:684-686 22. Hogan BL: Morphogenesis. Cell 1999, 96:225-233 23. Winnier G, Blessing M, Labosky PA, Hogan BL: Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995, 9:2105- 2116 24. Barlow AJ, Francis -West PH: Ectopic application o f recombinant BMP-2 and BMP-4 can change patterning o f developing chick facial primordia. Development 1997, 124:391-398 25. Yokouchi Y, Sakiyama J, Kameda T, Iba H, Suzuki A, Ueno N, Kuroiwa A: BM P-2/4 mediate programmed cell death in chicken limb buds. Development 1996,122:3725-3734 26. Zou H, Niswander L: Requirement for BMP signaling in interdigital apoptosis and scale formation. Science 1996,272:738-741 27. Scliultheiss TM, Burch JB, Lassar AB: A role for bone morphogenetic proteins in the induction o f cardiac myogenesis. G enes Dev 1997, 11:451462 28. W u LY, Li M, Hinton DR, Guo L. Jiang S, Wang JT, Zeng A, Xie JB, Snead M, Shuler C, Maxson RE, Jr., Liu YH: Microphthalmia resulting from MSX2-induced apoptosis in the optic vesicle. Invest Ophthalmol Vis Sci 2003,44:2404-2412 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29. Furuta Y, Hogan BL: BMP4 is essential for lens induction in the mouse embryo. Genes Dev 1998,12:3764-3775 30. Chang B, Smith RS, Peters M, Savinova GV, Hawes NL, Zabaieta A, Nusinowitz S, Martin IE, Davisson ML, Cepko CL, Hogan BL, John SW: Haplcinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure. BMC Genet 2001,2:18 31. Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving IP, Hogan BL: Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 1999,13:424-436 32. Massague J, Attisano L, Wrana JL: The TGF-beta family and its composite receptors. Trends Cell Biol 1994,4:172-178 33. Massague J: TGFbeta signaling: receptors, transducers, and Mad proteins. Cell 1996, 85:947-950 34. Piek E, Heldin CH, Ten Dijke P: Specificity, diversity, and regulation in TGF-beta superfamily signaling. Faseb J 1999,13:2105-2124 35. Mathura JR, Jr., Jafari N, Chang JT, Hackett SF, Wahlin KJ, Della NG, Okamoto N, Zack DJ, Campochiaro PA: Bone morphogenetic proteins-2 and -4: negative growth regulators in adult retinal pigmented epithelium. Invest Ophthalmol Vis Sci 2000, 41:592-600 36. He S, Wang HM, Ye J, Ogden TE, Ryan SJ, Hinton DR: Dexamethasone induced proliferation o f cultured retinal pigment epithelial cells. Curr Eye Res 1994, 13:257-261 37. Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM: ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996,62:155-169 38. Dunn KC, Marmorstein AD, Bonilha VL, Rodriguez-Boulan E, Giordano F, Hjelmeland LM: Use o f the ARPE-19 cell line as a model o f RPE polarity: basolateral secretion of FGF5. Invest Ophthalmol Vis Sci 1998,39:2744-2749 39. You L, Kruse FE, Pofal J, Volcker HE: Bone morphogenetic proteins and growth and differentiation factors in the human cornea. Invest Ophthalmol Vis Sci 1999,40:296-311 40. Paralkar VM, Nandedkar AK, Pointer RH, Kleinman HK, Reddi AH: Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen. J Biol Chem 1990,265:17281-17284 41. Buckley S, Shi W, Driscoll B, Ferrario A, Anderson K, Warburton D: BMP4 signaling induces senescence and modulates the oncogenic phenotype o f A549 lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol 2004,286:L81-86 42. Hjelmeland LM: Senescence of the retinal pigmented epithelium. Invest Ophthalmol Vis Sci 1999,40:1-2 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43. Hjelmeland LM, Cristofoio VI, Funk W, Rakoczy E, Katz ML: Senescence of the retinal pigment epithelium. Mol Vis 3999,5:33 44. Matsunaga H, Handa JT, Gelfman CM, Hjelmeland LM: The mRNA phenotype o f a human RPE cell line at replicative senescence. Mol Vis 3999, 5:39 45. Campochiaro PA, Hackett SF, Vinores SA, Freund J, Csaky C, LaRochelle W, Henderer J, Johnson M, Rodriguez IR, Friedman Z, et al.: Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells. J Cell Sci 1994, 107 ( Pt 9):2459-2469 46. Campochiaro PA, Hackett SF, Vinores SA: Growth factors in the retina and retinal pigmented epithelium. Prog Retinal Eye Res 1996,15:547-567 47. Liu J, Wilson S, Reh T: BMP receptor lb is required for axon guidance and cell survival in the developing retina. Dev Biol 2003, 256:34-48 48. Zhao S, Chen Q, Hung FC, Overbeek PA: BMP signaling is required for development of the ciliary body. Development 2002, 329:4435-4442 49. Miyazono K, Kusanagi K, Inoue H: Divergence and convergence of TGF -beta/BMP signaling. J Cell Physiol 2001,187:265-276 50. Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, Knaus P: The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002, 277:5330-5338 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 Bone Morphogenic Protein 4 (BMP4) As A Negative Regulator in Choroidal Neovascularization 3-1. Summary To study the role o f Bone Morphogenic Protein 4 (BMP4) in neovascular macular degeneration (CNV) using both in vivo and in vitro models. Expression of BMP4 under normal and pathologic CNV conditions was previouly investigated using imxnunohistochemistry. In vivo laser-induced choroidal neovascularization was studied using both normal C57/B6 and BMP4 haploinsufficient mouse models. Real-time PCR was applied to examine BMP4 expression change throughout the whole timecourse of laser-induced CNV progression in C57/B6 mouse model. The expression changes was also investigated using LacZ staining following the laser injury in the BMP4 haploinsufficient mouse model. Hematoxylin and Eosin staining was applied to study histology and flurosecein angiogram (FA) were performed to study the vascular maturity in response to laser injury in the haploinsufficient model. Choroidal endothelial cells (CEC) were isolated and reverse transcription-polymerase chain reaction (RT -PCR) was used to detect the expression o f BMP4 receptors in CEC cells under different extracellular matrix (ECM) coating. In vitro tube formation assay using bovine CEC was studied to examine the effects o f BM P4 on angiogenesis. Expression change o f BMP4 in response to both VEGF and T N Fa was also studied using real-time PCR. Immunohistochemistry assay revealed that BMP4 expression was completely inhibited in surgically removed CNV membranes. BMP4 expression was decreased following the laser injury in normal C57/B6 model. LacZ staining in BMP4 haploinsufficient mice showed BMP4 expression was initially decreased and later regained in the RPE surrounding the injury area. CNV 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phenotype demonstrated in both histology and fluorescent angiogram (FA). RT-PCR demonstrated abundant expression of BMP4 receptors (BMPR1A, BMPR1B and BMPRII) in CEC culture and BM PRII expression was dramatically increased when CEC cells were plated on fibronectin or collagen-coated surface. Bovine CEC ii vitro tube formation was inhibited in the presence o f BMP4. TNFa significantly decreased BMP4 expression while VEGF up-regulated BMP4 in RPE. O ur findings suggest that BMP4 may serve as a negative regulator that inhibits initial angiogenesis stage o f CNV pathogenesis. Modulation o f expression o f the BMP4 may provide a viable approach to control various processes involved in the progression o f age-related choroidal neovascularziation. 3-2. Introduction Choroidal neovascularization (CNV) is the frequent causes o f loss o f vision due to retinal damage and detachment and associated with several common retinal degenerative or inflammatory diseases, especially age-related macular degeneration (AMD).98’1 3 8 The pathogenesis of neovascular AMD is clearly multifactorial, with age, systemic heath, genetic, and environmental risk factors playing roles in onset and progression. During CNV, new blood vessels formed from choriocapillaris sprout beneath the retinal pigment epithelium (RPE) through defects in Bruchs membrane and in some cases they develop into the subretinal space.71’ 97 These neovascular structures, also known as choroidal neovascular membranes (CNVM), formed by the proliferating fibrovascular tissue are composed o f diversified cells such as RPE, vascular endothelial cells, fibroblasts, and macrophages. 71,139,98 CNV, commonly accompanied by AMD may result in subretinal hemorrhage often leading to desciform scarring. 69,98 Choriocapillaris and RPE are on the opposite sides o f Bruch's membrane and control transport in and out o f the retina. Normal functioning o f the RPE is crucial for the maintenance and survival of the choriocapillaris and photoreceptors.140 The accumulation of extracellular debris alters Bruch 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membrane composition and permeability. These changes may lead to impaired diffusion of waste products from and o f nutrients to the RPE. The degeneration and loss o f RPE was shown to lead to the atrophy o f the choriocapillaris and damage to retinal photoreceptors.62 The Disruption of the homeostasis between choriocapillaris and RPE may produce growth factors that stimulate the formation o f choroidal neovascularization (CNV) in AMD. RPE cells are one of the major components o f CNV membranes.'1,139 Uvama et al hypothesize that RPE cells promote the progression of CNV in the early stage of its development, however, in the late or involution stage of CNV, proliferating RPE enclose CNV and cause its regression.1 4 1 RPE cells in the CNVM of maculae with AMD showed positive immunostaining for VEGF, TGF-b, PDGF, and bFGF.142 It is necessary to identify additional angiogenesis-related factors in order to define the potential actions o f a protein in angiogenesis and characterize the specific neovascularization processes in the retina and choroids. Bone morphogenetic proteins (BMPs) are a large sub-family (more than 20 members) of the TGF - P superfamily, which includes TGF-Ps, activins/inhibins, and M ullerian inhibiting substance (MIS). BMP4 plays an important role in embryonic development. It has been implicated in various morphogenetic processes such as limb formation, neurogenesis, tooth formation, and other epithelial mesenchymal interactions in early embryos and skeletogenesis.143,144,145,146,147 In osteoblasts BMPs stimulate angiogenesis through the production o f VEGF-A and therefore, couple angiogenesis to bone formation.1 4 8 BMP4 is expressed in multiple tissues during development, and has critical roles in lens induction.35 Recent studies extended its roles into angiogenesis and ocular vascular modification. Heterzygous BM P4 knockout §m p4+l~ ) have abnormally arranged retinal blood vessels and abnormal persistence o f the hyaloid vasculature.36 In the mammalian eye, hyaloid vessels and the pupillary membrane (PM) nourish the immature lens, retina, and vitreous body during morphogenesis but are know to regress during the late stages of ocular development. The failure of 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. capillary regression in BMP4 haploinsufficient mice suggests that BMP4 may play a role in positive vascular remodeling. U nlike its well known effects in bone formation and embryonic development, the functional im portance o f BMP4 in pathological neovascularization is not clear, specially in the CNV pathogenesis. Therefore, we decided to study the functional roles o f BMP4 in endothelial biology an d pathobiology o f ocular angiogenesis by independent methods both in vivo and in vitro. One reproducible and quantifiable murine model o f CNV was developed.149 It involves focal laser disruption o f Bruch’s membrane resulting in choroidal blood vessels proliferating through resultant holes in Bruch’s membrane into the retina 2 weeks later. Applying the BMP4 heterozygous mice to the murine model we will be able to study the phenotypes through BMP4 loss-of-function. 3-3. Materials and methods 3-3-1. Animals BM P4]acme° mice used for expression analysis were created by Dr. Brigid LM Hogan36 and regenerated by Dr. Yihsin Liu at USC. All experiments were performed in accordance with the USC Animal Care and Use Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For all procedures, anesthesia was achieved by intramuscular injection of 50 mg/kg ketamine HCL (Fort Dodge Animal Health, Fort Dodge, IA) and 10 mg/kg xylazine (Phoenix Scientific, St. Joseph, MO), and pupils were dilated with topical 1% tropicamide (Alcon, Fort Worth, TX). 3-3-2. Human Samples Cryostat sections o f maculas o f two donors with CNV and one aged normal donor eyes were obtained from the Lyon’s Eye Bank of Oregan (Portland, OR). The study was conducted in accordance with the guidelines in the Declaration o f Helsinki for research involving human tissue. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M acular tissue sections from donors with early AMD and from aged control and drusen-containing eyes were identified by liistopathology, clinical records, or both. 3-3-3. Induction o f CNV Laser photocoagulation (532 nm, 200 nsV, 100 ms, 75 j dm; Ocuiight GL, Iridex, Mountain View, CA) was performed on both eyes o f each animal by a single individual masked to drug group assignment. Laser spots were applied in a standardized fashion around the optic nerve, using a slit lam p delivery system and a coverslip as a contact lens. The morphologic end point of the laser injury was the appearance o f a cavitation bubble, as sign thought to correlate with the disruption o f B ruch’s membrane. At different time points after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde for 30 minutes at 4°C. Eye cups obtained by removing anterior segments were washed three times in PBS, followed by dehydration and rehydration through a methanol series. Globes were dissected through the optic nerve and were processed for routine frozen sectioning. Six-micrometer serial sections were cut for hematoxylin-eosin staining or immunostaining. For immunostaining studies, a rabbit polyclonal antibody against CD31 (1:750, Dako, Carpinteria, CA) was used to identify vascular endothelium within CNV. CD31 stained endothelial cells in CNV were counted on the representative sections that were immediately adjacent to the thickest portion o f CNV. Adjacent sections were incubated without prim ary antibody and served as the negative control Flatmounts were examined with a scanning laser confocai microscope (TCS SP; Leica, Heidelberg, Germany). 3-3-5. Fluorescein Angiograms The effect o f BMP4 haploinsufficiency was evaluated by semiquantitative assessment o f late- phase (100-140 seconds after dye injection in mice) fluorescein angiography, as previously described. ! 50 Leakage was defined as the presence o f a hyperfluorescent lesion that increased in 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. size with time in the late-phase angiogram. Angiography was graded in a masked fashion by two exam iners using reference angiograms. When the two scores for a lesion did not coincide, the higher score was used. Discrepant scoring was observed on only 12 of 142 lesions and was never greater than one grade. Angiograms were graded as follows: 0, no leakage; 1, slight leakage; 2, moderate leakage; 3, prominent leakage. 3-3-6. 6-galactosidase Activity M ice were euthanized and eyes were immediately enucleated and fixed in 4% paraformaldehyde in PBS (pH 7.3) overnight at 4 °C. After fixation, samples were washed in PBS and incubated in 30% sucrose overnight at 4 °C. Thin sections (10 micron) were cut and stained in X-Gal staining solution for 1 hour at 37 °C as previously described (Manipulating M ouse Embryo, second edition, Cold Spring Harbor Laboratory Press). 3-3-7. Human and Bovine Choroidal Endothelial Cell Culture The choroidal endothelial cells were isolated as previously described,1 5 1 Fetal or bovine eyes were washed in PBS containing 5% penicillin/streptomycin and the cornea, lens, vitreous, retina and RPE were removed. Choroidal vessels were then dissected away from the sclera and digested in Trypsin followed by Collagenase/Dispase. The digest was washed several times in PBS/BSA and passed through a 70pm mesh filter followed by a 40pm mesh filter. Cells were spun down and re suspended in 500pl o f PBS/BSA. Cells were then incubated in PBS/BSA with mouse anti-human CD31 antibody for 10 minutes at 4*C and then washed in PBS. Sheep anti-mouse IgG beads (D ynabeads M-450; Dynal Biotech, Brown Deer, WI) were prepared and concentrated (Dynal Magnetic Particle Concentrator; Dynal Biotech, Brown Deer, WI). A ntibody-bound Dynabeads were added to cells and incubated for 20 minutes at 4°C. The Dynabeads with attached cells were then recovered using the Dynal magnet. The cell/bead suspension was plated onto fibronectin- coated plates and cells incubated in Endothelial Cell Basal Medium (Cambrex Bio Science, Walkersville, MD) 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-3-8. in Vitro Tube Formation M atrix gel (In vitro Angiogenesis Assay Kit; Ctiemicon, ,) plates were prepared in 96-well plates follow ing the manufacturer's instructions. Bovine choroidal endothelial cells (BCEC) (~80% confluent) were treated with trypsin, and 5 x 104 cells were seeded on the top o f plates with 1% C E C medium containing various concentration of BMP4 at 37“C for 24 h in a humidified atm osphere o f 5% COj. After incubation, five different fields in each culture were randomly o bserved with a pfaase-contrast microscope and photographed at X40 magnification. 8-3-9. RT-PCR T otal RNA was isolated from cultured RPE cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and the contaminating genomic DNA was removed with a kit (DNA- free; Ambion, Austin, TX). Reverse transcription was performed using 1 p g total RNA, oligo(dT)i5 primer and AMV reverse transcriptase (Promega, Madison, WI). PCR were performed as previously described. 129 The oligonucleotide primer sequences for human BMP4, BMPR-1A, BMPR-1B and BM PRII were also used as previously described.1 2 9 3-3-10. Relative Quantitative Real-Time RT-PCR Total RNA was isolated using RNA extraction reagent (TRIzol; Invitrogen Life Technologies, Carlsbad, CA) and the contaminating genomic DNA was removed with a kit (DNA-free; Ambion, Austin, TX). Reverse transcription was performed using 1 p g total RNA, oligo(dT)i5 primer (Promega Corp., Madison, WI), and AMV reverse transcriptase (Promega). Real-time PCR reactions were performed in triplicate with a sequence detection system (GeneAmp 5700; Applied Biosystems, Foster City, CA). Each 25 APCR reaction contained cDNA template, SYBR Green PCR M aster Mix (Applied Biosystems), and 167 nM gene-specific primers. Reaction conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of denaturation at 95°C for 15 seconds with annealing and extension at 60°Cfor 1 minute. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The primer sets were designed on computer (Primer Express software; Applied Biosystems). D etection o f product formation was set in the center of the linear portion o f PCR amplification. The cycle at which each reaction reached the set threshold (CT) was determined. Amplification efficiencies between primer pairs for genes o f interest compared with that for the L-32 reference gene was evaluated by amplifying a dilution range of cDNA template. A plot o f A CT on the y- axis and log (cDNA) on the abscissa generated a linear curve with slope < 0.01, verifying comparable primer pair amplification efficiencies between GAPDH and genes of interest. Relative multiples o f change in mRNA expression were determined by calculation o f AACT.15 Results are reported as mean difference in relative multiples o f change in mRNA expression ± SEM. 3-3-11. Statistic Analysis All statistical analyses were performed using SAS (SAS Institute, Cary, NC). Values reported in figures represent mean ± SEM. Each eye was treated as an independent event for statistical analysis o f corneal vessels. The Shapiro-W ilk statistic was used to test for normality o f the measurements within each group. If one or more o f the groups were found to follow a non-normal distribution, nonparametric statistics were used. For nonparametric distributions, The Kruskal- W allis test was used to determine overall differences between groups, and W ilcoxon rank sum was used to test for differences between two groups. For normal distributions, parametric tests used were analysis o f variance and paired Wests. For gene expression data, comparisons between groups were made using independent t-tests for equal or unequal variances. Accepted level of significance for all tests was<*= 0.05. 3-4. Results 3-4-1. BMP4 Expression Is Absent in Surgically Removed Human Choroidal Neovascularization Membrane CCNVM) Previously we have published that BMP4 expression is increased in surgically removed human dry AMD sections (paper submitted for publication). In this study, in contrary to what we have found 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in dry AMD, immunohistochemistry revealed that compared to age-matched normal eyes (Fig 1 A), BMP4 expression is completely absent in human CNV membranes (Fig IB). 3-4-2. BMP4 Is Down-regulated During Laser-induced CNV Progression Real-time PCR using nRNA extracted at different time points in C57/B6 mice revealed that BM P4 expression was initially decreased and slowly regained back at the end of two weeks following laser injury (Fig 2). BMP4 Haploinusfficient mice underwent the same injury was examined for lacZ expression. RPE cells surrounding the injury area lost the lacZ expression two days after the injury (Fig 3A) and around two weeks later RPE cells encapsulating the neovascularization area gained back BMP4 expression (Fig 3B). Therefore, BMP4 expression is consistent in both normal C57/B6 and BMP4 haploinsufficient mice. Figure 3-1: Immim ohistochemical Staining Immunohistochemical staining o f BMP4 in normal aging eye and eyes with CNV. IHC staining with BMP4 showed weak staining in non-dry AMD tissue section (A) but completely absent in RPE in CNV membrane (B) 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CN en ^4 <C % £ < L ) > ■ 4 d l C 3 S 30003 25003 20000 ' 15000 - 10000 - 500J- 0 - cortral Bhr 12hr 2 # r 3cfey 7 d a y 14ctey F ig u re 3-2: Quantitative Real-Time PCR Quantitative real-time PCR of BMP4 mRNA expression in surgically removed CNV membranes induced with laser photocoagulation in normal C57/B6 mice. BMP4 expression was dramatically decreased at 12 hr following the laser injury. The decrease continued until day 14 when the neovascularization was completely inhibited. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-3: Beta-gal Staining p-gal staining o f frozen CNV section o f eyes from BMP4?acZ "eo heterozygotes injured with laser photocoagulation to detect BMP4 expressions. RPE surrounding the CNV lesion lost BMP4 expression (red arrowheads) at w eekl (A) and surrounding RPE gradually gained back BMP4 expression as they encapsulate the lesion at week 2 (B). 3-4-3. BMP4 Haploinsufficiencv Promoted Neovascularization in Laser-Induced CNV Model To study how BMP4 is involved in the pathogenesis of CNV, haploinsufficient BMP4 mice and their littermate controls were used in a laser-induced CNV model. Fluorescein angiography (FA) photos at 2 weeks after laser injury revealed that BMP4 haploinsufficient mice tend to have significantly larger area o f fluorescent leakage in the photocoagulated lesions compared to control group (Fig 4A, B, C). There was also intense leaking in laser-induced lesions in the BMP4 haploinsufficient group, whereas there was minimal leaking in lesions in the control group. Sections of the choroid obtained from these laser-treated mice were examined at one week and two weeks post laser. The incidence o f CNV formation was determined by histochemical examination. At two weeks, the CNV membrane in haploinsufficient mice showed intense proliferation and migration o f RPE cells and neovascularization with a significantly larger area, in contrast, the smaller membranes in the normal littermate control were already circumvented with RPE cells and vessel regression already started (Fig 5 A, B). The vasculature in these CNV membranes was tested by immunohistochemisty with CD31 antibody. The vessel channels o f the CNV in haploinsufficient mice showed intense staining of CD31, in contrast to the much less staining in the CNV membranes o f littermate control (Fig 5C, D). 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-4: Fluorescein Angiogram of CNV Fluorescein angiogram o f CNV carried out at 2 weeks after laser photocoaguiation in one normal control mouse (A = 1 min, B = 3 min, C = 5 min) and two BMFAla c Z n e o heterozygotes (D, E, F, and G, H, I). Late phase (3 min and 5 min) revealed a more intense dye leakage beyond the laser scar in heterozygote eyes. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-5: H-E Stainning H & E staining of CNV lesion in normal and BMP4/ocZ"ra heterozygote mice two weeks after the laser injury. Compared to the weil-capsulated lesion in control (A), a much more severe neovascularization phenotype was observed in BM P4 haploinsufficient mouse (B). CD31 confocal staining o f same lesions (C, D) is consistent to the H& E observation. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-4-4. Preferential Expression of BMP4 Receptors in Choroidal Endothelium in vivo and Up- regulation o f BMPRII bv ECM in fCEC in vitro Previously we discovered that BMP4 was preferentially secreted by retinal pigmented epithelium (RPE) through basal side. The type I (BMPR1A and BMPR1B) and type II (BMPRII) receptors are required for BMP4 signaling. Immunofaistochemical staining o f BMPRII and BMPR1A and BM PR1B on normal human eye sections revealed that both RPE and choroidal endothelium expressed all three receptors (Fig. 6A). Additionally, choroidal endothelium demonstrated a more preferential expression pattern o f receptors toward the direction o f RPE. Using RT-PCR with primers specific for BMPR-1A, BMPR-1B and BMPRII, products of appropriated sizes were amplified from RNA from both primary RPE and CEC cell cultures as well (Fig. 6B). Additionally, h fCEC cells BMPRII was up-regulated to 2.1 and 2.8 fold by ECM components fibronectin and collagen-I, respectively (data not shown). 3-4-5. BMP4 Inhibits In Vitro Choroidal Endothelial Cell Tube Formation To study the effect o f BM P4 on vessel formation, an in vitro angiogenesis assay using bovine choroidal endothelial cells was applied. Fetal CEC cells were unable to form in vitro tubes and therefore, bovine CEC cells were applied. Co-incubation with BMP4 dramatically inhibited in vitro angiogenesis in a dose-dependent manner demonstrated by much less new sprouting cells and less vessel 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BMPR1A BM PRII BMP Mil F igure 3-6: RT-PCR RT-PCR for three BMP4 receptors (BMPR1A, BMPR1B, BMPRII) mRNA expression in both fetal RPE and CEC primary cultures on different coatings. All three receptors in RPE showed no difference on different coatings while BMPRII expression in CEC was elevated on fibronectin and collagen-1 coatings compared to plastic (non coating). BMPRIA and BMPRIB in CEC was not regulated by the ECM coating. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-7: Tube Formation In vitro bovine CEC tube formation was inhibited by BMP4 in a dose-dependent manner. Compared to control (A), The sprouting and branching o f new vessels were significantly suppressed by BMP4 at lng/m l (B), 5 ng/ml (C), and 10 ng/ml (C) concentration. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tubes presented in BMP4 treated groups compared to control group (Fig 7A, B, C, D). BMP4 treatm ent significantly prevented new endothelial cells sprouting from newly formed vessels, hence, we proposed that BMP4 may inhibit the sprout phase o f angiogenesis. 3-4-6. BMP4 Is Regulated by VEGF and TN Fa in fRPE Real time PCR using fetal RPE cells revealed that BMP4 expression is up-regulated by VEGF yet down-regulated by both TN Fa. The up- and down-regulation are both tim e- and dose-dependent (Fig 8). 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BM P4 140000 120000 100000 80000 60000 40000 20000 # F igure 3-8: Quantitative Real-Time PC R Quantitative real-time PCR o f BMP4 expression in fetal RPE in response to T N Fa (10 ng/ml) and VEGF (30 ng/ml) treatments at different time points. BMP4 expression was dramatically inhibited by T N Fa immediately at 3 hrs and the trend continued thereafter while VEGF enhanced the expression in an opposite direction. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 -5 . Discussion Previous studies in our laboratory have discovered that BMP4 produced by RPE unilaterally tow ards the Bruch’s membrand/choroid side is up-regulated in dry AMD yet in this study it was found completely absent in surgically removed CNV membranes. Using a laser-induced mouse m odel for CNV, we showed that BMP4 faaploinsufficiency dramatically interfered with CNV progression. Three BMP4 receptors were all detected in choroidal endothelial cultures and extracellular matrix (ECM) molecules significantly upregulated BMPRII expression in hCEC. We also demonstrated that BMP4 inhibited choroidal endothelial cell tube formation in vitro and BM P4 is regulated by inflammatory factor TN Fa and angiogenic factor VEGF in fetal RPE. In this paper, we discovered, in addition to roles in dry AMD, BMP4 mainly plays a negative role in the pathogenesis of CNV, the neovascular type of AMD. The choroid is playing a crucial role in the formation o f this CNV. In non-pathological situation, choroidal vessels are suppressed from penetrating choroidal capillary matrix and Bruch’s membrane and RPE. The observed preferential localization o f BMP receptors at the inner choriocapillaris, which is facing the basal side o f the RPE cells, is consistent with what we previously discovered that BMP4 was secreted by RPE cells torwards basolateral surface. These observations suggest that BMP4 is involved in a physiological paracrine relation between RPE and the choriocapillaris. In light of the known effects o f BMP4 on endothelial cells in vivo and in virto, 36 132 153 BM p 4 may heip control angiogenesis beneath the retina by modulating the choroidal endothelial cell functions together with other factors like VEGF 86 and anti-angiogenic PEDF 1 5 4 which are both produced by RPE to regulate angiogenesis. Increased thickness of Bruch’s membrane and relative impermeability to water-soluble com pounds have been reported in AMD. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 155 Increased BMPRII receptor expression in CEC ceils by ECM molecules suggests that abnormalities of the RPE ECM may help promote the angiogenic regulation o f BMP4 in AMD. Transgenic mice lacking a specific growth factor gene are very useful in investigating which growth factor is involved in CNV formation. Such models can be combined with the laser CNV m odel, which lias also been applied in rats and monkeys, where local destruction o f the RPE and Bruch s membrane by laser causes a CNV-like response. 156-1 d 7 Hapioinsufficiency o f BMP4 dramatically altered several aspects o f CNV production in those combined mouse models. BMP4 expression was lost in RPE cells surrounding the site of disruption o f Bruch’s membrane and later on resumed in proliferating RPEs as they come back for vacular involution. The failure of eliminating hyaloid vessels in BMP4 haploinsufficient mice indicates BMP4 may participate in vessel regression. 36 The resuming BMP4 expression by newly proliferating RPEs at later stage o f CNV vessel regression to arrest the growth of CNV further supported the inhibitory effect of BM P4 on endothelial cells. In addition, as RPE cells migrate to encapsulate the wound, they deposit early matrix compromised of collagens, fibronectin, and laminin. These ECM molecules could further promote the inhibitory effects by increasing BMPRII expression in CEC cells which will undergo apoptosis and vessel maturation. Based on all the studies, we speculate that under normal condition, BMP4 from RPEs inhibit choroidal endothelium from penetrating through Bruch’s membrane to maintain the anti-angiogenesis state in resting choroid; and that destruction of the RPE by laser invariably leads to loss o f BMP4 which no longer inhibits the invading choroidal endothelium and late-stage resuming BMP4 expression helps vascular involtion. In addition, a nuch more cellular proliferation and neovascularization phenotype was observed in haploinsufficient model along with more remarkable leakage o f fluorescent. This increased neovascularization again may be due to decreased angiogenesis inhibition. Further we demonstrated that BMP4 inhibited endothelial cells tube formation and slowed endothelial cell proliferation in vitro. This finding corrobrated and supported our findings in 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. haploinsufficient CNV models. It indicates that BMP4 inhibits angiogenesis in part by preventing endothelial cell tube formation and decreasing proliferation. However Regazzoni et al have reported that in their study type I collagen induces expression o f BM PRII in bovine aorta endothelial cell (BAEC) and addition of BMP4 increased 3H-thymidine incorporation. 158 The increased expression o f BMPRII in our study corroborated with theirs but the effect of BMP4 are contradictory. The discrepancy could result from several factors such as cell types, culture conditions, different experiment designs and working conditions. In spite o f the differences, both studies indicate BMP4 and its receptors are involved in angiogenesis. Inflammation and angiogenesis are key components o f multi-phase CNV development. Many growth factors involved in these events contribute to different stages of the evolution o f CNV. However, the framework o f the interaction and regulation between these factors in CNV remains largely unknown. TNFa released by newly arrived macrophages is responsible for the stimulation o f VEGF by RPE to initiate angiogenesis. 159 160 Here we discovered that TNF rapidly decreased BMP4 expression in RPE in vitro. These observations may indicate a possible initial synergistic relationship between increased angiogenesis stimulation and decreased inhibition. Loss of BMP4 may contribute greatly to the development o f neovascularization accompanied by the increased VEGF production. In addition, we observed the increased BMP4 production in RPE by VEGF, which may seem to counteract the inhibitory effect o f BMP4 as we proposed. Coupling VEGF w ith some anti-angiogenic factors may indicate an angiogenic balance swing from neovascularization to vascular involution. Because the netv/ork o f growth factor interaction is dependent on the stage o f angiogenesis within the CNV, the regulation o f BMP4 by TN Fa and VEGF could happen at different stages of CNV progression. In summary, we showed in our study that BMP4 inhibits angiogenesis in vitro and hapioinsufficiency o f BMP4 demonstrated more severe CNV phenotype and both inflammatory and angiogenic factors regulate BMP4 expression. It is the first time that BMP4 was studied in the 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. choroidal neovascularization pathogenesis and its involvement in multiple stages o f CNV may provide deeper insight into therapeutic development. 3-6. R eferences 1. Zarbin MA: Age-related macular degeneration: review o f pathogenesis. Eur J Ophthalmol 1998, 8:199-206 2. Campochiaro PA: Retinal and choroidal neovascularization. J Cell Physiol 2000, 184:301-310 3. Green WR, Enger C: Age-related macular degeneration histopathologic studies. The 1992 Lorenz E. Zimmerman Lecture. Ophthalmology 1993,100:1519-1535 4. Campochiaro PA, Soloway P, Ryan SI, Miller JW: The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration. Mol Vis 1999, 5:34 5. Grossniklaus HE, Hutchinson AK, Capone A, Jr., W oolfson J, Lambert HM: Clinicopathologic features o f surgically excised choroidal neovascular membranes. Ophthalmology 1994,101:1099-1111 6. Bressler NM, Silva JC, Bressler SB, Fine SL, Green WR: Clinicopathologic correlation o f drusen and retinal pigment epithelial abnormalities in age-related macular degeneration. Retina 1994,14:130-142 7. Liversidge J, Dawson R, Dick AD, Forrester JV: Uveitogenic epitopes o f retinal S antigen are generated in vivo via an alternative antigen-presentation pathway. Immunology 1998,94:271-278 8. Lutty G, Grunwald J, Majji AB, Uyama M, Yoneya S: Changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration. Mol Vis 1999,5:35 9. Uyama M: [Choroidal neovascularization, experimental and clinical study]. Nippon Ganka Gakkai Zasshi 1991,95:1145-1180 10. Kliffen M, Sharma HS, Mooy CM, Kerkvliet S, de Jong PT: Increased expression of angiogenic growth factors in age-related maculopathy. Br J Ophthalmol 1997, 81:154- 162 11. Gaha U, Gomes WA, Kobayashi T, Pestell RG, Kessler JA: In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev Biol 2002, 249:108-120 12. Tsumaki N, Nakase T, Miyaji T, Kakiuchi M, Kimura T, Ochi T, YosMkawa H: Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis. J Bone Miner Res 2002,17:898-906 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13. Minina E, Wenzel HM, Kresckei C, Karp S, Gaffieid W, McMahon AP, Vortkamp A: BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation. Development 2001,128:4523-4534 14. Pizette S, Niswander L: BMPs are required at two steps o f limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes. Dev Biol 2000,219:237-249 15. Brunet LJ, McMahon JA, McMahon AP, Harland RM: Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 1998,280:1455-1457 16. Deckers MM, van Bezooijen RL, van der Horst G, Hoogendam J, van Der Bent C, Papapoulos SE, Lowik CW: Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology 2002, 143:1545-1553 17. Furuta Y, Hogan BL: BMP4 is essential for lens induction in the mouse embryo. Genes Dev 1998,12:3764-3775 18. Chang B, Smith RS, Peters M, Savinova OV, Hawes NL, Zabaleta A, Nusinowitz S, Martin IE, Davisson ML, Cepko CL, Hogan BL, John SW: Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure. BMC Genet 2001,2:18 19. Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA: Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994, 35:101- 111 20. Takehana Y, Kurokawa T, Kitamura T, Tsukahara Y, Akahane S, Kitazawa M, Yoshimura N: Suppression o f laser-induced choroidal neovascularization by oral tranilast in the rat. Invest Ophthalmol Vis Sci 1999,40:459-466 21. Hoffmann S, Spee C, Murata T, Cui JZ, Ryan SI, Hinton DR: Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads. Graefes Arch Clin Exp Ophthalmol 1998,236:779-784 22. You L, Kruse FE, Pohl J, Volcker HE: Bone morphogenetic proteins and growth and differentiation factors in the human cornea. Invest Ophthalmol Vis Sci 1999,40:296-311 23. Marshall CJ, Kinnon C, Thrasher AJ: Polarized expression o f bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region. Blood 2000, 96:1591-1593 24. Sorescu GP, Sykes M, Weiss D, Platt MO, Saha A, Hwang J, Boyd N, Boo YC, Vega JD, Taylor WR, Jo H: Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response. J Biol Chem 2003, 278:31128-31135 25. Blaauwgeers HG, Holtkamp GM, Rutten H, Witraer AN, Koolwijk P, Partanen TA, Alitalo K, Kroon ME, Kijlstra A, van Hinsbergh VW, Schlingemann RO: Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. localization o f vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol 1999,155:421-428 26. Tombran-Tink I, Johnson LV: Neuronal differentiation o f retinoblastoma cells induced by medium conditioned by human RPE cells. Invest Ophthalmol Vis Sci 1989, 30:1700- 1 7 0 7 27. Bird AC: Bruch's membrane change with age. Br J Ophthalmol 1992, 76:166-168 28. Rakic JM, Lambert V, Devy L, Luttun A, Carmeliet P, Claes C, Nguyen L, Foidart JM, Noel A, Munaut C: Placental growth factor, a member of the VEGF family, contributes to the development o f choroidal neovascularization. Invest Ophthalmol Vis Sci 2003, 44:3186-3193 29. Tobe T, Takahashi K, Ohkuma H, Uyama M: [Experimental choroidal neovascularization in the rat]. Nippon Ganka Gakkai Zasshi 1994,98:837-845 30. Regazzoni C, Winterhalter KH, Rohrer L: Type I collagen induces expression of bone morphogenetic protein receptor type II. Biochem Biophys Res Commun 2001, 283:316- 322 31. Oh H, Takagi H, Takagi C, Suzuma K, Otani A, Ishida K, Matsumura M, Ogura Y, Honda Y: The potential angiogenic role o f macrophages in the formation of choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1999,40:1891-1898 32. Elner VM, Strieter RM, Elner SG, Baggiolini M, Lindley I, Kunkel SL: Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigment epithelial cells. Am J Pathol 1990,136:745-750 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4 Future Direction: Development of a Cell-type-specific Cre Transgenic Model and Its Potential Application for Site-Specific Mutation of BMP4 in the Retinal Pigment Epithelium (RPE) 4-1. Sum m ary This chapter describes a noval and reliable approach to study BM P4 function in age-related m acular degeneration (AMD) pathogenesis using site-specific somatic mutation of BMP4 or BMP receptors in RPE. As a proof o f principle prior to application to the RPE, we have succeeded in applying the Cre-loxP system for the study o f prostate cancer. This work was done by the author in the laboratory o f Dr. Pradip Roy-Burman and was published in Mechanisms o f Development 2001. A comprephensive account o f this work is detailed in this chapter. Future direction of how to apply Cre-loxP system in the ocular tissue is also provided. Recent advances in mouse genetics have enabled tissue- and time-specific gene targeting using the Cre recombinase. BMP4 homozygous mutation is embryonic lethal and heterozygous mutations have various ocular phenotypes. In order to study its specific role in AMD, it is necessary to generate time- and site-specific somatic mutations of BMP4 selectively in the RPE in mice. The author has previously generated a transgenic mouse line (PB-Cre4) that expresses the Cre recombinase under the control of the composite prostate-specific probasin (PB) promoter. The presence o f Cre in the prostate epithelium was determined by RT-PCR detection o f Cre mRNA and Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice. It is conclusively demonstrated that Cre expression is postnatal and prostatic epithelium-specific. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B esides the prostate gland no other tissues o f the adult PB-Cre4 mice demonstrate significant Cre expression except for a few scattered areas in the gonads and the stroma o f the seminal vesicle. By crossing the PB-Cre4 animals with Boxed retinoid X receptor a (RXRa) allelic mice, we dem onstrate that mice, whose conventional knockout o f this gene is lethal in emhryogeness, could be propagated with selective inactivation o f R X R a in the prostate and the inavtivation o f R X R a in the prostatic epithelium leads to the development o f preneoplastic lesions. The results o f recent studies have shown that the PB-Cre4 mouse line is a useful resource for the genetic-based studies on prostate development and prostatic disease. The conditional Cre-loxP system can be applied to any tissue in a cell-type restricted manner. Floxed BMP4 allele mouse line has been generated and as suitable Cre transgenic mouse lines or virus vectors become available, analysis of BMP4 function specifically in RPE at various stages for different duration can be fulfilled. Our future direction is to generate transgenic strains for producing conditional mouse mutants to investigate the function o f genes, specially BMP4 in RPE, opening new avenues in RPE cell biology and pathology. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-2. Introduction T h e ability to knockout genes in mouse embryonic stem cells has been widely used to study gene function in vivo. Although conventional gene knockouts have shed light on various parameters about the function o f many known genes during development,1 ’2 they have not achieved the same success in exploring the physiological and pathophysiological roles o f genes in mature animals. T his lim itation is due to the confounding and potentially lethal effects o f gene dysfunction during pre- or early post-natal development in standard gene targeting. This approach does not permit control over the timing of gene disruption or allow the subsequent study o f genes that lead to early em bryonic lethality or premature death. Furthermore, since conventional gene knockouts provide animals that inherit genetic deletions in all cell types, it is often difficult to exclude the possibility that abnormal phenotypes observed in adult animals arise indirectly from an underlying developmental defect3 or to define in w hich tissue the targeted gene acts. Conditional gene knockout techniques provide a means to circumvent some of the limitations of conventional gene knockout. The Cre-loxP recombination system 4 has become a powerful tool for conditional, cell-type and tissue-specific deletion o f genes and is limited only by the availability and specificity of the promoter. The bacteriophage site-specific DNA recombinase (Cre) 5 excises intervening DNA sequences located between two unidirectional ioxP recognition sequences (“ floxed” ), leaving one IoxP site on the linear DNA.6 Cre recombinase has been used successfully to delete endogenous genes or to activate transgenes in mammalian cell culture systems 7 as well as transgenic mouse models.8 Implementation of this strategy requires the creation o f conditional target alleles in mice also expressing Cre in a cell type-restricted 9 and /or inducible manner using tissue-specific l0 or inducible promoters.11 Recent studies focusing on m olecular genetics o f prostate cancer i2 ,1 3 are turning into a fruitful area o f investigation which might provide clues to the understanding the complex biology of prostate cancer. In this regard, it is o f paramount importance to derive a Cre recombinase 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transgenic animal system with robust expression of a biologically active Cre protein in a prostate epithelial cell-specific manner. To this end, we selected a rat probasin promoter (PB) to drive expression o f the Cre gene. The original PB promoter including bases -426 to +28 was shown to be sufficient to target gene expression specifically to the epithelial cells of the prostate in transgenic m ice.5 4 Using this minimum promoter to target gene expression o f SV40 large T and small t antigens, a mouse model (TRAMP) for prostate cancer was created.1 5 Subsequently, it was realized that the small PB promoter was lacking important enhancers that would facilitate high levels o f gene expression in the prostate. A large (L) fragment o f the PB promoter (-11,500 to +28) w as determined to be highly efficient for transgene expression in mice.1 6 This second generation LPB fragment was coupled to SV40 large T antigen (with a deletion to prevent the expression o f the small t antigen) which led to development o f new transgenic line o f prostate cancer.1 7 The LPB-Tag lines demonstrated a wide range o f tumor growth rates. In contrast to the rapid metastasis seen with TRAMP mice, the LPB-Tag transgenic lines rarely metastasized. Since the production o f the LPB promoter, a third generation promoter has been made to target high levels o f transgene expression specifically to the prostate in transgenic nice. This new composite PB promoter construct (ARR 2PB) is less than 500 bp in size, androgen and glucocorticoid regulated, prostate-specific and targets high level expression in transgenic m ice.1 8 Retinoids have attracted much interest for prostate cancer prevention and treatment. There is evidence that retinoids could effectively inhibit tumor growth and progression in various chemical- induced mouse prostate cancer models.19, 20 In studies with human prostate cancer cell lines, retinoids alone or with other chemotherapeutic agents reduced their clonal growth and tumorigenic potential. 2 1 Although clinical trials with retinoids for prostate cancer indicated only limited efficacy to date, it is, however, contended that improved pharmacokinetics and application of selective retinoid analogues might lead to abetter clinical outcom e.22,23’24 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A ctions of retinoids are mediated either by its unclear receptors or through receptor-independent mechanisms. There are two families o f RA receptors, RARs (a-, fl , and T) and RXRs («,B, and T )T5’ 26 The physiological consequences o f RAR and RXR inactivation were investigated via conventional knockout technology. It was reported that RAR'/ null mutant mice developed squamous metaplasia o f the prostate.2 Because mice lacking both RXRfi and R X R l were normal in term s of prostate morphology and function 28 and considering that the active RA receptor is indeed a heterodimer o f one RAR and one RXR,29 the critical RXR in prostate biology appears to be RXRa-. Moreover, to mediate multiple signaling pathways in the prostate, RXRjar may partner with other nuclear receptors, such as PPAR7 and vitamin D receptor, the ligands o f which have been shown to Inhibit prostatic cancer cell growth.29’30 Because conventional disruption o f the RXRa gene is embryonic lethal,3* ’ 32 we used our PB-Cre4 mice, which express a high level o f Cre recombinase specifically in the prostatic epithelium, to breed with floxed RXRa mice33 to selectively mutate the RXRa gene for a direct assessment o f the role o f RXRa in the prostate. We document here that RXRor is a critical gene function in m aintaining normal phenotype o f the gland because loss o f R X R a function results in developmental and functional abnormalities as well as preneoplastic lesions in the prostate. 4-3. M aterials and methods 4-3-1. Transaene Construction To construct a cassette for the production o f Cre recombinase transgenic mice, the N otlX hoI fragment o f probasin promoter containing two tandem repeats o f the androgen responsive region (ARR2 PB), was ligated into pCreSV which was digested w ith the same enzymes as above. The pCreSV vector contained the Cre cDNA, SV40 poly A sequence and splicing signal sequences in the backbone o f pBluescript SK plasmid. The final cassette is shown in Fig. IA. The fragment containing the above three regions was released by digestion with N o tl and Kpnl, isolated by 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. agarose gel, purified by Qiagen spin column and elirtip column according io the instruction from m anufacturer, and then used for transgenic injections. 4-3-2. Generation of PB-Cre Transgenic Mice T h e prom idear injection of the ARR2PB -Cre fragment into (C57BL/6 X DBA2) Fj hybrid mouse fertilized eggs and placement of the embryos into psuedo-pregnant females were performed at the N orris Cancer Center Transgenic Core Facility o f the University of Southern California. Potential founder animals were screened by PCR, and further confirmed by Southern blot analyses. Mouse tail DNAs (about 1 j1 g) were PCR amplified by 35 cycles on a thermal cycler using primers that span the SV40 poly A signal sequence (Fig. 1A). The primers used were 5'- CTTCrGTGGTGTGACATAATTGG-3' and 5'-GATGAGTTTGGACAAACCACAAC-3' and they produced a 500 bp amplification product. 4-3-3. Cross Breeding o f PB-Cre mice with R26R Reporter Strain and RXRa Floxed Strain The ARR2 PB-C1E mice were cross-bred with the reporter line, R26R (Soriano, 1999). In this system, production o f Cre results in the excision o f an intervening DNA segment resulting in expression o f LacZ protein. The homozygous R26R mice were used to breed with ARR2PB-Cre transgenic lines. All offspring were genotyped by PCR using primers specific for the Cre transgene and the R26R allele. The primers for the R26R reporter strain were a mixture o f three oligonucleotides: 5'-AAAGTCGCTCTGAGTTGTTAT-3', 5'-GCGAAGAGTTTGTCCTCAACC- 3' and 5'-GGAGCGGGAGAAATGGATATG-3' which amplified approximately 500 bp wild type and 250 bp mutant fragments. Another conditional allele selected was floxed RXRa allele (Chen et a l, 1998). The chromosomal R X R a gene was manipulated by homologous recom bination such that IoxP sites were introduced into the two introns surrounding the fourth exon of the gene, an exon which encodes an essential domain o f the RX R a protein; when the intervening sequence (including the fourth exon) is deleted, the gene is converted into a loss-of-function allele. The floxed allele of RXRa is a good 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. candidate for the test of Cre-mediated inactivation in post-natal growth and development o f the prostate. Consequently, we crossed the Cre mice with the floxed RXRa mice to assess the recombination of the conditional R X R a allele in the prostate epithelium. 4-3-4. RT -PCR The total RNA was isolated from prostate and other organs using Qiagen RNeasy Mini kit according to the instruction o f the manufacturer. Two micrograms o f RNA were reverse transcribed using ThermoScript reverse transcriptase (Gibco) and random primers. The RT reaction (2 p i) was used in a 50jjl 1 PCR reaction mixture. Thirty-five cycles o f PCR were perform ed in the presence of two specific primers from th e N-terminal region o f Cre cDNA sequence. The sequences o f these two prim ers were 5'-TTGCCTGCATTACCGGTCGATGCA-3' and 5'-GATCCTGGCAATTTCGGCTAT-3'. 4-3-5. X-aal Staining and Histology The lateral, dorsal, ventral and anterior lobes o f the prostate and other organs (liver, kidney, lung, heart, spleen, brain, salivary gland, skin, bladder, seminal vesicle, epididymas and testes) were dissected from R26R/Cre male mice, fixed and stained for 4 6 hours w ith X-Gal at 37 °C for P- galactosidase activity [Sanes et al., 1986]. In a separate experiment, the organs were frozen and embedded in O.C.T, and frozen sections were prepared and stained with X-gal for microscopic localization o f Cre activity. The female double transgenic mice were similarly aialyzed for the staining o f various tissues including gender-specific tissues such as, mammary glands, ovary and uterus. Embryos of 9 and 15 gestation days w ere opened, fixed, stained by the same procedure. To determine the pattern o f developmental expression o f Cre transgene, double transgenic male mice positive for Cre and lacZ were analyzed at weekly intervals from newborns through 2 weeks of age. The urogenital system was removed and stained by X-gal following the same procedure. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-3-6. DNA Preparation and PCR Analysis o f R X R a Alleles DNA was purified from various mouse tissues by overnight lysis at 55°C in lysis buffer containing proteinase K, followed by phenol/chloroform extraction and isopropanol precipitation. 0.5 jig o f genom ic DNA was used for each PCR reaction. Primers used for RXRa allele amplification were: 5'-ACCAAGCACATCTGTGCTATCT-3' and S'-ATGAAACTGCAAGTGGCCTTGA -3' and they produced 3 bands of approximately 1.5 kb, 1.7 kb and 500 bp corresponding to wild type, floxed and floxed out RXRa, respectively. 4-3-7. Tissue Preparations A 150-jx 1 solution o f 10 mg/ml BrdUrd (Sigma Chemical Co., St. Louis, MO) was injected i.p. 1 h before animals were sacrificed. The urogenital system was surgically isolated, and the individual prostatic lobes were dissected out under a dissecting microscope. Tissues for histopathological observation were fixed in 10% neutral buffered formalin (Surgipath, Richmond, IL) for overnight. Fixed tissues were processed and embedded in paraffin. Thin sections (5 p m) were produced and stained with H&E. Tissues for RNA assays were frozen in liquid nitrogen immediately after dissections until usage. 4-4. Results 4-4-1. Generation of ARR2PB-Cre4 Mice Transgenic animals resulting from the pronuclear injection o f the ARRjPB-Cre construct in Fig. 1A were evaluated by PCR analysis and the positives identified were confirmed by Southern blot hybridization (data not shown). Five founder animals were initially identified as having integrated the transgene. Germ line integration was found in animals 4, 5 and 6 and the transgene was passed to offspring in normal Mendelian ratios. Founders 8 and 12 were unable to transmit the transgene due to infertility and lack of germ line integration, respectively. The offspring from the three remaining founders were normal at birth and no abnormalities were observed through adulthood. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To determine if the transgene was appropriately expressed in the prostate, RT -PCR was performed on total RNA isolated from prostate tissue (Fig. IB). The three founder lines produced detectable levels o f Cre mRNA in the prostate, as evidenced by the expected 500 bp PCR product. The results were not due to any DNA contamination in the samples since no product was obtained in the absence o f reverse transcriptase. To determ ine f there was spurious expression o f the transgene, various other tissues (liver, kidney, ductus deferens, testes, seminal vesicles, heart, lung, spleen, and brain) from line 4 animals were examined for Cre mRNA expression by RT- PCR. There was no detectable Cre mRNA in these tissues. For illustration, the results obtained from testes, seminal vesicle, liver and kidney are shown in Fig. IB. Thus, the findings were consistent with the previously published tissue specificity o f the probasin promoter.14,16,1 8 These three founder lines were maintained for further analysis of Cre activity. 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-1: ARRjPB-Cre A, Structure o f the ARR2 PB-Cre-SV40 transgene construct. The plasmid contained the ARR2Pb composite probasin promoter followed by the cDNA for Cre and SV40 polyadenylation sequence. Arrows indicate the position o f the primers used in PCR-based identification of transgenic mice. B, RT -PCR detection o f Cre mRNA. Total RNA from the prostates o f founders 4, 5, 6 and nonprostatic tissues from line 4 mice was analyzed by RT-PCR RT-PCR was performed as described in the Experimental procedures either in the presence (+) or absence 0 of reverse transcriptase to control for DNA contamination. M, 100-bp marker. TE, testes; SV, seminal vesicles. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-4-2, Tissue-specific Cre recombination Activity T he ability to produce tissue-specific and cell-specific gene deletions is dependent on the level of the Cre protein and its subsequent recombination activity. In order to validate the extent and specificity o f Cre expression, each line was crossbred with the R26R line o f transgenic reporter m ice carrying a iox-stop transgene.j5 In the R26R allele, a constitutively active chromosomal gene was manipulated to insert the lacZ gene such that p-galactosidase protein is produced only following removal of a “staffer” fragment flanked by IoxP sites. The R26R gene is constitutively expressed in every cell o f the embryo and adult, but in the absence o f recombination does not encode a functional LacZ product. One important aspect regarding Cre-mediated recombination is that it is a unidirectional and cumulative process. With recombination occurring in a target cell, all progeny o f that cell will harbor the recombined allele, even when the promoter driving Cre expression is inactivated. Offspring from each founder were mated to the R26R mouse line and at 8 weeks o f age the urogenital system o f the double transgenics (lacZ+, Cre+ ) was stained for the presence o f LacZ activity using X-gal. Line 4 was the only line to produce sufficient amounts of Cre protein to result in recombination as determined by LacZ staining. This line was termed PB- Cre4 and was used in all subsequent experiments. We examined the expression pattern o f the R26R reporter allele crossed against the PB-Cre 4 transgene in several 8-week old male mice; all mice showed essentially the same pattern o f X-gal staining in the prostate (Fig. 2A), while Cre- negative littermate controls did not display X-gal reactivity (Fig. 2B). In the lateral lobe, almost complete (> 95%) labeling o f the prostatic epithelium was observed (Fig. 2C); approximately 50% of the epithelium o f the ventral prostate lobe (Fig. 2D), and about 10% o f the epithelium o f the dorsal and 5% of the epithelium of the anterior lobe, was stained (Fig. 2E, F). There was scattered fibromuscular stroma staining (approximately 1%) that was detected surrounding the ducts of the dorsal and anterior lobes (Fig. 2E, F), which was absent in the lateral and ventral lobes. To date, 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. w e have assessed the frequency o f recombination in animals up to S weeks o f age. Because the probasin promoter is active throughout adult male life, we expect that recombination events in all o f the prostatic epithelium will increase with further growth o f the animals. It is also expected that the onset and recombination frequency would be accelerated by treatment with androgens. To determine the tissue specificity o f Cre activity beyond that shown in Figure 2, 9 other tissues, namely, liver, kidney, lung, heart, spleen, brain, salivary gland, skin and bladder were removed from male or female double transgenic mice. In addition, seminal vesicle, epididymas and testes from the males, and ovary, uterus and mammary gland from the females were examined. Non prostatic tissues were negative for Cre activity w ith the exception of seminal vesicles, testes and ovaries. There was less than 5% blue staining in the stroma o f seminal vesicles, without any detectable epithelial staining (Fig. 3A). The age-matched control animals did not produce any X- gal reaction in the seminal vesicle (Fig. 3B). Since no Cre mRNA was detected in mature seminal vesicle o f the double transgenic mice, the discrepancy between X-gal staining and RT-PCR could be related to LacZ activation by recombination at an early time point in the development o f the seminal vesicles and lack o f continued Cre gene expression in this tissue in the mature transgenic mice. Similarly, it is noteworthy that although the gonads o f the adult PB-Cre4 mice tested negative for Cre mRNA expression by RT -PCR, a few isolated areas o f Cre activity were observed in the gonads o f the double transgenics. The testicular activity was characterized by staining of a few short segments o f individual tubules (Fig. 4A). However, it remains to be determined in which cells the recombination occurred. The activity in the testes appeared to be mostly extracellular staining rather than cytoplasmic (Fig. 4A, inset). The Cre activity, detected by Xgai, was restricted to a few sites in the ovary (Fig. 4C) which appeared to be w ithin (Fig. 4C, inset) oocytes but the number o f spots was far less than the num ber o f primary oocytes that reside in the ovary. 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The lack o f reactivity in the testes and ovary o f the Cre-negative age-matched ’ attenuates is illustrated in Fig. 4B and Fig. 4D, respectively. To further detect Cre expression in tissues other than the ones mentioned in the whole animal, double transgenic embryos of 9 and 15 gestation days were examined. No staining were observed, w hich provide additional strong evidence o f Cre specificity o f this model 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-2 Beta-gai Staining (3-gal staining o f mice harboring the Cre gene and lacZ reporter gene as well as Cre negative, age- matched control mice. Tissues from 8-week old double transgenic mice were used for detection o f Cre recombinase activity using X-gai staining (blue stain). W hole-mount staining o f the urogenital system o f a transgenic (A) and control mice (B). The individual lobes of the prostate are labeled: 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A, anterior lobes; D, dorsal lobes; L, lateral lobes; V, ventral lobes; other notations indicate DD. ductus deferens; SV, seminal vesicle; 3 , bladder and U, urethra. C, D, E, F, Cross-section of frozen tissues o f the lateral, ventral, dorsal and anterior prostate, respectively, stained with X-gal. Exclusive epithelial staining in lateral and ventral is emphasized in. insets in C and D, respectively. The presence o f staining in fibromuscular stroma in dorsal and anterior lobes is indicated by arrows in E a n d F an d emphasized in the corresponding insets. Ductus deferens showed staining in both double transgenic and control mice due to the presence o f endogenous P-galactosidase activity. Bar in A which also applies to B represents 1 mm; bar in C applying equally to D, E and F, denotes 0.1 mm and bar in inset o f C applies equally to all insets representing 0.01 mm. A B Figure 4-3: C re Activity in Seminal Vesicles Localization of Cre activity in seminal vesicles. Scattered staining was detected only in the fibromuscular stroma o f seminal vesicles o f the Cre/R26R double transgenic mice (A) but none in the control mice (B). The area selected for illustration in A is the most dense for staining which is not representative o f the average level o f staining in the seminal vesicles. Bar in A equals 0.05 mm. The magnification in B is identical to A. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F igure 4-4: C re Activities in Testes and Ovaries Detection of a limited amount of Cre activity in testes and ovaries. A, Whole mount of a testicle of a Cre/R26R double transgenic mouse showing highly focal staining in the seminiferous tubules. The inset at right shows a cross section o f an individual tubule and the associated staining. B, A matched control testicle and tubule (inset). C, W hole mount of an ovary o f Cre/R26R double transgenic mouse ovary showing patchy staining; the inset shows a cross section o f a single follicle with a X-gal positive oocyte, D, A matched control ovary and follicle (inset). The bar in A represents 1 mm which also applies to B. The bar in C, also applicable to D, represents 1 mm. The bar shown in the inset o f A, applicable to all insets, represents 0.05 mm. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-4-3. Onset and Extent o f Cre Activity During Postnatal Development To determine temporal expression o f Cre in prostate development, to date we performed X-gal staining on the urogenital system at weekly intervals from newborn animals to 2 weeks o f age. In the newborn, the prostatic buds begin the process o f branching morphogenesis, and it was found that Cre-mediated recombination was visible at this point in development (Fig. 5A). Several highly focal areas o f X-gai staining could be identified, and by one week of age, significant branching had occurred and the ventral lobe could be distinguished from the dorsal/lateral prostate (Fig. 5B). At this point, the ventral prostate had the most X-gal staining, but staining was still focal, much like in the ventral lobe o f the 8-week old mice. The dorsal/lateral prostate, which is much smaller in size, contained focal staining. By two weeks o f age, the ventral, lateral and dorsal lobes could be distinguished based on X-gal staining (Fig. 5C). The lateral prostate was completely stained indicating that by two weeks, the majority of epithelial cells have had recombinational events. Both the ventral and dorsal lobes had focal areas o f staining at this point in postnatal development. To determine when Cre-mediated recombination first occurs, it would now be necessary to examine appropriate primordia at various prenatal time points using the double transgenic mice. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-5: Postnatal Prostatic Cre Activity Cre expression during postnatal prostate development. X-gal reaction in the whole mount o f the urogenital system o f newborn (A), 1 week old (B) and 2 week old (C, D) double transgenic mice. Arrows in A point to evidence o f Cre recombinase activity in prostatic bud o f a newborn mouse. One week old mouse prostate (B) shows significant area o f focal staining in the ventral lobe (vp) and some activity in the dorsal/lateral prostate (dip). Two week old mouse prostate (C) displays strong staining in the lateral prostate (Ip) and focal staining in the dorsal (dp) and ventral lobes (vp). Whole mount staining o f the prostate o f a two week old double transgenic mouse is included for reference in which DD and B denote ductus deferens and bladder, respectively. Bar in A applies equally to B and C, representing 0.1 mm. Bar in D represents 1 mm. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-4-4. Evidence for the Utiiitv o f The Model: Conditional Mutation of R X R a alleles Since conventional knockout o f RX R a gene is lethal in embiyogenesis, we were interested in production o f double transgenic mice with both the Cre transgene and a conditional flexed (F) R X R a allele. When male Cre heterozygous PB-Cre4 mice were crossed with female homozygous floxed R X R a mice, the intact floxed allele was transmitted to 15/15 (100%) o f offsprings. However, if a female Cre heterozygous PB-Cre4 mouse was mated to a male R X R a homozygous mouse, the intact floxed R X R a allele was transmitted in only 8/17 (47%) o f the offsprings. The remaining 9 animals contained a floxed out (Fo) allele indicating that Cre mediated recombination occurred, resulting in disruption o f one RXRa allele. This could be due to expressed Cre protein in the egg because some Cre-negative progeny also showed recombination o f RXRa allele. To determine if sperm containing Cre transgene could initiate recombination o f floxed RXRa in the egg, Cre+/RXRa (F/W) male mice were bred to a RXRa (F/W) female. None o f the progenies contained floxed out RXRa allele in 39/39 (100%) animals. Thus, the localized Cre activity detected in the testes was not sufficient to initiate recombination o f either maternal or paternal copies o f the floxed RXRa The prostate specificity o f Cre recombination in the conditional RXRa mice was also confirmed. Male mice carrying the Cre transgene and one floxed RXRa were raised to maturity and analyzed for prostate specific RXRa recombination (Fig. 6). Total DNA from the indicated tissues were subjected to PCR analysis for the floxed RXRa gene and the only tissues w ith evidence of recombination were the dorsal/lateral prostate, ventral prostate and anterior prostate. Tail, epididymis, seminal vesicle, testes, bladder, liver, heart, kidney, spleen and muscle showed no detectable recombination. Male mice carrying the Cre transgene and two conditional RXRa alleles were raised to 3 months to date, demonstrating the utility o f the conditional approach for inactivating genes that would have embryonic lethal phenotypes in conventional knockouts. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Phenotypic changes in the prostate in the homozygous R X R a mutant animals of different age groups ranging from 1 to 15 months were investigated. Developmentaily, prostatic ductal branching appeared to be increased from the loss o f RXRa function. There was also a significant change in the profile o f secretory proteins in the RXRa mutant prostate relative to littemiate controls with intact RXRa allele. Histopathologically, homozygous JLTite-deficient prostates show ed multifocal hyperplasia as early as 4 months o f age. Lesions, which could be described as low-grade prostatic intraepitheilal neoplasias, were detected after 5 months. Subsequently, beginning at ~1Q months, high-grade prostatic intraepithelial neoplasias developed in some animals. The incidences of low-grade prostatic intraepithelial neoplasias and high-grade prostatic intraepithelial neoplasias among the animals 10-15 months o f age were 62 and 17%, respectively. The heterozygous mutant mice also developed sim ilar prostatic phenotypes but in a delayed manner, implying a role o f haploinsufficiency. Together, these results indicated for the first time th a t a major component of retinoid action in the prostate is mediated by a retinoid receptor, RXRa, the inactivation o f which in the prostatic epithelium leads to the development o f preneoplastic lesions. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F ig u re 4-6: PC R PCR genotyping o f R X R a alleles. Total genomic DNA from different lobes of the prostate and various other tissues from an 8 week old Cre +/RXRaF/W mouse was analyzed by PCR. PCR was performed as described in the Experimental procedures and alleles o f R X R a w ets differentiated by size. F, floxed; W, wild type; Fo, floxed out. AP, anterior prostate; VP, ventral prostate; DLP, dorsalateral prostate; SV, seminal vesicle. The band at approximately 800 bp is a nonspecific PCR artifact most pronounced in VP, and also detected in Cre negative prostate tissues (not shown). 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-5. Discussion In the present study, we developed a transgenic mouse model in which the Cre transgene is expressed in the prostate epithelium. By breeding with the R26R tester stein , conclusive evidence w as obtained for the prostatic epithelial-cell specific expression o f Cre in the transgenic mouse line PB-Cre4. The results o f the tissue specificity experiments Indicated that the vast majority of Cre activity was localized to the luminal epithelium of the mouse prostate lobes. There was no evidence o f Cre expression in the basal cells o f any o f the prostate lobes examined. The development o f the PB-Cre4 transgenic mouse line w ith a robust and tissue-specific expression of Cre recombinase allowed us to circumvent the embryonic lethality that is caused by conventional knockout o f the RXRa gene in the mouse. Our data from the Cre-loxP model that in the absence of RXRa, i.e., in the homozygous mutant animals, a substantial increase in ductal branching is induced in the prostate, most notably in the lateral and anterior lobes. A deficiency in RXRa* is also sufficient to drive proliferation in the prostatic epithelium to produce multifocal hyperplasia as early as 4 months of age. A stochastic pattern o f increased degree o f phenotypic abnormalities o f lesions, beginning with epithelial hyperplasia followed by presentations o f LGPINs and then by HGPINs is noted. Reproducible changes in secretory protein profiles in the prostatic lobes, mainly LP, DP, and VP, were also documented attributable to RXRa gene inactivation, although the nature o f the secreted proteins remains to be identified. As stated previously, analysis o f conventional knockout phenotypes are confounded by indirect effects between cell types. For example, an epithelial phenotype could be due to the loss of gene expression in the basal or stromal cells underlying the epithelium. By comparing the phenotypes o f both conventional and luminal epithelial cell knockout, one could determine if the phenotype is the result o f direct or indirect effects. By using primary cultures or tissue recombinants from 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conventional and luminal epithelial cell knockouts more elaborate dissections of cellular communication could be performed. W e presented evidence for the development o f a unique reagent, useful for the scientific community at large. This model will certainly be a resource to those who want to selectively m anipulate genes o f interest in the prostate epithelium during postnatal development o f this gland or tumorigenesis and progression to prostate cancer. For example, the model could immediately be explored for determining the involvement o f several potentially interesting genes (such as, FGFs, EGFR3, EGFR4, IGFI, gp 130, BRCAl, PTEN and STATE) in prostate biology and tumorigenesis. A ll o f these genes have been established as conditional alleles, and could be crossed with the PB- Cre4 mice. Additionally, those who may wish to embark on new strategies for isolation o f epithelial cells from the prostate using genetic-based approaches will benefit from the Cre model. RPE cells play critical roles in maintaining the homeostasis o f the retina and in controlling choroidal neovascularization. Mutations that cause loss o f function in RPE in humans are particularly devastating. RPE dysfunction is also involved in pathogenesis o f age-related macular degeneration, which is the major and increasing cause o f vision loss among the elderly o f the industrialized world. The requirement o f RPE in eye development and in retinal function and survival has been demonstrated by RPE ablation experiments using RPE-specific expression o f the diphtheria toxin in transgenic mice,36 as well as by the occurrence of retinal dystrophies in rodents or humans carrying mutations in genes expressed in the RPE (eg, RPE65?1' 38 RGR,39 and RLBP140). Cre-loxP system permits dissection o f the roles o f multifunctional genes in a particular tissue/cell type 4 1 ’ 42. It has been successfully used in dissecting gene function for kinesin-II in rod photoreceptors 4 1 and RXR-alpha in the RPE.10 Disruption o f gene function in RPE specifically and efficiently can provide clear cut information on the pathophysiology o f ocular disease related genes. Furthermore, we can control the target gene expression by using inducible gene targeting 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that allows the disruption o f a gene active in tissue specific sites at any time in the life o f a mouse. In this way the effects o f the genetic abnormality can be followed after the retina have fully developed and are anatomically, physiologically and biochemically normal. W hile Cre/loxP system marks a milestone in the field of mouse reverse genetics, full exploitation o f this system requires further improvements. Cre transgenic mice meet some o f those standard requirements, time consuming and costly breeding will have to be performed to combine the different alleles (especially when multiple conditional null alleles are being analyzed). Somatic gene transfer to introduce the recombinase into cells in vivo is, therefore, an appealing option. Adenovirus and lentivims gene delivery have been well studied in the retina. Replication-defective adenovirus vectors could be grown and concentrated to for transduction o f retinal cells. Subretinal injection of adenovirus results in efficient and stable transduction o f RPE cells (and photoreceptors). Because recombinant adenoviral DNA does not stably integrate into host chromosomal DNA, passenger gene expression is transient in ocular tissue. This may be of sufficient duration for specific short-term gene intervention, if the immune response can be further modulated. Lentivirus vectors have been prepared and tested successfully in rat retinas. Infection after a single inoculation spread throughout the retina and was stable for at least 3 months. The requirement for chromosomal integration to obtain gene expression suggests that lentivirus vectors should lead to long duration transduction. Thus, lentivirus vectors expressing Cre recombinase under tissue-specific promoter regulation could be applied for long term gene expression. Bone Morphogenetic Protein 4 (Bmp4) has been implicated in the regulation o f numerous processes throughout embryonic and postnatal vertebrate development. Embryos lacking a functional Bmp4 gene fail to form an allantois and die around embryonic day (E) 9.5, precluding the analysis o f Bmp4 functions during later stages of embryogenesis and postnatal life.44 To circumvent this problem and allow time and tissue-specific gene inactivation, Kulessa et al have successfully created a conditional allele o f the mouse Bmp4 gene by introducing Cre recombinase 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. recognition sites (loxP) into the Bmp4 locus. As a first step towards dissecting BMP4 function in R PE and ocular disease pathogenesis, we can generate RPE specific Cre mice with inducible system using a transgenic strategy or somatic delivery o f virus vectors carrying inducible Cre transgene expression. Nevertheless, an understanding of the early events controlling RPE signaling and degeneration in mice with an induced excision o f BMP4 may help to develop strategies for the prevention or delay o f human retinal degenerations. In summary, we have previously generated prostate specific Cre mice that are efficient in carrying out Cre mediated gene inactivation. The PB-Cre4 mice that we generated have been shown valuable for delineating the specific function o f widely-expressed proteins involved in prostate cancer pathogenesis. In light o f the observation we have gained experience in successfully generating Cre expression in non-ocular tissues, which will be very useful in designing conditional gene expression studies in ocular tissue, future studies will be carried out to generate transgenic mice expressing Cre in RPE-specific and tim e-dependent manner and to study in vivo function o f BMP4 in RPE using a conditional gene targeting strategy. 4-6. References 1. 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Greenbeig NM, DeMayo FJ, Sheppard PC, Barrios R, Lebovitz R, Finegold M, Angelopoulou R, Dodd JG, Duckworth ML, Rosen JM, et al.: The rat probasin gene promoter directs hormonally and developm ental^ regulated expression o f a heterologous gene specifically to the prostate in transgenic mice. Mol Endocrinol 1994, 8:230-239 15. Greenberg NM, DeMayo F, Finegold M I, Medina D, Tilley WD, Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM: Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A 1995,92:3439-3443 16. Yan Y, Sheppard PC, Kasper S, Lin L, Hoare S, Kapoor A, Dodd JG, Duckworth ML, Matusik RJ: Large fragment o f the probasin promoter targets high levels of transgene expression to the prostate o f transgenic mice. Prostate 1997, 32:129-139 17. Kasper S, Sheppard PC, Yan Y, Pettigrew N, Borowsky AD, Prins GS, Dodd JG, Duckworth ML, Matusik RJ: Development, progression, and androgen-dependence o f prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer. Lab Invest 1998,78:i-xv 18. Zhang J, Thomas TZ, Kasper S, M atusik RJ: A small composite probasin promoter confers high levels o f prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo. Endocrinology 2000,141:4698-4710 19. Chopra DP, W ilkoff LJ: Reversal by vitamin A analogues (retinoids) o f hyperplasia induced by Nmethyl-N-nitro-N-nitrosoguanidine in mouse prostate organ cultures. J Natl Cancer Inst 1977, 58:923-930 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20. Pollard M, Luckert PH, Spom MB: Prevention o f primary prostate cancer in Lobund- W istar rats by N-(4-hydroxyphenyl)retinamide. Cancer Res 1991, 5 i :3610-3611 21. de Vos S, Dawson MI, Holden S, Le T, Wang A, Clio SK, Chen DL, Koeffler HP: Effects o f retinoid X receptor-selective ligands on proliferation o f prostate cancer ceils. Prostate 1997, 32:115-121 22. Culine S, Kramar A, Droz JP, Theodore C: Phase II study o f all-trans retinoic acid administered intermittently for hormone refractory prostate cancer. J Urol 1999, 161:173- 175 23. DiPaola RS, Rafi MM, Vyas V, Toppmeyer D, Rubin E, Patel J, Goodin S, Medina M, Medina P, Zamek R, Zhang C, White E, Gupta E, Hait WN: Phase I clinical and pharmacologic study o f 13-cis-retinoic acid, interferon alfa, and paclitaxel in patients with prostate cancer and other advanced malignancies. I Clin Oncol 1999,17:2213-2218 24. Trump DL, Smith DC, Stiff D, Adedoyin A, Day R, Bahnson RR, Hofacker J, Branch RA: A phase II trial o f all-trans-retinoic acid in hormone-refractory prostate cancer: a clinical trial with detailed pharmacokinetic analysis. Cancer Chemother Pharmacol 1997, 39:349-356 25. Leid M, Kastner P, Chambon P: Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem Sci 1992,17:427-433 26. Giguere V: Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev 1994,15:61-79 27. Lohnes D, Kastner P, Dierich A, M ark M, LeMeur M, Chambon P: Function o f retinoic acid receptor gamma in the mouse. Cell 1993, 73:643-658 28. Krezel W, Dupe V, Mark M, Dierich A, Kastner P, Chambon P: RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable. Proc Natl Acad Sci U S A 1996,93:9010-9014 29. M angelsdorf D. J. UK, Evans R. M.: The retinoid receptors, Raven Press New York, 1994, pp 319-349 30. Kubota T, Koshizuka K, W illiamson EA, Asou H, Said JW, Holden S, Miyoshi I, Koeffler HP: Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent anti tumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 1998, 58:3344-3352 31. Kastner P, Grondona JM, Mark M, Gansmuller A, LeMeur M, Decimo D, Vonesch JL, Dolle P, Chambon P: Genetic analysis of RXR alpha developmental function: convergence o f RXR and RAR signaling pathways in heart and eye morphogenesis. Cell 1994,78:987-1003 32. Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM: RXR alpha mutant mice establish a genetic basis for vitam in A signaling in heart morphogenesis. Genes Dev 1994,8:1007-1018 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33. Chen J, Kubalak SW, Cfaien KR: Ventricular muscle-restricted targeting o f the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis. Development 1998,125:1943-1949 34. Bhatia-Gaur R, Donjacour AA, Sciavolino PJ, Kim M, Desai N, Young P, Norton CR, Gridley T, Cardiff RD, Cunha GR, Abate-Shen C, Shen MM: Roles for Nkx3.1 in prostate development and cancer. Genes Dev 1999,13:966-977 35. Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999,21:70-71 36. Raymond SM, Jackson II: The retinal pigmented epithelium is required for development and maintenance o f the mouse neural retina. Curr Biol 1995, 5:1286-1295 37. Gu SM, Thompson DA, Siikunari CR, Lorenz B, Finckh U, Nicoletti A, Murthy KR, Rathmann M, Kumaramanickavel G, Denton MJ, Gal A: Mutations in RPE65 cause autosomal recessive childhood-onset severe retina! dystrophy. N at Genet 1997, 17:194- 197 38. Marlhens F, Bareil C, Griffoin JM, Zrenner E, Amalric P, Eliaou C, Liu SY, Harris E, Redmond TM, Amaud B, Claustres M, Hamel CP: Mutations in RPE65 cause Leber's congenital amaurosis. Nat Genet 1997,17:139-141 39. Chen P, Hao W, Rife L, Wang XP, Shen D, Chen J, Ogden T, Van Boemel GB, W u L, Yang M, Fong HK: A photic visual cycle of rhodopsin regeneration is dependent on Rgr. Nat Genet 2001,28:256-260 40. Saari JC, Nawrot M, Kennedy BN, Garwin GG, Hurley JB, Huang J, Possin DE, Crabb JW: Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation. Neuron 2001,29:739-748 41. Marszalek JR, Liu X, Roberts EA, Chui D, Marth JD, Williams DS, Goldstein LS: Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Cell 2000,102:175-187 42. Xia CH, Roberts EA, Her LS, Liu X, Williams DS, Cleveland DW, Goldstein LS: Abnormal neurofilament transport caused by targeted disruption o f neuronal kinesin heavy chain KIF5A. J Cell Biol 2003,161:55-66 43. Mori M, Metzger D, Picaud S, Hindelang C, Simomrtti M, Sahei J, Chambon P, Mark M: Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium. Am J Pathol 2004, 164:701-710 44. Winnier G, Blessing M, Labosky PA, Hogan BL: Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995, 9:2105- 2116 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5 Conclusion M ost AMD cases may not be caused by a single gene defect, even if genes play a major role in determining susceptibility to the disease. Nonetheless, an important benefit o f identifying a gene that causes AMD (even if it were to account only for a small fraction o f the total patient population) or a condition that very strongly resembles AMD or just a particular aspect o f the disease is that one might then construct a biochemical pathway in which other causes o f the disease might be understood and various treatm ent strategies might be developed. The evidence summarized in this thesis may provide some insight into some biochemical pathways involved in AMD pathogenesis. And the evolution o f technologies such as microarray analysis may accelerate the identification o f genes conferring susceptibility to or protection against AMD and foster the development o f comp lex animal models exhibiting more than one gene defect. In this thesis, the focus was on the RPE regulation o f BMP4 in different forms o f AMD, how BMP4 regulates RPE function in vitro and how RPE cells respond to traumatic injury in the presence o f BMP4 haploinsufficiency. In the dry form o f AMD, In chapter 2, we thoroughly investigated BM P4 expression and its detailed location in different stages and forms of AMD. In the normal eye, the RPE forms an intact polarized monolayer that separates the vascular choroids and the photoreceptor of the neural retina, therefore, preferential secretion o f different factors is crucial to the RPE function. We established an in vitro RPE monolayer model to study the preferential secretion o f BMP4. Further, we created an in vitro RPE senescence model which enables us to demonstrate that for the first time that BMP4 inhibits RPE proliferation and facilitates their progression into senescence. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T he pathogenesis o f wet/neovascular form o f AMD Is more complicated and involves more than one ceil types. In chapter 3, we provided both in vivo and in vitro data suggesting that BMP4 is a m ajor inhibitor o f angiogenesis in wet AMD (CNV), including inhibition of CEC in vitro angiogenesis, more severe wet AMD phenotype in BMP4 haploinsufficient mouse model and regulation o f BMP4 by TNF and VEGP. Targeting the BMP4 signaling pathway involved in CNV m ay provide an alternative and complementary treatment approach which prevents CNV formation by stimulating RPE cells producing the anti-angiogenic growth factors, or by restoring the inhibitory effect o f the growth factor on the CECs. A n early portion o f m y PhD study was devoted to generate tissue-specific gene inactivation mouse model using Cre-loxP system. Tissue-specific inactivation of certain genes provide not only better understanding the pathogenesis o f altered tissue function and behaviors, but also stimulate the development o f specific therapeutic interventions targeting the specific intracellular pathways. We have successfully generated prostate epithilium-specific Cre mouse model and the model has been applied to generate various gene inactivation in the prostate, most o f which whole genome inactivation cause embryonic lethality. Despite the tissue difference, same concept and technology can be applied to the retina tissue and BMP4 inactivation in RPE can be fulfilled using either virus-mediated Cre or transgenic Cre mouse model. The results will provide strong information to dissect mediating pathways o f RPE activation in AMD and a potential therapeutic target in AMD (chapter 4). In summary, identification o f genes whose function or expression is altered in AMD remains critical for understanding and preventing AMD. Functional analysis o f the proteins encoded by those candidate genes, especially determining how they interact with the biochemical pathway in RPE cells, will be essential in establishing their role in AMD. This project successfully demonstrated that BMP4 plays critical roles in the regulation o f RPE during different stages of 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A M D . An understanding o f the mechanisms involved and how they are regulated provides insight in to the pathophysiology and potential therapeutic targets in AMD. 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLOGRAPHY Abrem ski K, Hoess R: Bacteriophage PI site-specific recombination. Purification and properties o f the Cre recombinase protein. 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Asset Metadata

Creator Wu, Jian (author)

Core Title Regulation of bone morphogenic protein 4 (BMP4) in age -related macular degeneration (AMD)

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Pathobiology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, pathology,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-341632

Unique identifier UC11340836

Identifier 3180361.pdf (filename),usctheses-c16-341632 (legacy record id)

Legacy Identifier 3180361.pdf

Dmrecord 341632

Document Type Dissertation

Rights Wu, Jian

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA