University of Southern California Dissertations and Theses

Regulation of inflammation and angiogenesis by Kaposi’s sarcoma-associated herpesvirus

(USC Thesis Other)

Regulation of inflammation and angiogenesis by Kaposi’s sarcoma-associated herpesvirus

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

Regulation of inflammation and angiogenesis by
Kaposi’s sarcoma-associated herpesvirus

by
Sanjna Mani

A Thesis Presented to the
FACULTY OF THE USC GRADUATE
SCHOOL UNIVERSITY OF SOUTHERN
CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
December 2016

Copyright 2016 Sanjna Mani
ACKNOWLEDGEMENTS
I would like to thank my family for their love, blessings and undying support, without
which I couldn’t have made it here. I would like to thank my Dad and Mom for being my
rock and my sister for reminding me of my capabilities every day.

Research is a team effort and involves time and effort from a number of people and I would
like to thank all of them here. Firstly, I would like to thank Dr. Shou-Jiang Gao for his
guidance and patience in mentoring me. Having had ups and downs during the time of my
research, he has constantly guided me with several ways to tackle tough situations. Having
had the opportunity to work in his lab he has given me the biggest gift of knowledge
working with a team of world class researchers and great minds. I would also like to thank
Dr. Axel Schonthal, without whom this thesis could not have taken shape. His
encouragement for me to continue on with a thesis despite trials and tribunals in the lab
and helping me learn that research, despite being challenging is something you never give
up on. I would like to thank Dr. Joseph Landolph, for taking the time to be a part of my
committee and being one of the people who saw me for my strengths. I can never forget
his words of encouragement and helping me keep my dreams alive.
It is indeed difficult to come into a new lab environment and having to learn every
technique from scratch. Therefore, my next bit of my gratitude is reserved for my lab
member and mentor Dr. Hong-Feng Yuan. Thank you for your patience and taking the time
out every single day to help me to learn something new. I will always strive to achieve your
work ethic.

2
To some of the most hardworking, ambitious individuals I have ever known, I would like
to thank all of my lab members Dr. Meilan He, Dr. Ying Zhu, Dr. Suzane Ramos, Dr.
Marion Guffaz, Dr. Fan Cheng, Brandon Tan, Tanvee Sawant, Ting-Ting Lee and Karthik
Vasan.

Finally, I would like to thank my friends Kiran Sriram, Vrishika Kulur, Ragini Ravi and
Pooja Sinha, for being a constant support and my family away from home.

3
TABLE OF CONTENTS

ACKNOWLEDGEMENTS 1
ABREVIATIONS 7
ABSTRACT 11
CHAPTER 1: INTRODUCTION 12
1.1 KAPOSI’S SARCOMA 12
1.2 KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS 13
1.3 KSHV GENOME AND ONCOGENIC FACTORS 13
1.4 KSHV AND HIV 15
1.5 ANGIOGENESIS- NORMAL PHYSIOLOGY 16
1.6 TUMOR ANGIOGENESIS 18
1.7 KSHV ANGIOGENESIS 19
CHAPTER 2 : CELLULAR FACTORS AFFECTING KSHV ANGIOGENESIS 21
2.1 DEGRADATION OF VE-CADHERIN 21
2.2 MATRIX METALLOPROTEASES 25
2.3 ANGIOPOIETINS 29
CHAPTER 3: HIV FACTORS INDUCING KSHV ANGIOGENESIS 35
3.1 SYNERGY OF HIV Nef WITH KSHV ONCOPROTEINS AND EFFECT
ON ANGIOGENESIS SIGNALING PATHWAYS 35
4
3.2 SYNERGY OF HIV Tat WITH KSHV ONCOPROTEINS AND
CCCCEFFECT ON ANGIOGENESIS SIGNALING PATHWAYS 43
3.2.1 HIV-1 Tat and KSHV ORF-K1 43
3.2.2 HIV-1 Tat and KSHV ORF-K2 48
CHAPTER 4 : KSHV GPCR's ROLES IN ANGIOGENESIS 52
4.1 kGPCR ACTIVATES SIGNALING IN ANGIOGENESIS 52
4.2 GRK2/CXCR2/AKT SIGNALING MEDIATED BY KSHV MIR-K3 55
DISCUSSION 61
BIBLIOGRAPHY 66

5
TABLE OF FIGURES

Figure 1 A map of the kshv genome including the lytic and latent genes 15
Figure 2 Types of primary vascular growth leading up to angiogenesis 18
Figure 3 KSHV induction of permeability in endothelial cells 23
Figure 4 (A) Immunofluorescence staining of VE-cadherin junctions that are
degraded from 0 hpi to 36 hpi 24
Figure 5 Western blots of VE-cadherin at various time points post-infection 24
Figure 6 KSHV viral genes required for disruption of adherens and increased
RRRRRRRR permeation 25
Figure 7 KSHV increases invasiveness of HUVEC cells 25
Figure 8 Western blots and quantifications of different subtypes of MMP1, 2
BBBBBBBB and 9 at different time points post-infection 30
Figure 9 Detection of mRNA expression of MMP-1, 2 and 9 by RT-qPCR in
BBBBBBBB mock and KSHV-infected HUVEC cells 31
Figure 10 Expression of Ang-2 following KSHV infection 33
Figure 11 KSHV- induced Ang-2 mediates angiogenesis of HUVEC cells 35
Figure 12 Synergistic effect of Nef and K1 affecting the PI3K/mTOR /AKT
pathway 40
Figure 13 Nef and K1 synergize in the CAM and nude mice models to induce
BBBBBBBB angiogenesis and tumorigenesis 41
Figure 14 miR-718 mediates K1 and Nef regulation of PTEN pathway 43
Figure 15 miR-718 mediates Nef- and K1-induced angiogenesis by targeting
PTEN to activate AKT/mTOR pathway 44
6
Figure 16 Cell proliferation, colony formation and microtubule formation assays
BBH K performed in both HUVEC and EA.hy926 cells to check for the effect
HJGLHG of Tat and K1 synergistic effect on angiogenesis 46
Figure 17 The effect of mir-891a-5p mediates Tat and Nef and inhibition of
IκBα 48
Figure 18 Inhibition of mir-891a-5p decreases Tat- and K1-induced angiogenesis
in vivo 49
Figure 19 Tat promotes angiogenesis through microtubule formation in vIL-6
MNJNUUH expressing cells 51
Figure 20 Tat regulates the PTEN/PI3K/AKT/GSK-3B pathway in vitro and in vivo 52
Figure 21 Luciferase reporter assays with overexpression of kGPCR and mGPCR 55
Figure 22 Staining of tumor cells to detect proliferation in KS lesions of
xenographic mice 57
Figure 23 miR-K3 transduced HUVEC cells and miR-K3 of KSHV in HUVEC cells
at different MOIs 58
Figure 24 Migration and invasion due to transduction of miR-K3 59
Figure 25 GRK2 down-regulation in presence of KSHV miR-K3 60
Figure 26 Invasion and migration assays for GRK2 and miR-K3 interaction 61
Figure 27 The signaling pathway of miR-K3, its effect on GRK2 and AKT
pathway 62

7

ABBREVIATIONS
1. AIDS: Acquired Immunodeficiency Syndrome
2. AKT: Protein Kinase B
3. Ang-2: Angiopoietin 2
4. AP: Activator Protein 1
5. BAC: Bacterial Artificial Chromosome
6. BALB/c: Albino Mouse Breed
7. BCBL: Body Cavity Based Lymphoma (B cell lymphoma cell line)
8. bFBF: Basic Fibroblast Growth Factor
9. CAM: chick embryo chorioallantoic membrane
10. CAT assay: Chloramphenicol acetyltransferase
11. CD3: Cluster of Differentiation 3
12. CDK: Cyclin Dependent Kinase
13. CsA: Cyclosporin
14. CXCR: Cysteine X cysteine chemokine receptor
15. DNA: Deoxy Ribonucleic Acid
16. EA.hy926: Endothelial Human derived cell line
17. EBV: Epstein-Barr Virus
18. EC: Endothelial Cells
19. ECM: Extra Cellular Matrix
8
20. EFG: Vector control
21. EGF: Epidermal Growth Factor
22. ELISA: Enzyme-Linked Immunosorbent Assay
23. Ets-1: E26 Transformation Factor-1
24. GPCR: G-Protein Couple Receptor
25. GRK: G Protein Couple Receptor Kinase
26. GSK-3 : Glycogen Synthase Kinase 3 Beta
27. H&E: Hematoxylin and eosin stain
28. HIV: Human Immunodeficiency Virus
29. hpi: hours post infection
30. HUVEC: Human Umbilical Vein Endothelial Cells
31. IL: Interleukin
32. IκBα: Inhibitor of Kappa Light Chain Gene Enhancer in B Cells, Alpha
33. KS: Kaposi’s Sarcoma
34. KSHV: Kaposi’s Sarcoma-Associated Herpesvirus
35. LANA: Latency-Associated Nuclear Antigen
36. MCD: Multicentric Cattleman’s disease
37. miRNA/miR: MicroRNA
38. MMP: Matrix Metalloproteases
39. MOI: Multiple of Infection
40. mRNA: Messenger Ribonucleic Acid
41. MSC: Mesenchymal Stem Cells
42. mTOR: mechanistic Target of Rapamycin
9
43. Nef: Negative Factor
44. NF- B: Nuclear factor kappa B
45. NFAT: Nuclear factor of activated T-cells
46. NIH3T3: Mouse Embryonic Fibroblast Cells
47. ORF: Open Reading Frame
48. P65: Transcription Factor (NF- B subunit)
49. PDGF: Platelet Derived Growth Factor
50. PDGF: Platelet Derived Growth Factor
51. PDI: Protein- disulfide isomerase
52. PEL: Primary Effusion Lymphoma (cell line)
53. PEL: Primary Effusion Lymphoma
54. PI3K: Phosphoinositide 3-kinase
55. PTEN: Phosphatase and tensin homolog
56. qPCR: Quantitative -Polymerase Chain Reaction
57. RNA: Ribonucleic Acid
58. RT-PCR: Reverse Transcription Polymerase Chain Reaction
59. RT: Reverse Transcription
60. RTA: Replication and Transcription Activator
61. SVEC: Endothelial Cells
62. TAF: Transcriptional Activation Factor
63. Tat: Trans activator of Transcription
64. TGF-b: Transforming Growth Factor Beta
65. TGN: Trans Golgi Network
10
66. Tie: EGF like domains
67. TNF: Tumor Necrosis Factor
68. UTR: Untranslated regions
69. UV: Ultraviolet irradiation
70. VE: Vascular Endothelial
71. VEGF: Vascular Endothelial Growth Factor
72. vFLIP: Viral Fas-associated inhibitory protein
73. vIL-6: Viral Interleukin 6

11
ABSTRACT

Kaposi's Sarcoma (KS), is a vascular tumor of endothelial cells caused by the infection of
herpesvirus Kaposi's Sarcoma Herpesvirus (KSHV). It is primarily found in HIV-infected
patients. KS is diagnosed by the KS lesions and bloody tumors on the skin. These tumors
have a complex vascular network that is primarily due to dysregulated angiogenesis.

Here in this thesis, we look at the effect of KSHV infection on angiogenesis and
inflammation. KSHV infection affects the adhesion molecules, VE-cadherin and induces
proteases like Matrix Metalloproteases (MMPs) and angiogenic factors like Angiopoietin-
2 (Ang-2) to degrade cell-cell junction and promote permeability and cell proliferation
leading to dysregulation of angiogenesis. We examine how HIV proteins, Tat and Nef,
interact with the KSHV-encoded proto-oncogenes, viral homologs of pro-inflammatory
cytokines and microRNAs (miRNA) to promote angiogenesis. By binding to 3' UTR
regions of certain genes, a miRNA synergizes with Tat and Nef proteins to induce the
release of several angiogenic factors and activate signaling pathways involved in cell
invasion, migration, angiogenesis, and tumorigenesis.

The KSHV-encoded G-Protein Coupled Receptor (kGPCR) activates the inflammatory
pathway to induce angiogenesis. KSHV miRNA again plays a role in regulating the
kGPCR downstream signaling that is essential in inducing angiogenesis. We also address
why and which KSHV miRNA are so heavily involved in regulating angiogenesis and its
complex signaling pathways.

12
CHAPTER 1
INTRODUCTION
1.1 KAPOSI’S SARCOMA

Kaposi’s sarcoma (KS) is a vascular tumor of endothelial spindle cells caused by infection
of Kaposi’s sarcoma-associated herpesvirus (KSHV). Histologically, KS tumor has some
hallmark features including: spindle cells, thin-walled vascular structures with slit-like
neovascular spaces, extravasated erythrocytes, infiltrating lymphocytes, and proliferating
spindle cells (Ellen G Feigal, 2000). KS often manifests as multifocal skin lesions, but
advanced disease is also involved with visceral tumors (Qian L G. W., 2008). Ther are four
clinical forms of KS:

Classic KS: Classic KS exhibits hemorrhagic sarcoma of the skin as well as sarcoma of
internal organs upon postmortem examination. It is described as an indolent tumor in older,
HIV-negative men of Mediterranean and Eastern European origins. The Classic form of
KS is mostly restricted to the skin (Dittmer DP, 2012).
Endemic KS: Also known as African KS, endemic to the sub-Saharan African region before
the emergence of HIV (Dittmer DP, 2012). It is an aggressive form of KS, indistinguishable
from others, and often seen in children (Dittmer DP, 2012).
Transplant KS: Also known as immunosuppressive KS, transplant KS is seen in patients
receiving immunosuppressive agents. The source of KSHV could be from the donor of
organ, the recipient of organ transplant or de novo exposure post transplantation when
immunosuppressive agents are given (Dittmer DP, 2012).
13
AIDS –KS: Also known as epidemic KS, AIDS-KS is found in HIV-positive patients and is
an AIDS-defining disease. AIDS-KS can be highly aggressive, and spreads quickly to
internal organs due to immune suppression (Dittmer DP, 2012). KS is the most common
cancer in AIDS patients.

1.2 KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8
(HHV8), is a human gamma herpesvirus. KSHV is associated with Kaposi’s sarcoma (KS),
primary effusion lymphoma (PEL), and a subset of Multicentric Castleman’s disease
(MCD). It has both a latent and a lytic phase of replication (Fukumoto, 2011).

The herpesvirus family has seven other members that infect humans, some of which are
associatd with other human diseases. KSHV is generally termed HHV8 among this family
of herpes viruses. KSHV is different from herpes type 1 and 2 of the herpes family, as it
causes a cancer called KS, PEL and MCD (Schulz TF).

1.3 KSHV GENOME AND ONCOGENIC FACTORS
KSHV is a large double-stranded DNA virus. The viral DNA is packaged in the viral
capsid, which is then surrounded the tegument, and finally is enclosed in a lipid envelope
derived in part from the cell membrane.
KSHV has a genome which is approximately 165,000 nucleic acid bases in length. It is a
Rhadinovirus and interestingly steals a lot of its genes from host cells. Complement-
14
binding protein, IL-6, BCL-2, Cyclin-D, a G protein-coupled receptor (GPCR), Interferon
regulatory factor, and Flice inhibitory protein (FLIP) are products of these genes. (Bu W,
2008).
After infection, the virus enters into lymphocytes via macropinosomes and it tends to
remain in a latent or quiet state. During latency, the virus genome, and host packaging
machinery work together to replicate the virus by using sequences within the terminal
repeats, as an origin of replication. The virus expresses the viral latency-associated nuclear
antigen (LANA) during this state. The virus uses the cellular replication, and LANA
connects the viral DNA to chromosomes during mitosis to ensure its segregation. LANA
can inhibit p53 and pRb tumor suppressor pathways. It also suppresses the expression of
viral lytic genes to promote viral latency.
The primary viral protein responsible for the switch from latent to lytic replication is the
open reading frame, ORF50, named Replication Transcriptional Activator (RTA).When
RTA is induced due to some signaling pathways, it, in turn, activates the synthesis of a
cascade of viral lytic genes leading to full replication of the virus.
15

Figure 1 A Map of the KSHV genome (Coscoy, 2007).
Various signals, such as inflammation, growth factors, and other gene inducers may
provoke the virus to enter into lytic replication. During this phase of replication, the viral
episome replicates in the form of linear DNA. These are packaged into virus particles and
expelled from the cells. During de novo infection, the virus enters the cells by endocytosis
and the viral capsid travels to the nucleus, where the viral genome is released for viral
replication. Thousands of virus particles can be made from a host cell, which results in
death of the host cell (Bu W, 2008).

1.4 KSHV AND HIV
KSHV infection has an oncogenic potential and culminates in KS in certain settings being
manifested along with HIV infection and AIDS. KSHV latent infection can cause
16
transformation of cells into malignant cells, whereas lytic infection, which expresses
KSHV pro-angiogenic genes, lyses the host cells, therefore, cannot transform cells. There
are a number of hypothesis that postulate the mechanisms of cellular transformation: The
Paracrine hypothesis is based on the presence of most latently-infected with sporadic
spontaneous lytic replication of a few cells that express early lytic genes including pro-
angiogenic genes (vGPCR, K1 and ORF45), which induce KS growth factors (VEGF, IL-
6, PDGF) to which synergize with viral factors, leading to deregulated angiogenesis
involving potential of neighboring cells (Cavallin LE, 2014). The Abortive hypothesis, on
the other hand, suggests that cells that are expressing early lytic genes do not undergo full
lytic replication. The expressed lytic genes also can cause autocrine and paracrine effects.
(Cavallin LE, 2014).The two hypotheses are supported by literature that shows the
paracrine nature of vGPCR-induced tumors of latent KSHV genes via paracrine
mechanisms.
These hypotheses can explain the high incidence of KS in HIV/AIDS populations.
Angiogenic HIV Transactivation of Transcription (Tat) and Negative Factor (Nef) proteins
in AIDS patients, can induce KSHV lytic reactivation and promote cell migration, invasion,
and angiogenesis in KS tumors (Cavallin LE, 2014).

1.5 ANGIOGENESIS- NORMAL PHYSIOLOGY

Angiogenesis is the growth of blood vessels from the existing vasculature. It occurs in both
healthy form for vascular generation and in disease. The adult vasculature is derived from
a network of blood vessels that is initially created in the embryo by vasculogenesis, which
17
is de novo from endothelial cell precursors termed angioblasts. Once vasculogenesis is
complete in the embryo, it is remodeled by the sprouting and branching of new vessels
from pre-existing ones in the process of angiogenesis in adults. This is also necessary for
capillary formation, serving for the diffusion exchange of nutrients and oxygen to tissues.
There are two basic types of angiogenesis- Sprouting angiogenesis and Intussusceptive
angiogenesis (Adair TH, 2010).
Sprouting angiogenesis is one type of angiogenesis in which sprouts of tubules composed
of endothelial cells, grow as a response toward an angiogenic stimulus. The mechanism of
this type of angiogenesis is enzymatically degrading the basement membrane, endothelial
cell (EC) proliferation, migration of ECs, tubulogenesis, vessel fusion, and pericyte
stabilization. This happens in response to hypoxic environments which can secrete
Vascular Endothelial Growth Factor (VEGF), a proangiogenic growth factor (Adair TH,
2010).
Intussusceptive angiogenesis is involved with the formation of blood vessels from pre-
existing vessels by a splitting process in which elements of interstitial tissue, invade pre-
existing vessels and forming vascular pillars that can expand to form a new blood vessel.
(Adair TH, et al., 2010). This type of angiogenesis is faster and highly efficient as it only
requires reorganizing of existing endothelial cells and not on immediate endothelial
proliferation or migration (Adair TH, 2010).

18

Figure 2 Types of primary vascular growth leading up to angiogenesis (Adair TH, 2010).

Angiogenesis is controlled by the balance of growth and inhibitory factors in healthy state.
When this balance is disturbed, it results in either too much or too little angiogenesis.

The “on” switches are angiogenic growth factors that stimulate blood vessel formation
while “off” switches are chemicals that inhibit blood vessel-formation, termed
angiogenesis inhibitors. Study of angiogenesis is important as it is involved with
cardiovascular diseases and cancer. Angiogenesis is the hallmark of many types of cancers
involving proliferation, migration and invasion (Karamysheva, 2008).

1.6 TUMOR ANGIOGENESIS

In the study of malignant tumors, angiogenesis is a key factor, more so in KS as it is
tumor with high amounts of vasculature. Hence, it is important to address how the tumors
spread and how inhibition of angiogenesis can be used therapeutically in cancer.

19
Some of the findings of Dr. Judah Folkman are that solid tumors up to 1-2mm in diameter
are able to obtain nutrients and oxygen for their growth and are avascular. He also found
that there is switching to angiogenesis by mobilizing surrounding blood vessels to begin
sprouting new capillaries that eventually help in the expansion of tumor mass. The
switching was proposed to happen due to the production of Tumor Angiogenesis Factors
(TAF).
He also stated that it is possible to block tumor angiogenesis by preventing TAF production
or by direct targeting of vascular endothelial cells. The other possibility is, that tumor
vessels and endothelial cells of tumor vessels, being different from endothelial cells of
mature vessels can be used as a therapy. This approach, however, can only prevent the
growth of the tumor mass but not eradicate tumor cells (Folkman J, 1971) (Folkman, 1974).
Weidner et al. who found that the prognosis for cancers was worse, the greater the degree
of angiogenesis (Weidner N, 1991) (Kerbal, 2000).

1.7 KSHV ANGIOGENESIS

It has been found that, KSHV can induce angiogenesis in an autocrine and paracrine action
through viral and cellular pro-angiogenic and inflammatory factors. KSHV infection of
cells leads to the expression of viral oncoproteins with transforming capabilities that
regulate signaling pathways which in turn can activate angiogenesis (Purushothaman,
2016). Accumulating evidence suggests that KSHV infection can induce tumorigenesis
through the complex interplay of several viral, cellular angiogenic, and inflammatory
markers. KSHV infection of endothelial cells that were cultured show KS infection, can
20
induce angiogenic phenotypes through higher secretion of pro-angiogenic factors,
including VEGF, IL-6, IL-8, MMPs, and Ang-2. In addition, KSHV-infected endothelial
cells grown on Matrigel have been shown to form tubules without any external growth
factors. It is also known that KSHV's latent and lytic proteins can synergize and modulate
cellular mechanisms to induce tumorigenesis. (Purushothaman, 2016).

21

CHAPTER 2
CELLULAR FACTORS AFFECTING KSHV ANGIOGENESIS
2.1 DEGRADATION OF VE-CADHERIN
Endothelial cells are found as a single-cell layer in underlying vascular and lymphatic
conditions. They serve as a barrier for the exchange of molecules between circulatory
system and tissues, and are thus selectively permeable. There exist several plasma
membrane junctions such as gap junctions, adherens junctions, tight junctions, etc. These
adherens junction consist of Vascular Endothelial-Cadherins (VE-Cadherin) and its
associated partners. VE-Cadherin is an adhesion molecule found between endothelial cells.
It is highly important in maintaining cell-to-cell contact. The mechanisms that regulate VE-
Cadherin adhesion and production are essential for vascular permeability and VEGFR
activity. VE-Cadherins play a crucial role in mediating endothelial permeability mediating
intravasation, extravasation and cancer-induced angiogenesis (Westweber, 2007).

A number of viruses that are pathogenic in nature have evolved to disrupt adherens
junctions. This compromises the integrity of endothelial cells that depend on the adherens.
As the endothelial barriers are compromised, it is easy for the virus to enter the basal
membrane and traffic within the vascular networks. Many studies have shown that KSHV
can induce an angiogenic and invasive phenotype in human primary endothelial cells, but
the mechanism has not been examined until recently.
22
Recent studies show that KSHV regulates of Human Umbilical Vein Endothelial Cells
(HUVEC) cells using a transwell permeability assay. HUVEC cells infected with KSHV
with an 80-90% infection rate 48 hours post infection (hpi). The permeability to serum
albumin, a common protein in blood, measured at various time points showed increased
permeability as early as 6hpi and increased fourfold by 24hpi. Inflammatory infiltrations
such as leukocytes also needed to be studied if they were permeable to the endothelial cell
layer. For this, U937 cells, a blood-derived monocyte cell line, showed similar results with
a 2.3 fold increase in permeability at 12hpi. Together, it was concluded that the KSHV
infection in endothelial cells has increased permeability of HUVEC cells to soluble
proteins and blood derived cells.

Figure 3 KSHV's permeability in endothelial cells.
(A) Serum albumin permeability at various time points post infection (B) U297 Cells have
increased permeability 2.3 fold as compared to mock infected cells (Qian L G. W., 2008).
Now that we know that, KSHV infection increases permeability, experiments were then
conducted to determine if it also affects membrane structure adherens junctions. Upon
23
immunofluorescence staining, it was evident that VE-Cadherins are tightly distributed
structures between normal adjacent cells. Upon KSHV infection, at 6 through 24hpi, there
was observed less intensity of the VE-Cadherins (Figure 4). As, expected, this shows that
the disruption in the VE-Cadherin structure is the reason behind the increased permeability
in KSHV infected endothelial cells. Upon examination of the protein levels of VE-cadherin
using western blots, it was observed that the level of VE-cadherin decreased by 60% at
4hpi. This was sustained at later time points such as 48hpi (Figure 5).

Figure 4 Immunofluorescence staining of VE-Cadherin junctions that are degraded from 0
hpi to 36 hpi (Qian L G. W., 2008)

Figure 5 Western blots of VE-Cadherin at various time points (Qian L G. W., 2008).
The degradation of VE-Cadherin, along with its reduced levels at early stages of KSHV
infection, suggests that the virus is involved in the degradation of adherens junctions during
24
the entry stage of infection. While normal endothelial cells that are confluent maintain
contact inhibition even in the presence of pro-angiogenic factors such as VEGF, upon
KSHV infection, there were degradation in adherens junctions in the confluent
monolayers.
To test the hypothesis that KSHV viral genes are required for degradation and permeation,
UV-KSHV which is KSHV inactivated by UV was used since UV radiation destroys the
viral gene function. Cells infected by UV-KSHV remained as the mock-infected control,
and did not show downregulation of VE-Cadherin protein.

Figure 6 KSHV Viral Genes are not required for disruption and permeation.
(A) and (B) Showing adherens junctions by VE-Cadherin staining in mock and KSHV Infected
cells (C) VE-Cadherin staining of endothelial monolayer of cells showing degradation at 6 hpi (D)
VE-Cadherin staining of UV-KSHV Infected endothelial monolayer of cells showing degradation
at 6 hpi. (Qian L G. W., 2008)
While the KSHV-induced degradation of VE-Cadherin and the degradation of adherens
junctions occurred at the early stage of infection, this experiment shows that it did not
25
require pro-inflammatory and pro-angiogenic cytokines or viral genes (Qian L G. W.,
2008).
2.2 MATRIX METALLOPROTEASES
Matrix Metalloproteases (MMPs) are a family of metalloproteinase enzymes that play a
major role in tissue remodeling and degradation of the extracellular matrix, such as
collagens, elastins, gelatin, etc. MMPs are excreted by connective tissue and pro-
inflammatory cells including endothelial cells (Verma, 2007).
The role of MMPs in angiogenesis is that they process angiogenic factors such as VEGF
and bFGF secreted by either inflammatory or tumor cells. The mature angiogenic factors
bind to their receptors of endothelial cells, which in turn activate them to secrete more
MMPs that degrade the ECM, and more matrix-bound VEGF and bFGF are secreted. This
continues as a constant loop. The MMPs not only degrade the ECM, they also remove the
site of adhesion between cells and cell-matrix receptors.
A number of MMPs, including MMP-1, 2, 3, 9, 19 have been detected in KS tumor cells,
and have a role in pathogenesis of KS tumors. The involvement of KSHV in expression
and secretion of MMPs has been unclear (Lockhart AC, 2003).
To see the effects of cell invasion on the Extra cellular matrix (ECM), HUVEC were
infected with recombinant KSHV BAC36 and the ECM invasion assay was performed.
Normal endothelial cells did not have any detectable invasiveness of the Matrigel, which
is supposed to emulate the basement membrane. However, in the presence of chemo
attractants, some normal HUVEC cells showed invasiveness. KSHV infection, on the other
26
hand, showed a 2.6 fold increase in number of cells traveling across the matrix barrier. The
course of time taken for the invasiveness was studied in mock and KSHV infected cells
across a 0-48 hpi. The invasiveness began as early as 4 hpi as compared to mock with
showed none. These results indicate an enhancement of HUVEC extracellular matrix
invasion by KSHV infection.

Figure 7 KSHV increasing invasiveness in HUVEC cells.
(A and B) Mock- or KSHV-infected HUVEC were assayed for their invasiveness in a Matrigel
layer at 24 hpi. (C) HUVEC invasiveness assayed at different time points post-KSHV infection
(Qian L X. J., 2007).
Cells secrete MMPs that digest the extracellular matrix and promote cell invasion during
KSHV infection, the time points after KSHV infection at which these MMPs are secreted
were studied.
27
Normal HUVEC cells, mock infected cells and KSHV cells were studied at 1, 6, 24 and
36hpi. In normal HUVEC cells, low levels of MMP-1 were secreted, which was slightly
elevated in mock cells and at 24hpi, the KSHV cells start to show a multi fold increase in
MMP -1 and MMP subtypes: pro-MMP, inter-MMP and active-MMP. Similar results were
observed in experiments for MMP-2 and its subtypes. Mock-infected KSHV cells showed
levels of MMP-2 from 1 hpi and increased progressively. KSHV infected cells showed a
3-fold increased secretion from 1hpi. One peculiar observation was that as both mock and
KSHV infected cells, pro-MMP-2 increased up until 36hpi, but KSHV infected cells show
less MMP after 24hpi. However, the inter and active subtype of MMP-2 showed higher
secretion levels after 24hpi and 36hpi in KSHV infected cells.
On a study of MMP-9 secretion MMP-9 and its subtypes, pro, inter and active MMP-9
were increased in mock-infected cultures at 24 and 36hpi. While the inter-MMP-9 levels
remained similar in KSHV infected cells, at the 24 and 36hpi time points, the pro and active
levels of MMP-9 increased tremendously as compared to the mock infected cells. Thus
KSHV infection increased the secretion of MMPs-1,2 and 9.
28

Figure 8 Western blots and Quantifications of different subtypes of MMP1, 2 and 9 at
different time points.
(A)(B)(C) Western blot analysis of Pro, Active and Inactive forms of MMP 1, 2 and 9 (D)(E)(F)
Intensity of increase of secretion in MMP 1, 2 and 9 at various time points (Qian L X. J., 2007).
When MMP-1, 2 and 9 transcripts were studied, using RT-qPCR, it was again seen that
while the expression of MMP-2 transcript slightly increased, both MMP-1 and 9 showed
both higher secretion and expression levels due to KSHV infection.
29

Figure 9 Detection of mRNA expression of MMP-1, 2 and 9 by RT-qPCR in mock and KSHV
infected HUVEC cells (Qian L X. J., 2007).
It was concluded that KSHV infection enhanced the invasion of cells in the Matrigel assay.
This is due to the increased secretion and expression of MMPs 1, 2, 9 in HUVEC cells.
Due to this, there is invasiveness of the cells both paracrinally and autocrinally. This
demonstrates a role of MMPs in cell invasion during de-novo viral infection of KSHV
(Qian L X. J., 2007).
2.3 ANGOIPOIETINS
Angiopoietins are endothelial-specific growth factors that, along with VEGF, promotes
vascular development and angiogenesis. VEGF enhances angiogenesis by promoting
endothelial cell proliferation, and Angiopoietins (Ang) are required for vascular blood
vessel remodeling, sprouting and maturation. Ang-1 stabilizes blood vessels and promotes
adhesive interaction between endothelial cells. It is an agonist of the Tie-2 receptor. Ang-
2 works conversely and induces detachment of mural cells to destabilize the existing blood
vessels, which is necessary for new blood vessel formation. Ang-2 is an antagonist of the
Tie-2 receptor.
30
Ang-2 expression is upregulated by cytokines, growth factors such as VEGF, angiotensin,
TNF-  etc., Ang-2 has only limited expression in normal tissue but strongly expressed
during remodeling of vasculature including tumor-driven angiogenesis. Ets-1 is an
important transcriptional factor that regulates expression of Ang-2. Several of the KSHV
proteins vIL-6, VGPCR and several of the inflammatory cytokines VEGF, bFBF, IL-6, IL-
8, TNF-  etc., directly induce angiogenesis.
The mechanism and effect of KSHV infection in Ang-1 and Ang-2 was investigated by
examining the expression of Ang-2 in HUVEC cells after infection. The KSHV infected
cells showed an increased Ang-2 secretion at 12hpi and peak at 52 hpi; This was in contrast
to the control of mock-infected cells, which showed no expression. The Ang-2 mRNA
expression was also studied and KSHV infected cells show increased expression and mock
cells showed no mRNA expression.
31

Figure 10 Expression of Ang -2 with KSHV infection with latent and lytic genes.
(A) Western blots showing secretion of Ang-2 after KSHV infection at different time points. (C)
mRNA Expression levels of mock and KSHV infected cells for Ang-2, latent and lytic genes (Ye
F, 2007).
The results were also confirmed in clinical samples of KS lesions that were stained for
detection of Ang-2. 23 of 27 samples were found to be Ang-2 positive for strong intratumor
vessels. The majority of the samples also showed positive results for LANA, confirming
infection. With the absence of LANA, Ang-2 was also negative in the lesions which
32
correlate with the mRNA experiments (Ye F, 2007). Thus upregulation of Ang-2 Could be
mediated by KSHV lytic and latent genes: vFLIP and LANA.
In order to determine the role of Ang-2 in angiogenesis, Matrigels were introduced
subcutaneously into mice for easy removal of pellets for observing angiogenesis. The two
groups were Sup- KSHV and Sup-Mock. Histological examinations of Sup-KSHV
revealed both micro and macro-vessels that stained positive for VE-Cadherin, indicating
they are endothelial cells. Sup-Mock infected cells showed very few vessels. The Matrigel
specimens were observed for hemoglobin content, which was found to be 30% higher in
Sup-KSHV than Sup-Mock. This shows that KSHV in HUVEC can induce angiogenic
effect by a paracrine mechanism. Further analysis of Matrigels with anti-Ang-2 antibody
in Sup-KSHV, reversed these effects. Also, Sup-Mock infected cells was supplemented
with human Ang-2 that showed blood vessel formation. Sup-KSHV, Sup-KSHV plus
antibody, Sup-Mock and Sup-Mock plus supplemented with Ang-2 Matrigels were
compared for hemoglobin levels, Sup-KSHV plus antibody, Sup-Mock with Ang-2 and
antibody showed low levels compared to Sup-Mock
This indicated that KSHV-induced Ang-2 was necessary for the induction of angiogenesis.
33

Figure 11 KSHV- induced Ang-2 angiogenesis in HUVEC cells.
(A) Matrigel pellets from in-vivo angiogenesis assays showing micro and macrovascular vessels
(B) Histological staining of Matrigel pellets using supplement Ang-2 and anti-Ang-2 antibody (C)
VE- Cadherin staining of pellets to confirm angiogenesis in EC cells (D) Hemoglobin content assay
of all illustrations (A), (B) and (C) (Ye F, 2007).

Since Ang-2 mRNA was upregulated in the presence of KSHV, KSHV transcriptional
activation of the Ang-2 promoter was examined. KSHV activated the Ang-2 promoter
reporter pEBS1. Subsequently, it was found that KSHV activated Ang-2 promoter at 12
and 52 hpi which was consistent the Ang-2 mRNA expression. Since KSHV is activates
the Ang-2 promoter, a promoter deletion analysis at these time points were conducted.
34
These results indicate that both AP-1 and Ets1 are involved in KSHV induction of Ang-2
and subsequently, angiogenesis. This was confirmed by using inhibitors of the upstream
pathways of Ets1 and AP-1. Thus KSHV activates AP-1 and Ets1 to induce Ang-2
expression which is mediated by both AP-1 and Ets 1(Ye F, 2007).

35

CHAPTER 3
HIV FACTORS INDUCING KSHV ANGIOGENESIS
We know that the interaction between HIV and KSHV has promoted and played an major
role in the development of KS. The pathogenesis of AIDS-KS is a topic that needs to be
looked into, as it is largely unknown and is one of the most aggressive forms of KS.
KSHV Infection alone is not sufficient for development of KS, and one of the co-factors
in question is HIV-1. According to the literature, HIV-1 promotes the initiation and
progression of KS through several mechanisms- secretion of proteins and inflammatory
cytokines to induce immunosuppression. This includes HIV-1 Tat and HIV-1 Nef.
3.1 SYNERGY OF HIV Nef WITH KSHV ONCOPROTEINS AND
EFFECTS ON ANGIOGENESIS SIGNALING PATHWAYS
HIV-1 negative factor (Nef) is a 27 kDa protein produced in early HIV infection and
translated from many spliced viral mRNAs. It can interact with a number of cellular factors
to induce changes in cellular signaling and gene expression to promote viral replication
and immune suppression. Nef is released from HIV-infected cells and is constantly present
in the blood plasma of HIV infected patients. Nef is also known to be found in pulmonary
arterial endothelial cells of AIDS patients. Thus, it might be interesting to determine if Nef
might be induced by KSHV infection and regulate angiogenesis.
36
miRNAs are small amino acid sequences that regulate gene expression by targeting their
UTRs. They are noncoding in nature but act post-transcriptionally to regulate the
expression of genes. Generally, they bind to their complementary sequences in the 3' UTR
region of the target gene. which can lead to its degradation or downregulation of protein
translation.
The KSHV genome encodes for 12 precursor miRNA, in the latency associated region
which then matures to 25 miRNAs. These are named as KSHV-miR-K1-12. They have
high expression levels in latency and highly functional in the viral life cycle.
The KSHV miRNA regulate host genes to control angiogenesis, apoptosis, cell cycle,
cytokine production, immune evasion, metastasis of KS tumors which means they can
mediate migratory and invasive behavior of tumor cells. Recent studies have been
speculating a greater involvement of these KSHV miRNAs in latency, immune evasion,
and pathogenesis of KSHV. However, their roles in KSHV and HIV induced angiogenesis
has only recently been studied.
K1 is an oncogene of KSHV that is known to induce angiogenesis. The ORF-K1 protein
transforms HUVEC, involved in inhibition apoptosis, promotion of cell proliferation,
angiogenesis, and tumorigenesis. K1 is also known to activate PI3K/AKT/mTOR pathway,
NF- B and AP-1 signaling pathway that leads to VEGF, bFGF, TNF-a, IL-6, and IL-8
secretion. ORF-K1 also induces expression of MMPs in endothelial cells, promoting
metastasis. The synergistic effect of Nef with K1 has already shown to activate the
PI3K/AKT pathway. To confirm this, western blots showed soluble Nef protein or K1
alone increased the levels of P13K, AKT and mTOR in HUVEC cells and EA.hy926 cells.
37
K1 and Nef, synergized with each other and further increased the levels of the
phosphorylated proteins. Nef and K1 levels were studied with respect to PTEN, an inhibitor
of P13K and found that it was present in decreased levels, which is consistent with the
results. The effect of Nef and K1 on the proliferation of cells through AKT activation was
studied in the same two cells types. This was seen to increase the cell proliferation rate
using cell counting kit assays when both K1 and Nef synergized. The plate colony
formation assay was also used to determine cell proliferation in EA.hy926 cells and noted
an increase when the two oncoproteins were expressed together.

38
Figure 12 Synergistic effect of Nef and K1 affecting the PI3K/mTOR/AKT pathway.
(A)Western blot analysis of PTEN, P13K, AKT and mTOR expression with K1 HUVEC cells in
the presence of Nef (B) Cell proliferation using HUVEC and EA.hy926 cells for the synergistic
effect of K1 and Nef (Soluble and ectopic) (C) Plate colony assay with similar conditions as (B).
(D)(E) Matrigel assay of tube formation in HUVEC cells and EA.hy926 cells respectively treated
as described in (B) (Xue M, 2014).
To observe the angiogenic effects of the two proteins, a tube formation assay was done in
HUVEC cells as well as EA.hy926 cells. This was done for both ectopic and soluble Nef.
39
Matrigel plug assays in Chrorio allantoic Membrane (CAM) and nude mice models were
also performed in vitro. K1 and Nef protein synergized with each other in both HUVEC
and EA.hy926 cells to promote angiogenesis.

Figure 13 Nef and K1 synergize in the CAM and nude mice models to induce angiogenesis
and tumorigenesis.
(A) Nef promoted, K1 induced HUVECS showing angiogenesis (B) Quantification of results from
(A). (C) Angiogenesis also observed in EA.hy926 cells. (D) H&E staining and ICH staining of
CAM tumor-induced be EA.hy926
40
The CAM tumors with Nef and K1 expression, subjected to H&E staining showed high
vascularization in EA.hy926 cells. Western blots of K1 or Nef alone showed high levels of
PI3K, AKT, and mTOR and higher when Nef and K1 Synergize. There was also decreased
levels of PTEN in both cases. Thus, even in in-vivo models, K1 and Nef synergizes
promote angiogenesis through PI3K/AKT/mTOR pathway.

Phosphate Tensin (PTEN) is involved in angiogenesis. In order to study it, a microarray
screening was done to find the miRNAs regulated by Nef and K1. What was seen were
cellular miRNAs that were upregulated in HUVEC cells by both Nef and K1, individually
and together which indicates that it could regulate the expression of PTEN. Bioinformatics
examination identified 9 miRNAs that have targeting sites at PTEN 3' UTR. Out of the
nine, only the cellular hsa-miR-718 significantly inhibited PTEN 3' UTR luciferase
reporter activity. As a confirmation, HUVEC and EA.hy926 cells had increased miR-718
expression in the presence of both K1 and Nef. Another luciferase reporter assay confirmed
the inhibition of PTEN 3'UTR by miR-718.
41

Figure 14 miR-718 mediating K1 and Nef regulation of PTEN pathway.
(A) miRNA array analysis (B) Luciferase assay of PTEN 3'UTR binding against miRNAs detected
in (A). (C) Study the of effect of miR-718 on Tat and Nef (D)Luciferase reporter assay for activity
of PTEN in the presence of MIr-718,Tat and Nef (E) (F) & (G) Western blots showing expression
level changes in PTEN protein in the presence of miR-718 (Xue M, 2014).

To confirm the effect of miR-718 on PTEN, western blots were run, these showed
inhibition of PTEN in HUVECS, and mutant miRNA-718 negative sequences did not show
an effect.
However, there is an involvement of miR-718 in Nef and K1 induced angiogenesis was the
question to be answered soon by tube formation assays. HUVEC with K1and soluble Nef
along with an inhibitor of PTEN miR-718, showed the blocked tube formation thus
inhibiting angiogenesis function.
In-vivo, CAM models were also used to examine the same and the results were consistent
with in vitro. Most importantly, western blots run showed suppressed miR-718 with its
inhibitor, in HUVECs transduced with both Nef and K1 increased PTEN expression. this
is consistent with AKT and mTOR levels dropping.
42

Figure 15 miR-718 mediated Nef- and K1-induced angiogenesis by targeting PTEN to
activate AKT/mTOR pathway.
(A) Matrigel assay performed for HUVECS transduced with K1 and incubated with Nef for tube
formation (B) Quantification of results of (A). (C) Blood vessels expressed (D) Quantification of
(C), counting the number of vessels expressed (E)Western blot of PTEN, AKT, mTOR in the CAM
tumor tissues, negative control denoted by 1 (F) Inhibition of mi-718 leads to reduced effect on
angiogenesis induced by K1 and Nef (G) Matrigel plugs analyzed for hemoglobin level in the 6
tumor groups (Xue M, 2014).

Together, these data indicated that, by targeting PTEN, miR-718 mediates Nef- and K1
induced angiogenesis and tumorigenesis by activating AKT/mTOR signaling (Xue M,
2014).

43
3.2 SYNERGESIS OF HIV Tat WITH KSHV ONCOPROTEINS AND
EFFECT ON ANGIOGENESIS SIGNALING PATHWAYS
3.2.1 HIV-1 Tat and KSHV ORF K1
HIV-1 Tat is a protein that activates gene expression of HIV by binding to HIV-1 terminal
repeats. Tat is abundant in spindle cells of AIDS-KS lesions and known to promote the
growth of endothelial cells in AIDS patients. Studies have shown that Tat activates KSHV
lytic replication and increases the effects of oncogenic proteins like vGPCR and vIL-6 for
tumorigenesis. Tat promotes tumorigenesis by synergizing with TNF-A, bFGF, and other
cytokines.
To understand the synergistic relationship between HIV-1 Tat and ORF-K1 in angiogenesis
caused by KSHV, cell proliferation, colony formation and tube formation assays were
performed. The proliferation rates of Tat and K1 were studied separately and together in
both HUVEC and EA.hy926 cells and found that the two synergized to increase cell
proliferation rate. Colony formation assays performed showed, K1 alone increased
colonies in EA.hy926 cells and synergized with Tat. Microtubule formation showed K1 or
TAT Increasing tube formation about two fold whereas together, the tube formation
increased six fold. This trend was seen in both types of cells.
44

Figure 16 Cell proliferation, Colony formation and Microtubule formation assays performed
in both HUVEC and EA.hy926 cells to check for the effect of Tat and K1 synergistic effect on
angiogenesis.
(A) Cell Proliferation graph in HUVEC and EA.hy926 cells induced K1 with soluble Tat (B) Plate
colony formation assays for Tat or/and K1 treated as in (A). (C) Microtubule formation assay of
K1 and Tat induced in HUVEC and EA.hy926 cells. (Yao S, 2015).
In order to understand the mechanism of Tat and K1 synergysis, key molecules associated
with angiogenesis were studied. Both Tat and K1 seemed to degrade IκBα which negatively
regulates VEGF. This suggests that Tat and K1 reduced the expression of VEGF by
activating NF- B pathway.
One or several of the miRNAs could have an effect in regulating IκBα. A microarray
analysis was done to screen the miRNA in Mock, Tat, K1 and Tat and K1 expressing cells.
The miRNAs in the cells expressing K1 and Tat, showed greater expression level than the
mock group. Among the upregulated miRNAs, eight putative targeting sites in IκBα's 3'
UTR region. A luciferase assay showed miR-323a-3p, miR-421, miR-550b-3p, miR-891a-
5p, miR-1284 and miR-2113 inhibited IκBα's 3' reporter. Transfection of miR-891a-5p
inhibited the endogenous IκBα expression. Expression of miR-891a-5p inhibited the
45
activity of IκBα 3′UTR reporter but not that of the control reporter. It was confirmed that
Tat, K1 and both increased miR-891a-5p in HUVEC and EA.hy926.
KSHV infection also increased the level of miR-891a-5p in HUVEC. A study of miR-
891a-5p showed inhibiting activity of the IκBα 3′UTR reporter and the endogenous IκBα
expression in HUVEC. As a result, miR-891a-5p enhanced the activity of an NF-κB
reporter. IκBα expression level was upregulated by a miR-891a inhibitor. Bioinformatics
analysis was conducted to identify one miR-891a-5p binding site in the IκBα 3′UTR region.
Mutation of this site reduced the miR-891a-5p inhibitory effect on endogenous IκBα
expression. It also inhibited the IκBα 3′UTR reporter activity as expected in HUVEC cells.
These data suggest that miR-891a-5p targets IκBα since neither Tat nor K1 inhibited the
expression of IκBα mRNA as seen earlier.

46

Figure 17 The effect of miR-891a-5p on Tat and Nef and inhibition of IκBα.
(A) Thermal analysis of eight miRNAs to see expression levels in EA.hy926 cells (B) Inhibition
of IκBα 3'UTR reporter activity by miRNAs (C) The influence of miRNAs on expression of
endogenous IκBα (D) Luciferase activity of IκBα 3′UTR reporter (E) miR-891a-5p fold change
under effect of Tat, K1 or both in HUVEC and EA.hy926 (F) (G) Tat, Nef and both showing higher
expression level of miR-891a -5p (H) Western blots to confirm miR-819a-5p inhibiting activity of
IκBα reporter (I) Bioinformatics to Identify binding site of miR-819a-5p on 3'UTR of IκBα (J)
Mutation of the site to see loss in inhibition of IκBα (K) Activity of IκBα 3′UTR under mutated
miR-819a-5p (Yao S, 2015).
Finally, the effect of K1 and Tat synergistic effect and miR-819A-5p expression was
studied in vivo. In CAM mouse models, the HUVEC cells with Tat and Nef were
transfected with miR-891A-5p. The in vitro and in vivo results were similar with the
inhibitory function of miR-891a-5p resulted in suppressed angiogenic effect induced by
K1 and Tat.
47
In the tumors isolated from Matrigel plug assay, ELISA results showed decreased p65
binding to NF-κB due to the presence of sponge miR-891a-5p. Western blotting results
showed that the miR-891a-5p inhibited degradation of IκBα that is caused by K1 and Tat
in EA.hy926 cells. Thus, the above data suggests that miR-891a-5p mediates K1 and Tat
induced angiogenesis by targeting IκBα to activate the NF-kB pathway (Yao S, 2015).

Figure 18 Inhibition of miR-891a-5p decreasing Tat- and K1-induced angiogenesis in vivo.
(A) Inhibitory effect of miR-891a-5p inhibitor on angiogenesis in CAM model (B) Quantification
48
of the results in (A). (C) Inhibitory effect of miR-891a-5p sponge on angiogenesis in nude mice
(D) The hemoglobin level of the Matrigel plug treated as in (C) (Yao S, 2015).

3.2.2 HIV-1 Tat and KSHV ORF K2
On analyzing the KSHV genome obtained from DNA libraries, the open reading frame
(ORF) K2 was identified as a viral homolog of hIL-6 was identified; its gene product was
named vIL-6 (Tosato, 2011). vIL-6 promotes cell proliferation, cell survival, KSHV
immune evasion and other signaling pathways. Additionally, vIL-6 can induce the
secretion of cellular IL-6 and VEGF to promote cell proliferation, angiogenesis, and
tumorigenesis.
NIH3T3 cells expressing KSHV vIL-6 or Tat induce angiogenesis. When vIL-6 and Tat
are co-expressed, they further enhanced angiogenesis. This effect was also observed in
human endothelial cells. Western blots showed a high level of Tat with increasing MOI.
The two stable clones of NIH3T3, 4E3 and E6 not effected by Tat expression. An MTT
assay showed high proliferation of vIL6-expressing cells in the presence of Tat. A soft agar
assay showed colonies of Tat transduced 4E3 cells more than mock cells. Simlar enhanced
effects were observed with vIL-6 and Tat expressing cells. A tube formation assay also
confirmed that Tat transduced 4E3 cells was significantly more than mock cells. VEGF
levels were examined, all to confirm Tat promotes proliferation and angiogenesis in vIL-6
expressing cells.
49

Figure 19 Tat promoting angiogenesis through microtubule formation in vIL-6 expressing
cells.
(A) Soft agar assay of 4E3 cells and control (B) Plate Colony formation assay E6 and control (C)
Matrigel tubule formation assay 6hpi and quantified results. (D) Western blot showing VEGF
expression in 4E3 cells (Zhou F, 2013).
In-vivo, studies were performed which was consistent with the above results in CAM
Models where the presence of soluble Tat and vIL-6 increased tumorigenesis and
angiogenesis.
To further understand the mechanism of Tat promoting vIL-6 expression and its
involvement in angiogenesis and tumorigenesis, the PI3K/PTEN/AKT/GSK-3  was
observed. It was seen in vitro that Tat did not affect the expression of vIL-6 without the
expression of KSHV RTA. But Tat or vIL-6 alone increased a little but together increased
more of the activation of PI3K and AKT in NIH3T3 cells.
50
In In-Vivo CAM model, 4E3 cells also saw the reduced expression of PTEN in Tat or vIL-
6 expressing cells with an increase in phosphorylated AKT.

Figure 20 Both in-vitro and in-vivo analysis of PTEN/PI3K/AKT/GSK-3  pathway by Tat.
(A) Tat was not synergizing with vIL-6 without the presence of RTA (B) Phosphorylation levels
of PTEN, PI3K, AKT and GSK-3  IN 4E3 cells of Tat or vIL-6 (C) Phosphorylation of AKT and
GSK-3  in vivo of 4E3 cells in CAM model (Zhou F, 2013).
Tat enhanced vIL-6 activation of PI3K. When inhibited, it led to the inhibition of the
downstream phosphorylated products of AKT. When AKT was inhibited, it reduced the
activation of GSK-3 . Overexpression of PTEN inhibited Tat enhanced angiogenesis and
also decreased the phosphorylated forms of AKT induced by vIL-6. As seen previously,
PI3K /AKT and GSK-3 mediate the Tat expressed angiogenesis. GSK-3 might mediate
Tat-induced enhancement of angiogenesis. This was seen as GSK-3 inhibited Tat,
decreasing both angiogenic index and tumorigenesis. Together these results suggest that
51
Tat synergizes with vIL-6 to induce angiogenesis and tumorigenesis through the activation
of PI3K/AKT whereas PTEN act as inhibitors as seen in-vitro (Zhou F, 2013).
52
CHAPTER 4
KSHV GPCR ROLES IN ANGIOGENESIS
4.1 kGPCR OVER mGPCR ACTS AS ACTIVATION SIGNALING IN
ANGIOGENESIS
Human gamma herpesviruses include Epstein-Barr virus and KSHV. Murine Gamma
HerpesVirus 68 (MHV68) is genetically related to both EBV and KSHV. Mice with
endothelial cells expressing kGPCR, developed angiogenic lesions similar to KS. The
GPCR of all herpesvirus are functionally different. KSHV kGPCR is an active signaling
molecule and independent of ligand binding. However, the MHV68 mGPCR requires a
ligand for signaling. On comparing the two, kGPCR activates signaling led to gene
expression of NF- B, NFAT and AP-1 transcription factors. kGPCR expression led to an
elevated expression of NFAT dependent reporter whereas mGPCR had no detectable effect
in luciferase reporter assay. Similarly, kGPCR, but not mGPCR, upregulated gene
expression of NF-κB and AP-1 transcription factors. Western blotting showed that mGPCR
and kGPCR were expressed at similar levels under similar conditions in 293 cells. Murine
NIH3T3 cell line with high expression of MHV68 showed higher activation of NF- B NF-
B and NFAT from the kGPCR, but in mGPCR, that was not the case although high
expression levels were expected. Thus the results show that kGPCR, but not mGPCR,
activates NF-κB, NFAT, and AP-1 transcription factors, signaling events for
tumorigenesis.
53

Figure 21 Luciferase reporter assays with overexpression of kGPCR and mGPCR.
(A) To detect NFAT (B)To Detect NF-kB (C) To detect AP-1 (D) Western Blotting of kGPCR and
mGPCR expression in 293 cells (E)(F) NF-kB and NFAT activated by kGPCR over mGPCR in
yH68 expressing cell line NIH3T3 (Zhang J Z. L., 2015).
It was studied if infection by MHV68.kGPCR can induce tumors in BALB/c mice for in
vivo confirmation. The mice were infected with either wild-type MHV68 as control or
MHV68.kGPCR as study subject; 15 each. Three weeks post-infection, mice were injected
with cyclosporine (CsA) to inhibit T cell response. Of the mice infected with
MHV68.kGPCR and treated with CsA, five mice displayed malignant conditions of organs
including lungs, liver and subcutaneous compartment.
54
Over a period of six months, one of the mice infected with MHV68.kGPCR and treated
with CsA died. Though there was found to be no skin lesion, there were vascularized liver
nodules. Four more mice treated with CsA showed evidence of subcutaneous tumors 5
months into infection; no skin lesion was observed, but the tumors were found to be highly
vascularized consistent with the theory that kGPCR induces angiogenesis in endothelial
cell types. H&E staining of the tumor cells showed spindle-shaped structure, and infiltrated
with high level of erythrocytes along with a large numbers of immune cells in the tumor
region. kGPCR scattered through small sections of tumor lesions shown in IHC staining,
which is consistent with the in-vivo results of kGPCR involvement in KS tumors. CD31,
an endothelial cell marker was examined with antibodies against it for the activity of
endothelial cells. High levels of CD31 were expressed, and they appeared to be undergoing
proliferation as the cells were connecting with each other. A marker for proliferating cells
Ki67 stained positive in most of the tumor cells.

55

Figure 22 Staining of tumor cells to detect proliferation from KS lesions of xenographic mice.
(A) Spindle shaped structure and erythrocytes with H&E staining (B) kGPCR expression levels (C)
CD31 marker showing interconnection between endothelial cells (D) ki67 marker for proliferation
of cells (Zhang J Z. L., 2015).

Hence, we can conclude that these studies show kGPCR promotes angiogenic lesions in
infected mice. Although MHV68 encodes its own GPCR homologue, it is not sufficient to
induce KS-like lesion or tumors in infected mice. As seen above, kGPCR, but not mGPCR,
induced tumor formation in a xenograft nude mouse model. Thus the two may have similar
cell surface expression but behave differently in intracellular localization, kGPCR
promoting tumorigenesis and angiogenic lesions (Zhang J Z. L., 2015).

4.2 GRK2/CXCR2/AKT SIGNALING MEDIATED BY MIR-K3
CXCR2 is a gene from the CXC chemokine receptor family which binds IL-8 and has
shown to mediate its angiogenic effects in the intestinal microvascular endothelial cells.
(Cannon, 2006). It has been reported that cellular miRNAs facilitate migration and
invasiveness in endothelial cells. The viral miR-K3 regulates the viral life cycle and also
expressed in high levels in KS lesions. This suggests a potential role in KS pathogenesis.
To study the involvement of miR-K3, HUVEC cells were transduced with lentivirus
expressing miR-K3 at different MOI. At MOI 2, the transduced cells as well as, miR-K3
56
expressing cells in KSHV (BAC16) infected HUVEC showed the same level of expression.
But at MOI 8, miR-K3 expressing cells showed massive increase of miR-K3 expression.

Figure 23 miR-K3 transduced HUVEC cells and miR-K3 of KSHV in HUVEC cells at
different MOI (Hu M, 2015).
miR-K3 was transduced for transwell migration and matrigel invasion assays showed
higher levels of migration and invasion as compared to the vector control. Besides these
assays, studies for cell proliferation, cell cycle and plate colony for changes due to miR-
K3 were futile. In order to detect several cytokines that are related to cell migration and
invasion, qPCR was performed. There were upregulated transcripts of MMPs1, 9 and 10,
and inflmmatory cytokines IL-6 and IL-8 in the presence of miR-K3.
57

Figure 24 Migration and Invasion due to transduction of miR-K3.
(E-G) Transwell Migration and invasion assay with transduced miR-K3 (I) Upregulation of
cytokines and MMPs (Hu M, 2015).

Since miRNAs usually bind to their target genes to induce the degradation of the transcripts
or inhibit the translation of protein, miR-K3 was studied to predict its binding site and as
predicted, it was found to be in the 3'UTR of GPCR Kinase 2 (GRK2). By qPCR and
Western blotting, it showed that the protein of GRK2 and mRNA of GRK2 were both
down-regulated in miR-K3-expressing HUVEC cells. In KSHV infected HUVEC, mRNA
and protein levels of GRK2 were decreased as expected. Compared to normal skin tissue,
KS lesions were found to be less GRK2- positive cells in IHC staining.

58

Figure 25 GRK2 down-regulation in the presence of KSHV miR-K3.
(A)(B)(C) GRK2 being downregulated in the presence of miR-K3 (D)(E) mRNA and protein
expression of GRK2 is downregulated in KSHV HUVEC (F) Immunohistochemistry of GRK2
positive cells (Hu M, 2015).
To establish the role of GRK2 in miR-K3 induced migration and invasion of HUVEC,
HUVEC cells expressing miR-K3 were transduced with lentivirus GRK2. The over
expression of GRK2 drastically reduced cell migration and invasion due to miR-K3. Thus
KSHV infection and miR-K3 expressing HUVEC can increase cell migration and invasion
but reduce the overexpression of GRK2.

59

Figure 26 Invasion and Migration assays for GRK2 and miR-K3 interaction.
(A)(B)(C) The cell migration and invasion detection in HUVEC expressing miR-K3;
overexpressing lentivirus-GRK2 (D) Western blot confirming the suppression of lentivirus-GRK2
by miR-K3 (Hu M, 2015).

Additionally, overexpression of KSHV miR-K3 in HUVEC reduced the expression of
GRK2 while a knockdown of GRK2 in HUVEC cells by itself was sufficient to increase
cell invasion and migration. Thus, the miR-K3 is involved in targeting GRK2 for migration
and invasion. Overexpression of GRK2 downregulated CXCR2. Higher levels of CXCR2
was observed in miR-K3 expressing cells as GRK2 was downregulated. This confirms that
a CRCX2 mediated miR-K3 and targeted GRK2 function to increase cell migration and
invasion. The AKT signaling pathway could also promote the same. As GRK2 was
overexpressed in miR-K3 expressing HUVEC, there was a dramatic decrease in AKT
activation and reduced CXCR2 levels. Cell migration and invasion assays were done with
miR-K3 expressing HUVEC and knocking down AKT which showed no signs of migration
or invasion. Most importantly, there was a blocked induction of MMP1, 9 AND 10, IL6,
IL-8 during this knockdown in miR-K3 induced HUVEC. Thus, together
GRK2/CXCR2/AKT signaling is mediated by miR-K3 and promotes migration and
60
invasion of cells of endothelial origin and would be interesting to delineate its role in
angiogenesis and tumorigenesis (Hu M, 2015).

Figure 27 The action of miR-K3 and its effect on GRK2 and AKT Pathway (Hu M, 2015)

61
DISCUSSION

The VE-Cadherin and other adhesion molecules are important for cell-cell contact. VE-
Cadherin is one of the major components of adherens junctions implicated in endothelial
cell monolayer of vasculature. A number of viruses are known to disrupt these adherens
and compromises endothelial cells integrity giving way to permeability, extravasation,
migration and angiogenesis in virus induced cancers. Studies conducted in HUVEC cells
showed increase in permeability to soluble proteins and blood derived cells by KSHV. The
infection of KSHV increases permeability and there is increase in disruption in VE-
Cadherin which was found to happen in the early stages of infection. The viral genes were
found to not participation in this VE-cadherin disruption. KSHV infection of these
endothelial cells alter their permeability by disruption of KSHV infected endothelial
confluent monolayers. Signaling pathways regulate the components of these adherens
factors, function and permeability. Many pro-inflammatory and pro-angiogenic factors like
TNF-, IL-6, IL-8 and VEGF mediate viral permeability through the endothelial cells. The
mechanism of disruption is still unclear since it was found to be in early phase of infection
in this study. Since there is no pro-inflammatory or proangiogenic cytokines or viral gene
involvement in the early stages of infection, the hypothesis is that the mechanism maybe
be occurring in later stages of infection.

The MMP family of genes produce proteolytic enzymes that degrade the ECM and leads
to the release of angiogenic factors VEGF and bFGF. KSHV infection increases the
invasiveness of HUVEC cells by KSHV infection. There was also a study of the MMP-12,
9 subtypes whose levels ion secretion increased during KSHV infection. It is still not
62
understood, the mechanism, conditions and effects of MMPs that are secreted during
KSHV infection.

Ang-2 is a protein derived from the Ang-2 gene and are known to be closely associated
with VEGF. We know that VEGF promotes cell proliferation for angiogenesis. Ang-1
promotes this cell growth. However, Ang-2 is seen to work conversely and destabilize new
blood vessel formation. Ang-2 was expressed highly and could be involved in destabilizing
vasculature during early KSHV infection. KSHV induced Ang-2 is necessary for
angiogenesis by destabilizing blood vessels. KSHV activates AP-1 and Ets1 to induce Ang-
2 expression where both AP-1 and Ets1 together act in late stages of infection. These can
further upregulate VEGFs, MMPs and cytokines to accelerate tumor angiogenesis. It would
be interesting to examine the effects of Ang-1 in angiogenesis during KSHV infection and
how the two antagonize each other upon infection.

We have seen the cellular factors’ involvement in KSHV infected endothelial cells by
increasing permeability, disruption cell-cell-junctions and increase in MMP and Ang-2 to
destabilize the vascular network and subsequently up regulate many more KSHV proteins
such as vIL-6, vGPCR and several of the inflammatory cytokines VEGF, bFBF, IL-6, IL-
8, TNF that can induce angiogenesis.

Interactions between KSHV and HIV’s proteins and pro-inflammatory factors, have been
studied extensively in KS. Here we study how these proteins of HIV synergize with KSHV
proteins were examined.
63
K1 is an oncogene of KSHV that is known to induce angiogenesis. The ORF-K1 protein
transforms HUVEC, involved in inhibition apoptosis, promotion of cell proliferation,
angiogenesis, and tumorigenesis. It also activates the PI3K/AKT/mTOR pathway, NF- B
and AP-1 signaling pathway that leads to VEGF, bFGF, TNF-a, IL-6 and IL-8 secretion.
ORF-K1 also induces expression of MMPs in endothelial cells, promoting metastasis.

The synergistic effect of HIV Nef and K1 has not been well studied. The results showed
that there is a good synergistic effect between the two proteins in increasing cell
proliferation and tubule formation both in vivo and in-vitro. To understand the mechanism
of the synergy PTEN and its effect on the PI3K/AKT/mTOR pathway were examined.
miRNA 718 was one of the 8 miRNAs that were targeting PTEN. Inhibition of the PTEN
miRNA showed blocked tubule formation and angiogenesis function, consistent with in
vivo models.

K1 can also interact with HIV Tat, and has shown to activate lytic replication and increases
the effects of oncogenic proteins like vGPCR and vIL-6 for tumorigenesis. A synergistic
effect between Tat and K1 was found and showed to have increased cell proliferation and
angiogenic microtubules formation was higher when they synergize. Some studies
suggested that K1and Tat degraded I B  which negatively regulates VEGF. miR-891a-5p
inhibited I B  expression. The synergistic effect of Tat and K1 also showed increase in
miR-891a-5p. miR-891a-5p was further increased by both Tat and K1, and inhibited I B 
to activate the NF B pathway and induces angiogenesis.
Tat also synergizes with K2 whose product is homologous to vIL-6. This vIL-6 can induce
64
the secretion of cellular IL-6 and VEGF to promote cell proliferation, angiogenesis and
tumorigenesis. Synergy between Tat and vIL-6 expressing cells was seen in the form of
increased cell proliferation, and VEGF levels were higher. The synergism also showed
increase in tubule formation indicating the potential for angiogenesis. Tat and vIL-6 was
also seen to have an effect on the PI3K/AKT/GSK-3  pathway. They increased AKT by
PTEN inhibition that has the potential to induce angiogenesis whereas GSK-3  acts as an
antagonistic the PI3K/AKT pathway.

Another important pro-angiogenic factor is the protein vGPCR. kGPCR was found to
activate gene expression of NF B, NFAT and AP-1 Transcription Factors more than the
comparator mGPCR (Murine gamma Herpes Virus GPCR). This show that although the
receptors can be the same, they are functionally very different. kGPCR is independent of
ligand binding and mGPCR dependent of ligand binding to activate the same pathway and
induce angiogenic signaling. The study of infecting mice with HV68.kGPCR led to an
observation of KS skin lesions and the presence of highly vascularized tumors. However,
we still need to know whether kGPCR expressed in HUVEC cells can cause KS lesions
and angiogenesis on its own and needs to be studied in other endothelial cell types.
The viral miR-K3 regulates the viral life cycle and also expressed in high levels in KS
lesions. This suggests a potential role in KS pathogenesis. miR-K3 was studied and found
that migration and invasion assays showed higher levels of migration and invasion. To
detect the cytokines involved in cell migration and invasion, there was also up regulation
of the DH gene that codes for MMP and also IL-6 in the presence of miR-K3. This miR-
K3 was found to bind to the 3’UTR of GPCRK. Thus, the expression of GRK2 was
65
decreased when cells were infected with HIV. Overexpression of GRK2 downregulated
CXCR2. Higher levels of CXCR2 was observed in miR-K3 expressing cells as GRK2 was
downregulated. This confirms that a CRCX2 mediated miR-K3 and targeted GRK2 with
respect to cell migration and invasion. The knockdown of AKT in the miR-K3 expressing
cells showed miR-K3 regulates the GPCR/CXCR2/AKT pathway.
The theme here is that there are several viral factors that are able to degrade, permeate,
migrate and invade host cells. This causes the activation of several inflammatory and pro-
angiogenic signaling pathways that produce more pro-angiogenic and factors in circulation
and allow angiogenesis to manifest. The common factor among all the above studies is that
KSHV miRNAs are somehow able to regulate angiogenesis through the synergy of HIV
and KSHV proteins. The 3' UTR region to which they bind and regulate certain genes for
angiogenesis to occur has been identified for some miRNA.
Although all precursor and mature miRNA K1-12 have been identified, studies are still
underway in finding which miRNA is responsible for cell migration, invasion and tubule
formation leading to angiogenesis. One could develop a targeted viral miRNA inhibitors
and identify other diagnostically relevant methods of targeting these miRNAs, as a way to
block angiogenesis and preventing the formation of blood vessels, cut off blood flow,
nutrient and oxygen supply to the cells, thereby eliminating KS tumors.

66

BIBLIOGRAPHY
1. Adair TH, M. J. (2010). http://www.ncbi.nlm.nih.gov/books/NBK53242.
2. Bu W, P. D. (2008). Identification of Direct Transcriptional Targets of the
Kaposi's Sarcoma-Associated Herpesvirus Rta Lytic Switch Protein by
Conditional Nuclear Localization. Journa of Virology, 82 (21).
3. Cannon, M. (2006). KSHV G Protein-coupled Receptor Inhibits Lytic Gene
Transcription in Primary-effusion Lymphoma Cells via p21-mediated Inhibition
of Cdk2. Blood, 107(1), 277-284.
4. Cavallin LE, G.-C. P. (2014). Molecular and Cellular Mechanism of KSHV
Oncogenesis of Kaposi's Sarcoma Associated with HIV/AIDS. Condit RC.
5. Coscoy, L. (2007). Immune Evasion by Kaposi's Sarcoma-Associated
Herpesvirus. Nature Reviews Immunology, 391-401.
6. Dittmer DP, R. K. (2012). Treatment of Kaposi Sarcoma-Associated Herpesvirus-
Associated Cancers. Frontiers in Microbiology, 3:141.
7. Ellen G Feigal, A. M. (2000). AIDS-Related Cancers and Their Treatment.
8. Folkman J, M. E. (1971). Isolation of Tumor Factor Responsible for
Angiogenesis. J Exp Med, 133, 275-288.
9. Folkman, J. (1974). Tumor Angiogenesis Factor. Cancer Research, 34.
10. Fukumoto, H. (2011). Pathology of Kaposi's Sarcoma-Associated Herpesvirus
Infection. Frontiers in Microbiology, 2.
11. Hu M, W. C. (2015). A KSHV microRNA Directly Targets G Protein-Coupled
Receptor Kinase 2 to Promote the Migration and Invasion of endothelial Cells by
Inducing CXCR2 and Activating AKT Signaling. PLoS Pathogens, 11(9).
67
12. Karamysheva, A. (2008). Mechanisms of Angiogenesis. Biochemistry Moscow,
751-762.
13. Kerbal, R. S. (2000). Tumor Angiogenesis: Past, Present and Near Future
Carcinogenesis. Cancer Biology, 21 (3): 505-515.
14. Lockhart AC, e. a. (2003). Reduction of Wound Angiogensis in patients Treated
with BMS-275291, a Broad Spectrum Matrix Metalloproteinase Inhibitor.
Clinical Cancer Research, 9:00-00.
15. Pravin Kumar Purushothaman, T. U. (2016). KSHV-Mediated Angiogenesis in
Tumor Progression.
16. Qian L, G. W. (2008). Kaposi's Sarcoma-Associated Herpesvirus Disrupts
Adherens Junctions and Increases Endothetlial permeability by Inducing
Degradation of VE-Cadherin. Journal of Virology, 82(23), 11902-11912.
17. Qian L, X. J. (2007). Kaposi's Sarcoma-Associated Herpesvirus Infection
Promotes Invasion of Primary Human Umbilical Vein Endothelial Cells by
Inducing Matrix Metalloproteinases. Journal of Virology, 81(13), 7001-7010.
18. Schulz TF, M. P. (n.d.). Kaposi's Sarcoma-Associated Herpesvirus (Human
Herpesvirus 8). Humor Tumor Viruses, 87-134.
19. Tosato, S. S. (2011). Viral Interleukin-6: Role in Kaposi's Sarcoma-Associated
Herpesvirus-Associated Malignancies. Journal of Interferon & Cytokine
Research, 31(11), 791-801.
20. Verma, R. P. (2007). Matrix Metalloproteinases (MMPs): Chemical-Biological
Functions and (Q)SARs. ChemInform.
68
21. Weidner N, S. J. (1991). Tumor Angiogenesis and Metastasis - Correlation in
Invasive Breast Carcinoma. N Engl J Med, 324, 1-8.
22. Westweber, D. (2007). VE-Cadherin: The Major Endothelial Adhesion Molecule
Controlling Cellular Junctions and Blood Vessel Formation. Arteriosclerosis,
Thrombosis and Vascular Biology, 223-32.
23. Xue M, Y. S. (2014). HIV-1 Nf and KSHV Oncogene K1 Synergistically Promote
Angiogenesis by Inducing cellular MiR-718 to regulate the PTEN/AKT/mTOR
signaling pathway. Nucleic Acids Research, 42(15), 9862-9879.
24. Yao S, H. M. (2015). MiRNA-891a-5p Mediates HIV-1 tat and KSHV Orf-K1
Synergistic Induction of Angiogenesis by Activating NF-κB signaling. Nucleic
Acids Research, 43(19), 9362-9378.
25. Ye F, B. D. (2007). Kaposi's Sarcoma-Associated Herpesvirus Promotes
Angiogenesis by Inducing Angioprotein-2 Expression via AP-1 and Ets1. Journal
of Virology, 81(8), 3980-3991.
26. Zhang J, Z. L. (2015). Recombinant Murine Gamma Herpesvirus 68 Carrying
KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice. PLoS
Pathogens, 11(6).
27. Zhou F, X. M. (2013). HIV-1 tat promotes Kaposi's Sarcoma-Associated
Herpesvirus (KSHV vIL-6-Induced Angiogenesis and Tumorigenesis by
regulating PI3k/PTEN/AKT/GSK-3β signaling pathway. PLoS ONE, 8(1).
e53145.

Abstract (if available)

Abstract Kaposi's Sarcoma (KS), is a vascular tumor of endothelial cells caused by the infection of herpesvirus Kaposi's Sarcoma Herpesvirus (KSHV). It is primarily found in HIV-infected patients. KS is diagnosed by the KS lesions and bloody tumors on the skin. These tumors have a complex vascular network that is primarily due to dysregulated angiogenesis. ❧ Here in this thesis, we look at the effect of KSHV infection on angiogenesis and inflammation. KSHV infection affects the adhesion molecules, VE-cadherin and induces proteases like Matrix Metalloproteases (MMPs) and angiogenic factors like Angiopoietin-2 (Ang-2) to degrade cell-cell junction and promote permeability and cell proliferation leading to dysregulation of angiogenesis. We examine how HIV proteins, Tat and Nef, interact with the KSHV-encoded proto-oncogenes, viral homologs of pro-inflammatory cytokines and microRNAs (miRNA) to promote angiogenesis. By binding to 3' UTR regions of certain genes, a miRNA synergizes with Tat and Nef proteins to induce the release of several angiogenic factors and activate signaling pathways involved in cell invasion, migration, angiogenesis, and tumorigenesis. ❧ The KSHV-encoded G-Protein Coupled Receptor (kGPCR) activates the inflammatory pathway to induce angiogenesis. KSHV miRNA again plays a role in regulating the kGPCR downstream signaling that is essential in inducing angiogenesis. We also address why and which KSHV miRNA are so heavily involved in regulating angiogenesis and its complex signaling pathways.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

An essential role of argininosuccinate synthase 1 in Kaposi’s sarcoma-associated herpesvirus-induced cellular transformation

PDF

Kaposi's sarcoma associated herpes-virus induces cellular proxi expression to modulate host gene expression that benefits viral infection and oncogenesis

PDF

Regulation of acute KSHV infection by SIRT1

PDF

Viral and cellular N⁶-methyladenosine and N⁶,2'-O-dimethyladenosine epitranscriptomes in the Kaposi’s sarcoma‐associated herpesvirus life cycle

PDF

FoxO1 suppresses Kaposi's sarcoma-associated herpesvirus lytic replication and controls viral latency

PDF

Epigenomic analyses of Kaposi's sarcoma-associated herpesvirus

PDF

Single cell gene expression analysis of KSHV infection in three-dimensional oral epithelial tissue cultures

PDF

Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression

PDF

Targeting vessel maturation: an anti-angiogenesis based cancer therapy

PDF

Molecular targets for treatment of glioblastoma multiforme

PDF

Kaposi's sarcoma‐associated herpesvirus‐Bcl-2 mediated regulation of NM23-H2 is required for KSHV lytic replication

PDF

Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer

PDF

Discovery of mature microRNA sequences within the protein- coding regions of global HIV-1 genomes: Predictions of novel mechanisms for viral infection and pathogenicity

PDF

MGMT in emergence of melanoma resistance to temozolomide

PDF

Musashi-2 promotes c-MYC expression through IRES-dependent translation and self-renewal ability in hepatocellular carcinoma

PDF

Exosomal inhibitory activity of clinically approved tertiary amines for the treatment of Kaposi’s sarcoma tumorigenesis

PDF

Study of protein deamidation in innate immune signaling

PDF

Lymphatic cell environment promotes sustained KSHV lytic replication and viral maintenance

PDF

Improving adeno-associated viral vector for hematopoietic stem cells gene therapy

PDF

Placental growth factor mediated transcriptional and post-transcriptional regulation of hemeoxygenase-1

Asset Metadata

Creator Mani, Sanjna (author)

Core Title Regulation of inflammation and angiogenesis by Kaposi’s sarcoma-associated herpesvirus

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2016-12

Publication Date 11/18/2016

Defense Date 10/24/2016

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

Tag AIDS,angiogenesis,HIV,inflammation,KSHV,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Gao, Shou-Jiang (committee chair), Landolph, Joseph (committee member), Schonthal, Axel (committee member)

Creator Email sanjnama@usc.edu,sanjnamani@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC11256187

Unique identifier UC11256187

Identifier etd-ManiSanjna-4937.pdf (filename)

Legacy Identifier etd-ManiSanjna-4937

Dmrecord 323305

Document Type Thesis

Format theses (aat)

Rights Mani, Sanjna

Internet Media Type application/pdf

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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu