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Altered interaction of human endothelial cells to the glycosylated laminin

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Altered interaction of human endothelial cells to the glycosylated laminin

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Content ALTERED INTERACTION OF HUMAN ENDOTHELIAL CELLS
TO THE GLYCOSYLATED LAMININ
By
Bill Subin
A Thesis Presented to the
Faculty of the Graduate School
University of Southern California
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Biochemistry and Molecular Biology)
December 1994
Copyright 1994 Bill Subin
UNIVERSITY O F S O U T H E R N CA LIFO R N IA
THE G R A D U A T E S C H O O L
U N IV E R S IT Y P A R K
L O S A N G E L E S . C A L IF O R N IA 0 0 0 0 7
This thesis, written by
B i l l S u b i n
under the direction of h.i.$,..Thesis Com m ittee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
Master of Science
Dtan
Date... J T .. 9a.. 1994
THK5IS COMMITTEE
Chair mam
DEDICATION
To my wife and my parents, as well as my great teacher Dr. Marcel
Nimni.
1 1
ACKNOW LEDGMENTS
I am grateful to my research advisors, Dr. Marcel E. Nimni and Dr. Zoltan
Tokes for their inspiration and guidance throughout my graduate studies. I
would also like to thank them for providing me access to an excellent
working environment.
I sincerely appreciate and thank Drs. Vijay Kalra, Nino Sorgente, Tai-Lan
Tuan, and David Cheung for serving as the consultants for my thesis and
guidance committees.
During the years o f 1991-1994 at USC I benefited greatly from interacting
with many colleagues and friends. In particular I like to acknowledge Dr.
Anthony Sank for his kind and attentive guidance. I also want to thank
Natasha Perelman, Judy Zhou, Delia Ertl, Nadereh Asemanfar and my
classmate Bo Han for their help in guiding me through the many laboratory
techniques, and our secretary Melinda Soto for administrative assistance.
Special thanks go to my beloved family, my parents Moo-Lin Su and Wen
Ma, my wife Helen HH Zou, for their love, support and patience. Without
their encouragements this thesis would not be possible.
TABLE OF CONTENTS
Dedication
Acknowledgments
Abstract
Introduction
1. Overview
2. Nonenzymatic glycosylation
3. The effect of glycosylated laminin on endothelial cellular
adhesion and proliferation
4. Expression of laminin receptors— integrin p on endothelial cells
5. Regulation of growth factor on adhesion
6. Research strategy
Materials and Methods
1. Purification of laminin
2. Removal of contamination
3. Nonenzymatic glycosylation of laminin
4. Isolation of Human Umbilical Vein Endothelial Cells from
Diabetic and Nondiabetic Patients
5. Coating ELISA plate
6. EC adhesion assay
7. The effect of TGF p on EC adhesion
8. EC proliferation assay
9. Detecting human Integrin pi on the Surfaces of EC
Results
1. Quantitation of glycosylation by ^H-glucose
2. Effect of glycosylated laminin on EC adhesion
3. Effect of TGF p on EC adhesion 26
4. Effect o f glycosylated laminin on EC proliferation 3 1
5. Expression of integrin P 1 42
Discussion 47
Conclusion and Future Study 51
References 52
V
List of Figures and Tables
Figures
1. The chemical reactions of nonenzymatic glycosylation 5
2. Removing of unknown contamination by chromatography I 5
3. Tritiated glucose incorporation in glycosylation of laminin I 7
4. Phase-contrast micrography of normal HU VEC 22
5 Fluorescent micrography of HUVEC stained with factor VIII 22
6. Fluorescent micrography of HUVEC indicated Di-LDL uptake 23
7. Reduction of adhesion of HUVEC on glycosylated laminin 28
8. Reduced adhesion of nondiabetic and diabetic HUVEC on
glycosylated laminin 30
9. Effect of TGF P on the adhesion of HUVEC 33
10. Micrography of nondiabetic HUVEC in adhesive assay
after TGF p treatment 35
11. Micrography of diabetic HUVEC in adhesive assay
after TGF p treatment 37
12. Dose response of TGF p to EC adhesion 39
13. Dose response o f TGF p to EC adhesion on glycosylated
laminin 41
14. Effect o f glycosylated laminin on HUVEC proliferation 44
15. Fluorescent patterns of the different groups of HUVEC
conjugated to integrin p i. 46
vi
ABSTRACT
Although both endothelial cells (EC) and glycosylation of the basement
membrane (BM) have received much attention recently, little is known of
how human diabetic endothelial cell respond to glycosylated laminin, one
of the major components of BM. Purified human laminin was glycosylated in
vitro with D-[6-^H] glucose incorporated. Human umbilical vein endothelial
cells (HUVEC), from diabetic and nondiabetic subjects, were evaluated for
their ability to adhere and proliferate on a surface precoated by either
glycosylated or nonglycosylated laminin; the effect of TGF P and expression
of integrin p I o f these cells were also examined. The results of these studies
show that EC respond to the glycosylated laminin with a reduction in
adherence, proliferation, and a reduction in the expression of integrin pi on
their cellular surfaces. Although glycosylation inhibited adherence of normal
and diabetic cells, diabetic cells were affected to a greater extent. In view of
these Findings, the delayed healing of diabetics may be the result of
glycosylated proteins in the wound which slow the proliferation and
adherence of endothelial cells, and these defects could be amenable to further
clinical therapeutic measures.
vii
INTRODUCTION
1. Overview
Microangiopathy is one of the major causes o f morbidity in patients
with diabetes mellitus. The pathogenesis o f diabetic microangiopathy
remains enigmatic, despite the acquisition o f a great deal of knowledge
concerning functional, biochemical, and morphological changes in the
vasculature of diabetic patients. These characteristic changes include
increased regional vascular flow, increased microvascular permeability,
capillary basement membrane thickening, and the presence of
microaneurysms and neovascularization.
Since endothelial cells are responsible for blood vessel growth as well
as for bringing nutrients and eliminating metabolic waste from tissues, it is
reasonable to assume that EC play a critical role in the development of
microangiopathy. Although in vitro studies have shown abnormalities of
fibroblasts and arterial smooth muscle cells from diabetic animals and
humans, little is known about the specific biochemical abnormalities in the
vascular endothelial cells, and no studies are avaibable on possible
abnormalities of capillary endothelial cells (EC) from diabetic humans
(Kwok 1989).
It is well established that nonenzymatic glycosylation is a major
mechanism by which hyperglycemia modifies the structure and function of
various macromolecules, especially macromolecules with relatively long half-
lives. Among them, basement membrane components are of major interest
because their alteration may be a critical event contributing to diabetic
microangiopathy (Shaklai 1984). Two major basement membrane (BM)
glycoproteins, laminin and type IV collagen have been shown to be
structurally altered in the presence of high glucose, and these alterations
which result in defective interations may have profound influences on the
molecular architecture of the BM (Tsilibary 1988 and Charonis 1990). In the
vasculature BM components interact not only with each other but also with
the EC that line every vessel, and these interations are crucial to various EC
properties and ultimately to the structural and functional integrity of the
vascular wall (Haitoglou 1992).
Since nonenzymatic glycosylation of components of the extracellular
matrix (ECM) could alter their structure and function, and in turn could alter
the functions of EC, we have studied cell adhesion and proliferation of
HUVEC from diabetic and nondiabetic subjects in response to
nonenzymaticaly glycosylated laminin.
2. Nonenzymatic Glycosylation
Nonenzymatic glycosylation (NG) involves the attachment of free
sugars to certain amino acid residues of proteins. It occurs under
physiological conditions and has been considered as a tracer of time (aging).
However, NG is also closely related to diabetes mellitus because
hyperglycemia promotes increased NG of circulating proteins such as
hemoglobin and albumin, thus allowing glycohemoglobin or glycoalbumin to
assess diabetic control; hyperglycemia is also responsible for the
glycosylation o f tissue proteins (collagen, fibronectin and laminin), very likely
contributing to the pathogenicity of the chronic complications o f diabetes
(Cohen 1986).
2
Proteins with long-half-lives are more susceptible to severe structural
and functional alterations as primary targets of nonenzymatic glycosylation.
The nonenzymatic glycosylation of hemoglobin is considered as a model
reaction relevant to the pathogenesis of certain complications o f chronic
diabetes. Many of the proteins studied are found in tissues typically involved
with diabetic complications, such as BM, aorta, lens, and peripheral nerves.
With mounting evidence that NG can alter the structure and /or function of
proteins (Table 1), the hypothesis that the reaction participates in the
development of diabetic complications has received wide attention. At the
same time it has become increasingly clear that hyperglycemia per se exerts a
deleterious influence on several metabolic processes implicated in the
pathogenesis of diabetic angiopathy, including basement membrane synthesis
and turnover, the polyol pathway, and myo-inositol metabolism. It has been
reported that glycosylated hemoglobin has a lower affinity for oxygen,
glycosylated albumin has a decreased ability to bind to bilirubin and cis-
parinaric acid, and glycosylated lens crystallins may aggregate and cause
opalescence (Charonis 1990). Such functional change may be the result of
either a direct binding of glucose onto lysine residues or subsequent cross-
linking between modified and unmodified lysine residues (Charonis 1992).
Nonenzymatic glycosylation involves a condensation reaction between
carbohydrate and free amino groups at the NH2-terminus or e-amino groups
of lysine residues of proteins. The reaction is initiated with the attachment of
the aldehyde function of acyclic glucose to a protein amino group via
nucleophilic addition, forming an aldimine, also known as a Schiff base
(Figure 1).
3
Table 1. Some Consequences of Excess Nonenzymatic Glycosylation of
Proteins
Proteins
Hemoglobin
Serum albumin
Low-density lipoproteins
Lens crystallins
Collagen
Fibronectin
Myelin
Laminin
Consequence
Increased oxygen affinity
Increased transendothelial transport
Decreased receptor-mediated uptake
and degradation
Aggregation
Decreased solubility, abnormal cross
links; immunogenicity
Decreased ligand binding
Macrophage recognition
Decreased self-assembly, abnormal
cross- links; altered structure and
function
4
{CHOH)4
NH2
CVI20H (CHOHJ4
Protein
+ H O
2
Glucose
Reversible
K
CH20H
Schiff Base
K _
jr
Reversible
NH
* M CH^
C,
Irreversible Protein
(CHOHI3
Protein Cross-link CH20H
in Advance Glycosylation
End Products
Amadori Product
Figure 1. The steps leading to nonenzymatic glycosylation are reversible until
the formation of Amodori products. The final products of this reaction is the
advance glycosylation end products (AGE), which formed cross-link with
other protein.
5
This intermediate product subsequently undergoes an Amadori
rearrangement to form a 1-amino-1-deoxyfructose derivative having a stable
ketoamine linkage, which in turns can cyclise to a ring structure. This
biomolecular condensation of free saccharide with protein constitutes
therefore a mechanism by which proteins undergo postribosomal modification
without enzymatic interaction. These early glycosylation products on
proteins with long half-lives undergo a slow, complex series of chemical
rearrangements that give rise to advanced glycosylation end products (AGEs)
(Vlassara 1992).
AGEs accumulate in vascular tissues with aging and at an accelerated
rate in patients with diabetes; and may to be linked to tissue damage due to
their ability to cross-link proteins. In addition to collagen and laminin, AGEs
attach to and cross-link plasma proteins, enhancing protein deposition,
impairing interaction with macromolecules and stimulating macrophages to
secrete cytokines which influence endothelial cells and fibroblasts (Vlassara
1992).
Laminin is a large heterotnmeric complex of approximately 8.5x10^
D., shaped in the form of an asymmetric cross. Formed by the association of
1 a and 2 p chains, there are many isoforms of laminin that appear to have
tissue-specific distributions. At least tow isotypes of the a chain and three
short P chain variants exist. As with virtually all extracellular matrix
molecules, laminin is a multidomain molecule, which interacts with a variety
o f integrin and nonintegrin receptors, as well as other BM components (Beck
et al., 1990). This large glycoprotein, found almost exclusively in BM, is
involved in many interactions with other BM macromolecules, e.g., type IV
6
collagen, heparan sulfate proteoglycan, entactin, and with itself (Timpl 1986).
It also has the ability to interact with various cell surface molecules.
Because of these numerous and complex associations with most other,
laminin may be a crucial factor in maintaining the structural and functional
integrity of basement membrane (Charonis, 1990). Therefore the study of
laminin alterations in diabetic conditions can lead to better understanding of
the molecular mechanisms operating under these conditions
3. The Effect of Glycosylated Laminin on EC Adhesion and
Proliferation
In multicellular organisms, cells are surrounded by an extracellular
matrix, a complex milieu composed of some or all of the following: structural
macromolecules, attachment factors, signals for cells to spread, migrate,
proliferate, or synthesize specific products, a selective filter system, possibly
even signals that specify cell death or a radical change in their phenotype.
The composition of the extracellular matrix varies with its role and location
and changes with development, aging, neoplastic transformation, etc.
Glycoproteins, including laminin, fibronectin, tenascin, thrombospondin, and
other cell adhesion molecules, have been localized to a variety of extracellular
matrices. These are large, complex, multidomain molecules with numerous
biological activities. The influence of the lysine-linked carbohydrate moieties
of ECM glycoproteins on cellular interactions has not been extensively
studied (Robert 1993).
Since laminin is an important extracellular matrix protein, it is
important to define how EC respond to an altered laminin and in the context
7
o f diabetes how EC respond to glycosylated laminin, such as may occur in
diabetic hyperglycemia. A report on the response of bovine EC to
glycosylated proteins has been published (Haitoglou 1992); however it is not
known how human EC, specifically HUVEC from both diabetic and
nondiabetic individuals respond to glycosylated laminin.
4. Expression of Human Integrin p i on the Surface of EC
EC form a continuous layer lining the luminal surfaces of blood
vessels and represent the physical barrier between the blood and the
surrounding tissue. The mechanical and functional integrity of this barrier is
assured by cell-cell interaction among EC as well as by the adhesion o f EC to
the basement membrane. Thus the adhesive properties of EC are important
for the maintenance of vessel wall integrity which allows selective passage of
nutrients. On the luminal side these cells form a nonthrombogenic surface,
whereas on the basal side they adhere to the basement membrane. The
endothelial BM is a complex structure made up by interactions among
macromolecules such as laminin, type IV collagen, type V collagen,
fibronectin and proteoglycans (Timpl 1989). Some of tne basement
membrane macromolecules mediate EC adhesion, and laminin is thought to
be of particular importance to the adhesion between cells and BM (Languino
1989).
Various types of cells have been shown to have laminin receptors that
belong to the integrin family. Integrins are heterodimeric proteins that consist
o f a and P subunits. Integrin a 3 p i, a 6 p l and a l p l have each been
implicated as laminin receptors. EC express the a 5 p i,a 3 p i, and a 2 p i
integrins (Albelda, 1989). The a 2 p l is the major integrin on EC, and it is
8
responsible for EC adhesion to laminin, and with lower affinity to collagen
and fibronectin (Lampugnani 1991). That EC bind to laminin via integrin-
mediated interactions is suggested by the inhibition of EC attachment to
laminin by an antibody directed against the p i integrin subunit (Languino
1989).
5. Regulation of EC adhesion by TGF p
Cell migration, homing, and settlement during tissue formation, repair,
tumor invasion, and metastasis are guided by a complex set of adhesive
interaction between cells and extracellular matrices. The adhesive behavior
o f a cell is determined in part by the type and number of adhesion receptors
that it expresses, and the type of extracellular matrix that it produces and with
which it interfaces. In addition to providing physical support, adhesive
interactions are a major conduit for intercellular regulation of cell function
and phenotype. The possibility that the cell adhesion as well as the
composition o f extracellular matrices may be regulated by growth and
differentiation factors has been shown the action of TGF p on many cell types
(Massague 1990).
TGF p is a multifunctional growth factor with wide ranging and often
opposite effects on many cellular processes. The effect of TGF p on the
production and remodelling of extracellular matrix and thus alteration of
matrix composition, cellular adhesion and cell-cell interactions is well
documented (Roberts 1990). The action of TGF p on normal mesenchymal,
epithelial, and lymphoid cells, as well as various tumor cell lines, generally
leads to up-regulation of cell adhesion. This action is mediated in concert by
enhanced synthesis and deposition o f extracellular matrix components,
9
decreased pericellular proteolysis, and modification of the repertoire of cell
surface adhesion receptors ( Massague 1990).
However, TGF p inhibits endothelial cell adhesion to human
lymphocytes as well as to monocytes (Gamble 1991). It has also been
reported that the ability of endothelial cells to respond to TGF p by altering
their adhesion is lost with prolonged culture of the cells. Although earlier
studies have shown that TGF p regulates cellular adhesion, what the role of
TGF p in regulating HUVEC adhesion to extracellular matrices is, and how
this regulation is affected by nonenzymatic glycosylation of laminin are still
unknown.
6. Research strategy
The objectives of our studies were to determine whether HUVEC from
diabetic individuals have intrinsic characteristics different from those of
cells from normal subjects; and how nondiabetic and diabetic HUVEC
responded to nonenzymatically glycosylated laminin; and whether TGF P
modified adhesion of diabetic and nondiabetic endothelial cells on either
normal or glycosylated laminin. The approach used in these studies was to: a)
isolate HUVEC from nondiabetic and diabetic subjects (insulin dependent
type); b) glycosylate human laminin; c) measure adhesion of normal and
diabetic HUVEC cultured on surfaces coated with either laminin or
glycosylated laminin; d) examine proliferation of normal and diabetic
HUVEC cultured on surfaces coated with either laminin or glycosylated
laminin; e) examine adhesion of both cells following treatment with TGF P;
and 0 evaluate the expression of integrin p i by these cells.
10
MATERIAL AND METHODS
1. Purification of laminin
Purified laminin (20mg/ml) was a gift of Dr. Hynda Kleinman (National
Institute of Dental Research, NIH, Bethesda, MD). Before use, laminin was
dialyzed at pH 7.4 at 4°C for 2 days against a 200 fold volume of phosphate-
buffered saline (PBS), containing 10 mM FDTA, 50 ug/ml
phenylmethylsulfonyl fluoride (Sigma Chemical Co), 50 ug/ml N-
ethylmaleimide (Sigma Chemical Co), and 0.02% NaN^ (USB. Ohio) After
dialysis the laminin solution was centrifuged at 20,000 imp for 30 min. at 4°C
to remove aggregates. The final concentration of the solution used for
nonenzymatic glycosylation was 200 ug/ml.
2. Removal of contaminants from radioactive and cold glucose by
chromotography
It is known that radioactively labeled glucose from several
manufactures contains impurities that bind covalently to proteins, such as
collagen, fibronectin, basic myelin protein, bovine serum albumin, and
hemoglobin, simulating nonenzymatic glycosylation (Trueb 1980).
In order to correctly quantitate NG, Timpl's method ( Timpl 1979) to
remove impurities was modified by adding cold glucose to the reaction. The
unknown contaminants were removed from radioactive as well as
nonradioactive glucose, thus greatly increasing the accuracy o f the
quantitation o f NG (see results of glycosylation) in our study.
The commercially available solution of D-[6-^H] glucose, (Amershan
Life Science, Arlington, IL) was dried under a stream of nitrogen and
U
dissolved in PBS containing lOmg/ml bovine serum albumin (BSA) (Sigma
Chemical Co), and lOmM EDTA at pH 7.4. The reconstituted radioactive
glucose, 450 uCi D-[6-^HJ, was incubated with 1.55 molar D-[+]-glucose
(Sigma Chemical Co), 20 mg BSA (Sigma Chemical Co), and 1 ml deionized
water at 37 °C for 3 days in the dark with occasional shaking.
The incubation mixture was chromatographed on a Sephadex G -15
column (1x25 cm), equilibrated and eluted with PBS. G15 was boiled in
distilled water and poured into the 1x25 cm filter column, allowed to settle;
and the column was then washed with 50 ml of washing buffer (PBS-10 mM
EDTA, pH 7.4). The glucose mixture was loaded on the column equilibrated
with washing buffer and eluted at a flow rate of 1 ml/min with the same
buffer. After all the fractions (0.5ml) were collected using a fraction collector,
aliquots were assayed for radioactivity in a scintillation counter and for
protein (BSA) at 280 nm in a spectrophotometer.
The radioactivity and protein peaks could be separated (Figure 2). The
overlap in the two peaks was considered as the area of the unknown
contaminants bound to BSA.. The fractions of radioactive glucose from the
large peak were collected, lyophilized and diluted at the desired
concentration.
3. Nonenzymatic glycosylation of laminin
In vitro nonenzymatic glycosylation of laminin was performed using a
modification of Charonis1 method (Charonis 1992). Laminin at a
concentration of 200 ug/ml was incubated in the dark at 29°C with occasional
shaking for 60 hours in the presence of various concentrations o f D-(+)-
12
glucose from 0, 10, 100 to 200 mM in PBS, pH7.4, containing 450 p Ci of
radioactive glucose in a total volume as 10 ml.
Glycosylation was stopped by dialyzing the samples at 4°C against 0 1
M phosphate buffer, pH 7.0. The glycated proteins were further incubated
with 200 molar excess of sodium borohydride diluted in 0.2 M NaOH for 10
minutes at room temperature and another 50 minutes at 4°C . All the samples
were exhaustively dialyzed against 0 1 M phosphate buffer, pH7 0, then
against 1 M urea in 0.1 M phosphate buffer pH7.0, to remove nonspecifically
bound radioactivity. Urea was removed by extensive dialysis against PBS
containing 10 mM EDTA, pH7.4. Samples were then aliquoted, and stored at
-20°C until use. To quantitate NG, aliquotes were acetone precipitated, and
radioactivity determined by liquid scintillation using a Beckman LS6000IC
(Fullerton, CA). . NG in the final laminin preparation was expressed as cpin/
pg laminin (Figure 3).
4. Isolation of HUVEC from Diabetic and Nondiabetic Patients
A Definition of nondiabetic and diabetic EC.
Fro this study nondiabetic EC are defined as cells obtained from
umbilical cords from subjects 1) with no chronic illnesses eg. diabetes,
jaundice or vascular diseases, and had a blood glucose level* 80 + 20 mg/di,
2) who received no medication and 3) who were non-smokers. Diabetic EC
are obtained from umbilical cords of diabetic mothers who 1) suffered from
insulin-dependent diabetes for at least 1 year, and had a blood glucose level*
148 + 26 mg/dl, 2) who received no other medications chronically, except
insulin and 3) who were non smokers. * Patients' blood glucose levels were
obtained from the medical records.
Figure 2. Radioactivity and 280nm absorption of the fractions of ^H-glucose
chromatographed on G-15 Sephadex. A solution of 1.55 M D-[+]-glucose,
containing 450 pCi ^H-glucose was incubated at 37°C for 3 days and
chromatographed on Sephadex G-15 (1x25 cm). Fractions (0.5ml) were
collected and the radioactivity in each fraction was estimated by liquid
scintillation. The radioactivity in fractions 15 to 18 coeluting with the peak of
absorption at 280 nm (fractions 14 to 19) indicate the fractions of BSA that
bind the unknown contaminants in ^H-glucose and cold glucose. Fraction 21
and 39 was collected and lyophilized for in vitro NG.
14
Fractions
Absorption
-0.2
< > 111111 , i
Figure 3. In vitro nonenzymatic glycosylation of laminin. Purified laminin
(200 ug/ml in PBS pH 7.4) was incubated with 0, lOmM, lOOmM and
200mM D-[+} glucose, respectively; as well as with the same ratio of
radioactive glucose. After 60 hrs incubation the reaction was terminated and
radioactivity estimated by liquid scintillation. Results are expressed as cpm /
ug laminin.
16
H-Glucose incorporation (cpm) I u g Laminin
800
700
600
500
400
300
200
100
250
200
150
50 100
Glucose Concentration (mM)
F ig u r e 3
1 7
B HUVEC isolation
EC were harvested from human umbilical veins and cultured according
to a modification of the Jaffe's method (1973). Fresh umbilical cords were
obtained from non-diabetic and diabetic mother (who had delivered at
Woman's Hospital of Los Angeles County Medical Center USC). The cords
were wrapped in saline-moistened 4" x 4" sterile gauze, placed at 4°C and
brought to our laboratory within 12 hours The external surface of the cords
was rinsed with 70% ethyl alcohol and all clamp marks and needle sites were
removed. Both ends of the umbilical vein were attached by Luer connectors
and flushed twice gently with 20 ml of Medium 199 (Gibco, Grand Island,
NY). The vein was then distended with 10 ml of 0.05% trypsin/1 mM
ethylene diaminetetraacetic acid (EDTA) (Gibco, Grand Island, NY) in
phosphate-buffered saline (PBS) prewarmed to 37 ° c , and the entire cord
was incubated in a water bath at 37 °C for 10 minutes. After incubation, the
vein contents were flushed twice with 20 ml of medium 199 twice. The vein
was filled with 8 ml of 0.1% collagenase (CLS, Worthington Biomedical,
Freehold, NJ) in Ca^+ and Mg2 + free PBS, and the cord was incubated at 37
°C in a water bath rocking at 120 cycle per minute. After 20 minutes
incubation the vein contents were flushed with 20 ml o f medium 199 into a
50-ml tube, and centrifuged at lOOOrpm for 5 minutes at room temperature.
The cell pellet was suspended in 5 ml medium 199 containing 10% fetal
bovine serum (FBS), plated onto a 20-cm2 Nunclon tissue culture plate
(Irvine Scientific, Irvine, CA ) and incubated at the 37 °C in a humidified
atmosphere of 5 % CO 2 -95% air.
C Culture o f EC
18
Two days after isolation the dishes were washed with medium 199, and
the endothelial cells were fed with complete medium (M l99 with 10 % FCS,
2,000 units of heparin, 30 mg of L-glutamine, 10 mg o f EC mitogen, 250 ug
amphotericin, 250 ug sodium desoxyction, 5,000 units penicillin G sodium
and 5,000 ug streptomycin). Subsequently the medium was changed every 3
days .
The cells were examined by phase-contrast microscopy (Figure 4), and
percentage of confluence was estimated. The cells were passaged when
approximately 80 % confluent using 0.05% trypsin -I mM EDTA,
centrifuged at 1,000 rpm for 5 minutes and resuspended in complete medium
before plating at a density o f 3,000 cells/cm^ in the 100 mm Nunclon plates .
Cell viability was assessed during all passages by trypan blue dye exclusion.
All cells used in our studies were from passages 1 through 5.
D EC characterization.
EC were identified by their typical cobblestone morphology at
confluence under phase-contrast microscopy (Jaffe 1973) as well as by the
presence of Factor VIII and acetylated LDL.
a). Presence of Factor VIII. The presence o f von Willebrand factor
(factor VIII antigen) on EC was detected using a rabbit antihuman von
Willebrand antibody (Dakopatts a/s 42, Produktionsvej, Glostrup, Denmark).
EC were trypsinized and plated onto glass coverslips in 35 mm Nonclon petri
dish at 50,000 cells per well. EC cultured on glass coverslips for at least 48
hours were washed with PBS and fixed with cold methanol for 5 minutes at -
20° C. Fixed cells were washed 3 times with PBS over a period of 30
19
minutes, followed by a 30 minutes incubation with an anti-vWF antibody at
room temperature in a humidified atmosphere. Anti-vWF (Boehringer
Mannheim Cor., Indianapolis, IN) was dissolved in distilled water at O.lug /
ul, and further diluted 1 : 10 in complete medium. The cells were washed 5
times with PBS over a period 30 minutes and incubated with anti-mouse
FITC-IgG antibody (diluted 1:40 in the complete medium) at room
temperature in a humidified atmosphere . After 30 minutes the coverslips
were washed with PBS, covered with mounting medium and placed onto the
glass slides The slides were examined with an Olympus microscope
equipped with epifluorescence (Figure 5).
b). Uptake of Di-LDL. To examine the uptake of acetylated low-density
lipoprotein (LDL) (Biomedical Technologies Ins., Stoughton, MA) EC
cultured on the glass coverslips were incubated in complete medium
containing Di-Acetyl-EDL (10 ug/ml) at 37 0 C in a humidified atmosphere of
5% CO 2. After 3 hours the medium was removed, EC were washed 3 times
with PBS, fixed in 3% formaldehyde in PBS for 20 minutes at room
temperature, observed and photographed with an Olympus microscope
equipped with epifluorescence (Figure 6).
5. Coating ELISA plates with laminin
Flat bottom 96 wetl ELISA plates were coated with the various
laminin solutions (normal laminin and laminin glycosylated in 10, 100 and
200 mM glucose) by placing 50 ul laminin solutions (50ug/ml in PBS-1%
BSA) into each well (at least 6 wells in a group), and dried at the room
temperature in a laminar flow hood overnight. If not used immediately the
coated plates were stored at -20°C. Before the experiment the wells were
20
coated with 1% BSA in PBS for 30 minutes in order to coat any surface not
coated by laminin. As a control one set of the wells was coated only with
BSA in PBS.
6. Adhesion assay
Nondiabetic and diabetic EC at approximately 80% confluency were
detached with 0.05% trypsin-EDTA, centrifuged, resuspended in medium 199
containing 2% BSA, plated at 5000 cells per well which precoated with either
glycosylated or nonglycosylated laminin, and incubated for 2 hours at 37 °C
in 5% CO 2 humidified atmosphere. After incubation, the unattached cells
were washed out with medium 199 and the remaining cells were fixed with
3% formaldehyde and stained with 0.5% toluidine blue.
Adherent cells were counted either manually by using the phase-
contrast microscope or spectrophotometrically by measuring the absorption of
toluidine blue, and expressed as percentage of total number plated (%
attached cells = number of counted cells/ total cells plated x 100% ).
7. The effect of TGF p on adhesion
To examine the effect of TGF P on the adhesion o f diabetic and
nondiabetic EC, the same adhesion assay described above, was used except
that: a). EC with 80% confluency were plated onto 35 mm Nonclon dishes at
10,000 cells /dish in complete medium, b). After overnight culture the
medium was changed to medium 199 containing 2 % FBS and 0.2% BSA
without EC mitogen, c). After 12 hours the medium was changed to medium
199 containing 10 % FBS, or 2 % FBS and TGF p (5ng/ml), or 2% FCS and
incubated for an additional 12 hours before estimating adhesion.
21
Figure A Phase-coiurasi micrography o f normal 1IIJV1X' (magnification
lOOx). 1 3ji
* ;
$
m
. J
*
v ^ '
M H u i
■i
H
m m
n
0* W < V
* J
t / ■
* m
Figure s fluorescence micrograph ol noim.il M l VI ( stamcd uilli factor
VIII ( 1 0 0 \ )
igure 6. Fluorescent micrograph ofllUVFC’ incubated with Di-A cetyl-FD L
d). Alteratively after overnight culture in 2% FCS-M199 the cells were
cultured in the presence of 1 and 5 ug/ml o f TGF p j in medium 199
containing 2% FBS for 12 hours, then adhesion was estimated on either
laminin or glycosylated laminin.
8. Proliferation assay
96 well ELISA plates were precoated with 50 ul of 50 ug/ml o f either
laminin or glycosylated laminin ( with 200 mM glucose) per well. It was
followed by countercoating with 0 1 % BSA in PBS.
Proliferation of EC was assessed by tritiated thymidine incorporation.
EC were plated in 200 ul of complete medium at a density of 5000 cells/well
o f ELISA precoated with either glycosylated or nonglycosylated laminin.
After 24 hours the medium was changed to complete medium containing
pHJ-thymidine (5 uCi/ml ) (ICN Radiochemical, Irvine, CA, s.a. of
2.0mCi/mmol). After an additional 24 hours incubation the cells were washed
with PBS, followed by treatment with 5% cold trichloroacetic acid (TCA) to
precipitate macromolecules. The precipitate was washed 3 times, with ice-
cold 5% TCA and solubilized with 0.2 M sodium hydroxide (NaOH) for 1
hour. An aliquot o f the solubilized cell layer (100 ul ) was mixed with on
equal volume o f 0.5 M hydrochloric acid (HCI) and 4 ml of scintillation
counting fluid ( Beckman, Brea, CA) and radioactivity was determined with a
Beckman LS6000IC scintillation counter. Another aliquot o f the solubilized
TCA precipitate was used for protein determination. Results are expressed as
cpm/ug protein.
9. Estimation of Human Integrin p i on EC Surface
24
Early passage ( 1 to 4 ) of ECs grown to 80% confluency on the
Nunclon dish (about 2 x 10^ cells) were gently scraped and centrifuged at
1,000 rpm for 5 minutes.
Cells were washed twice with the cold buffer ( 2 % BSA in medium 199 with
0.02% NaN 3 ), centrifuged at 4000 rpm for 4 minutes at 4°C, resuspended in
0,5 ml of the M199-BSA medium and incubated with a 1:20 dilution of goat
IgG 20 ug/ml at 4°C to block Fc fragments After 30 minutes the cells were
washed with M199-BSA medium followed by incubation with a 1:200
dilution of a mouse anti-human integrin p i monoclonal antibody ( Gibco,
Grand Island, NY). After 30 minutes incubation at 4°C, the cells were
washed twice with M199-BSA and incubated with FITC-conjungated goat
anti-mouse Ig G (1:75), followed by two washes with M199-BSA. The
samples were sorted using a fluorescent activated cell sorter (FACS Starplus,
Becton Dickson, Mountain View, CA) which counted 5000 cells
automatically. Controls included cells treated with either first or second
antibody alone (Forsyth and Talbot 1991).
25
RESULTS
1. Quantitation of glycosylation by ^H-glucose
Incorporation of tritiated glucose showed that radioactive glucose
bound to laminin with increasing concentration of glucose. As expected NG
o f laminin was found to be dependent on the concentration of glucose. There
is a linear relationship between NG and glucose and this relationship is
clearly seen in Figure 3
2. Effect of glycosylated laminin on EC adhesion
When normal and diabetic EC were cultured on surfaces coated with
either laminin or glycosylated laminin for 2 hours, both cells types showed
decreased adhesion on glycosylated laminin. In the Figure 7 the results
showed that: 1) diabetic cells attached significantly lesser than normal cells,
on normal laminin as well as glycosylated laminin (P<0.001); 2) adhesion of
normal cells reduced on glycosylated laminin compared to adhesion on
normal laminin (reduction = % of attached cells on laminin - % of attached
cells on glycosylated laminin, 12.5 + 7,5%); and 3) adhesion of diabetic cells
was significantly reduced on glycosylated laminin compared to that on
laminin (reduction is 43.1 + 5.8%, P<0.001). In addition to differences in cell
adhesion, diabetic EC did not spread out as much as nondiabetic cells
morphologically (Figure 8).
3. Effect of TGF P on EC adhesion
In the presence of 10 % FBS diabetic and nondiabetic EC attach
equally well. In the presence of 2 % BSA and no serum there is a statistically
significant decrease in adhesion in both diabetic and nondiabetic cells, but the
decrease in attachment is more pronounced in diabetic cells (PO .O l).
26
Figure 7. Effect of glycosylated laminin on the adhesion of nondiabetic and
diabetic EC. EC were plated on surfaces precoated with either glycosylated
laminin or laminin, incubated for 2 hours and percentage of attached cells wer
determined. Significant differences between the bars of EC and DC (diabetic
EC, P<0.001) on laminin, and between the bars of DC on laminin or glycated
laminin (P<0.001) can be clear seen.
27
% Attached Cells
100
80 -
60 -
40 -
20 -
□ EC
I I DC
P<0.001
Laminin Glycated Laminin
F ig u r e 7
A H
Figure 8. Adhesion o f nondiabetic and diabetic HUVEC on nonglycosylated
or glycosylated laminin. EC were plated on surfaces precoated with either
laminin or glycosylated laminin, incubated for 2 hours and stained with
toluidine blue. A shows attached nondiabetic cells on normal laminin and B is
those cells on glycosylated laminin. C represents attached diabetic cells on
normal laminin and D on glycosylated laminin It is evident that fewer cells
attached on the glycosylated surface. Furthermore diabetic cells did not
appear to spread out on either surfaces.
29
!■' i t j u r o H
The addition o f TGF 0 decreases attachment further, but the difference
between TGF p treated cells and the 2 % albumin treated cells is not
statistically significant (Figure 9). It is also interesting that not only diabetic
EC lost their spreading ability as described earlier, but also nondiabetic EC
did not spread out well after being treated with TGF p compared to the cells
cultured in 10% FCS (Figure 10 and 11).
In Figure 12 it is evident that nondiabetic cells adhere significantly less
in the presence of TGF p (P<0.01); whereas diabetic cells show only a
decrease in adhesion at high (5ng/ml) TGF p concentrations. When cells were
plated on glycosylated laminin, a further decrease in adhesion of nondiabetic
as well as diabetic cells occured in the presence o f 5ug of TGF p (P<0.01),
but not in the presence of 1 ng TGF p (Figure 13).
4. Effect of glycosylated laminin on the incorporation of thymidine
We have previously shown that diabetic EC have a slightly higher
proliferation rate than nondiabetic EC (Sank 1994). In this study we plated
EC on plastic surfaces coated with glycosylated and nonglycosylated laminin,
and measured ^H-thymidine uptake. After 48 hours of culture diabetic as well
as nondiabetic EC exhibited a lower rate of thymidine incorporation when
cultured on glycosylated laminin. From the data it is clear that the diabetic
cells are more susceptible to glycosylated laminin, thus showing a greater
decrease in proliferation than nondiabetic cells (Reduction o f proliferation^
rate of tritiated thymidine incorperation o f EC on normal laminin-rate o f it on
glycosylated laminin / rate of it on normal laminin x 100%, diabetic EC 19.2
% vs normal EC 9.8 % ) (Figure 14).
31
Figure 9 Effect of TGF p on the adhesion o f nondiabetic and diabetic EC.
EC were cultured in 1% BSA in M l99 with TGF P, or BSA in M l99 only,
or 10% FCS in M l99 for 12 hours after starving. They were plated onto 96
ELISA plate and incubated for 2 hours. In the serum free group diabetic cells
reduced the adhesion significantly compared to the control group (*P<0.01).
Addition of TGF p to diabetic cells reduced the adhesion further, but the
difference between the serum free and TGF p treatement groups of diabetic
cells is only marginally significant (*P<0.05).
32
Cell Numbers
3000 -|
2500 -
2000 -
1500 -
1000 -
500 -
CD Nondiabetic
H~al Diabetic
BSA TGF +BSA
F ig u r e 9
10%FCS
t i
Figure 10. Micrograph of attached nondiabetic EC. The cells were fixed by
3% formaldehyde and stained by 0.5% toluidine blue (lOOx). A shows the
attached nondiabetic cells treated with 10% FCS; B is those cells treated with
0.2% BSA and C is those cells treated with 5ng/ml TGF p.
34
35
Figure 11. Micrograph of attached diabetic EC. The cells were fixed by 3%
formaldehyde and stained by 0.5% toluidine blue (lOOx). A shows the
attached diabetic cells treated with 10% FCS; B is those cells treated with
0.2% BSA and C is those cells treated with TGF p.
36
&
*
■ '* V .
* ' V \ #
■% » - i
1
,T „jf'r> .- ..-\, '
£.:$$.*!•/ * * ■ * W
J-JV1 - u ' . • t , « . * - » t
* , ': .< ■ > * < * » > . , , ; • • .(♦■ •ft ' • ^ * - # y .‘ ■ fi*'™ if\Z
I ' I ■ 1 I I t I ' I 1
Figure 12, The effect of different concentration o f TGF pi on diabetic and
nondiabetic HUVEC adhesion. EC were treated with TGF p i (0 ng/ml, 1
ng/ml and 5 ng/ml in media) for 12 hours before adhesion was estimated.
There is a trend that nondiabetic as well as diabetic EC showed reduced
adhesion with increasing TGF P concentrations; however no statistically
differences were observed except for nondiabetic EC at 5ng/ml o f TGF p
(P<0.01).
38
C ell Number
1200
1000
800
600
400
i i l
200
Control 1 ng/ml 5ng/ml
TGF p
F ig u r e 12
3 9
Figure 13. The effect of glycosylated laminin on HUVEC adhesion with TGF
p i treatment, a). Adhesion of nondiabetic EC and b) adhesion of diabetic
EC, plated on laminin (□ ) or glycosylated laminin (■). TGF p i, reduces
adhesion o f both diabetic and nondiabetic EC on glycosylated laminin; the
reduction was statistically significant at the dose of 5 ng/ml o f TGF p
(*P<0.0l).
40
C ell Number
1200
1000 -
800 -
600 -
400 -
200 -
0 -
1000 -
800 -
600 -
4 0 0 -
200 -
0 -
IP
Nondiabetic EC
* P<0.01
: ' 4
4
. ; 3
m
m
|J j§ |
Wm
'M
s.
Diabetic EC
m m .
Laminin
Glycated Ln
_L
i M
Control 1ng/ml
TGF -Pj
5ng/ml
F ig u r e 1 3
41
5. Expression of Integrin (31
The fluorescent intensity o f the control cells treated with only the
second antibody was considered as the basal level o f fluorescence intensity,
thus only those cells with a fluorescence intensity beyond this level were
considered positive for the presence of integrin p i (Figure 15). The data
processed by the computerized analysis program o f the FACS, showed the
fractions (percentage) of Ab-p] integrin positive cells among the population
of each sample (5,000 cells) are: nondiabetic EC 72.9 j_ 10.9 %, and
diabetic EC - 56.1 + 16 % (n-5).
In diabetic EC the fraction of integrin p i positive cells was about 20 %
less than that of nondiabetic EC. Although ANOVA test showed no
statistically significant difference at the 95 % confidence level, there is a
marginal difference between the two cell types in the expression o f this
laminin receptor subunit. ( P < 0.058).
42
Figure 14. ^H-Thymidine incorporation of nondiabetic and diabetic HUVEC.
Diabetic and nondiabetic EC were incubated with ^H-thymidine and
complete medium for 24 hours on either laminin or glycosylated laminin; and
3-H-thymidine incorporation of both EC was measured. □ represents
thymidine incorporation by nondiabetic EC, ■ thymidine incorporation by
diabetic EC.(*p<0.05).
43
H -T hym idine Incorporation (cpm ) I u g Protein
Nondiabetic
Diabetic
1 -
C O
Ln
Glycosylated Ln
F i g u r o 14
44
Figure 15 Fluorescent patterns o f different groups of HUVEC.
Both nondiabetic and diabetic EC were incubated with primary Ab-p]
integrin and secondary anti-mouse Ig G antibodies, and sorted by FACS. 16a:
Pattern of negative control (no primary antibody); 16b: pattern of nondiabetic
EC; 16c and 16d show different patterns of diabetic EC groups.
45
o 4
T ' 1 . 1 i. «
CVL
«
A
1
I
U
i
i
e
o
f U
U*ttn * > t n ^
i i
, I I O
v i ta
• i iu‘
003
: 30-JUL-93
Tlwc: 1 2 : 3 7 : 5 0 _JV h . 9i S t a r t Time: 1 2 :5 5 :3 9
aao
V I T T ? W M
1 Q tlO
4
«
9
L
F ig u r e 15
46
DISCUSSION
Nonenzymatic glycosylation is one of the main mechanisms by which
hyperglycemia affects the structure and function of various macromolecules
(Roberts 1993 ). NG is considered a major pathological event in diabetes
mellitus. NG in vivo occurs when proteins are subjected to increased glucose
concentration over a long period of time. In our in vitro experiments
significant differences in glycosylation were observed in the presence of
different glucose concentrations, suggesting that glucose concentration may
be an important variable in protein glycosylation in vivo. The binding o f sugar
to laminin is consistently increased with increased glucose concentration; and
as will be mentioned later, glycated laminin affects the cellular properties of
nondiabetic and diabetic EC.
The technique of nonenzymatic laminin glycosylation has been
described previously (Charonis 1992). In this study, we have taken into
consideration contaminants present in the commercially available glucose
which might interact with laminin in an nonspecific manner These
contaminants were removed by pretreating a mixture of ^H-glucose and
glucose with BSA, followed by chromatography to separate BSA from the
mixture. In this way, we have shown that the binding o f glucose to laminin
was clearly dependent on glucose concentration. The highest amount of
glucose incorporated was -14 mol /mol laminin. It has been shown that after
NG, laminin undergoes various alterations of its cruciform shape, and its
ability to self-assemble is decreased. Our results suggest that glucose-
induced glycosylation is in part (although not exclusively) responsible for the
changes in structure and the ability of laminin to polymerize after NG
(Haitoglou 1992 , Charonis 1990).
47
Although incorporation o f tritiated glucose in NG in vitro is a well
established technique, unknown contaminants can represent 5 to 20 percent of
the radioactivity (Timpl 1979, Trueb 1980, Charonis 1992), and have to be
removed by gel filtration. However, it should be made clear that such
contaminants can be present in nonradioactive glucose as well, and that those
contaminants can bind to proteins, leading to possible errors in studies when
using varying glucose concentrations in vitro. In order to avoid these
nonspecific effects we chromatographed the nonradioactive glucose in the
same manner as radioactive glucose to remove possible contaminants. The
results of our glycosylation studies showed a linear binding relationship
between glucose and laminin.
In this study, evidence is provided that human diabetic and nondiabetic
EC undergo changes in their interactions with nonenzymatic glycosylated
laminin. The altered interactions resulted in impaired cellular adhesion and
spreading. BM macromolecules, and extracellular matrix components in
general, are important elements in the regulation of cellular phenotype (Bissel
1982) and are crucial in determining cell shape and induction of specific gene
expression in several cells (Ben-ze'ev 1988). NG can change the structure
and function o f laminin and type IV collagen (Charonis 1992), which in turn
mediate changes of cell shape and interfere with growth and differentiation.
Cell adhesion and proliferation are essential events for tissue repair and
angiogenesis (Lorenzi 1991). Glycosylated laminin did not seem to affect
significantly either cell adhesion or growth, as determined by thymidine
incorporation, of HUVEC from nondiabetic origin. On the other hand,
diabetic cells, which showed no significant differences from nondiabetic cells
48
either in cell morphology, cell adhesion or growth on normal laminin. were
sensitive to laminin glycosylation and exhibited reduced cell adhesion as well
as reduced cell growth. Early passage cells (second and third passage) were
used in our experiments to minimize phenotypic changes which might occur
during in vitro culture.
Adhesion and spreading are very early events in the interaction
between cells and extracellular matrices, therefore, these early binding events
may be very critical in determining the extent and nature of such interaction.
Although it had been suggested that EC behavior may be altered by exposure
to a high glucose concentration, this report provides evidence that the altered
behavior is not a direct effect of glucose but rather a consequence of
nonenzymatic glycosylation of matrix proteins subsequent to the increase in
glucose. In diabetes such high concentrations of glucose would lead to
nonenzymatic glycosylation of basement membrane proteins and thus altered
functions of EC.
Our findings also provided evidence that endothelial cells have
decreased adhesion and cell growth on nonenzymatically glycosylated laminin
or when treated with TGF p. These differences in cell adhesion and growth
were not detectable under normal culture conditions, i.e., in 10 % FBS and on
laminin. Therefore, the change in diabetic phenotype of HUVEC, ie., cell
adhesion and growth on laminin, seemed to be manifested by the alteration of
laminin due to high glucose or by the treatment of TGF p i. Differences in
cell adhesion were also observed in F1UVEC exposed to TGF pi in serum
free culture condition. HUVEC, of either diabetic or nondiabetic origin, are
very sensitive to serum deprivation as shown by a decrease in cell adhesion
49
on laminin. TGF p i, which modulates EC migration and differentiation
(Vlassara 1992), further reduced the adhesion of diabetic cells on
glycosylated laminin.
Integrins, heterodimeric membrane proteins consisting o f an a and a P
subunit, are responsible for various aspects of cell adhesion to extracellular
matrices (Cagliero 1991, Defilippi 1991 ). Alpha 2-beta I and alpha 3-beta 1
are the major receptors for laminin in endothelial cells ( Languino 1989 ) In
our studies using flow cytometry analysis, diabetic cells showed a marginally
reduced population o f beta 1 positive cells ( P<0.058 ). Although TGF p has
been shown to increase synthesis of a2 and pi in microvascular endothelial
cells ( Enenstein 1992, Defilippi 1992 ), the functional significance of this
increase is not known. Human integrin p i, is the main functional receptor
subunit of laminin. In this study expression of integrin p i on diabetic EC
detected by FACS is marginally decreased compared to that on nondiabetic
EC and this decrease was reflected by decreased adhesion o f diabetic EC on
laminin coated surfaces. However additional studies need to be done , to
define whether the receptors may be functionally abnormal.
50
CONCLUSION
In conclusion our studies have shown that nonenzymatically
glycosylated laminin interferes with cell attachment, spreading and thymidine
uptake of normal as well as diabetic vascular endothelial cells. These effects
are more pronounced in the diabetic cells suggesting that vascular diabetic
endothelial cells have intrinsic defects that accentuate the effect of a
glycosylated substrate. In fact, an analysis of the membrane receptor pi
integrin, demonstrated a decreased level in diabetic cells. This lower level
may be responsible for the decreased attachment of diabetic cells The
decreased cell attachment of vascular diabetic cell can be accentuated by
treating these cells with TGF p i, suggesting that TGF p i may modulate
surface receptors on endothelial cells.
Therefore, the phenotypic changes of diabetic HUVEC, apparent either
when the basement membrane components are modified or by growth factor
treatment such as TGF p, may contribute to the pathology seen in diabetes
mellitus.
The details of this signal transduction between the NG of the matrix
and the cellular components need to be further studied.
51
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56
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Biochemical analysis of somatic mutations in steroid 5alpha-reductase type II in prostate cancer

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Fibroblast growth factors and notch signaling in a diethoxycarbonyl dihydrocollidine-induced hepatic progenitor cell liver injury model

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Amelogenin domains in a self-assembly process

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A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells

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Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

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Functional analysis of single nucleotide polymorphisms (SNPs) in the 5' regulatory region on the SRD5A2 gene

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Generation of mutant tissue inhibitor of metalloproteinases-2 (TIMP-2) in the baculovirus expression system

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A model for the mechanism of agonism and antagonism in steroid receptors

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Cyclophilin C is a candidate protein to interact with saposin B using the yeast two-hybrid system

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Dual functions of Vav in Ras-related small GTPases signaling regulation

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Construction and characterization of RRP6 deletion in Saccharomyces cerevisiae

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Three-dimensional functional mapping of the human visual cortex using magnetic resonance imaging

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Implementation aspects of Bézier surfaces to model complex geometric forms

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Differential effect of ethanol and r-sulforaphane on regulation of heme oxygenase-1 in endothelial cells

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Development and secretions of salivary glands using mouse models

Asset Metadata

Creator Subin, Bill (author)

Core Title Altered interaction of human endothelial cells to the glycosylated laminin

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 1994-12

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

Tag health sciences, pathology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Nimni, Marcel E. (committee chair), Kalra, Vijay K. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-5224

Unique identifier UC11357701

Identifier 1376518.pdf (filename),usctheses-c18-5224 (legacy record id)

Legacy Identifier 1376518-0.pdf

Dmrecord 5224

Document Type Thesis

Rights Subin, Bill

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