(Circulation. 1999;99:2883-2891.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Distinct Patterns of Transforming Growth Factor-ß Isoform and Receptor Expression in Human Atherosclerotic Lesions
Colocalization Implicates TGF-ß in Fibrofatty Lesion Development
From Baker Medical Research Institute and Alfred Hospital, Melbourne, Victoria, Australia, and Cardiology Research Center, Moscow, Russia (A.K., V.S., E.T.).
Correspondence to Dr Alex Bobik, Baker Medical Research Institute, PO Box 6492, Melbourne 8008, Victoria, Australia. E-mail alex.bobik{at}baker.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Some animal studies suggest that transforming growth factor-ß (TGF-ß) protects vessels from atherosclerosis by preventing intima formation, but others indicate a role in vessel proteoglycan accumulation and lipoprotein retention. To distinguish between these possibilities in humans, immunohistochemical studies were performed examining the coexpression of TGF-ß isoforms and the TGF-ß receptors ALK-5 and TßR-II in aorta during the various stages of atherosclerotic lesion development.
Methods and Results—The spatial relationships between TGF-ß1, TGF-ß3, ALK-5, and TßR-II expression were compared in aortic segments from 21 subjects. Nonatherosclerotic intima contained predominantly TGF-ß1, low concentrations of TßR-II, and barely detectable amounts of ALK-5. In contrast, fatty streaks/fibrofatty lesions contained high concentrations of both TGF-ß isoforms. Smooth muscle cells (SMCs), macrophages, and foam cells of macrophage and SMC origin contributed to these high levels. These lesions also contained high, colocalized concentrations of ALK-5 and TßR-II. Despite fibrous plaques containing TGF-ß1, its receptors were at detection limits. We found no evidence for truncated TßR-II expression in either normal intima or the various atherosclerotic lesions.
Conclusions—TGF-ß appears to be most active in lipid-rich aortic intimal lesions. The findings support the hypothesis that TGF-ß contributes primarily to the pathogenesis of lipid-rich atherosclerotic lesions by stimulating the production of lipoprotein-trapping proteoglycans, inhibiting smooth muscle proliferation, and activating proteolytic mechanisms in macrophages.
Key Words: atherosclerosis • lesion • immunohistochemistry • cells • receptors
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Three mutually compatible hypotheses have been put forward to explain the development of atherosclerotic lesions. The "response-to-retention" hypothesis emphasizes the importance of atherogenic lipoprotein accumulation in the subendothelial region,1 whereas the "oxidation" hypothesis emphasizes the importance of oxidative modifications,2 both leading to recruitment of macrophages and other inflammatory cells.1 2 The "response-to-injury" hypothesis proposes vascular smooth muscle cell (SMC) proliferation as a key event in the formation and progression of atherosclerotic lesions.3
One potential link between these hypotheses is the involvement of transforming growth factor-ß (TGF-ß). The extent of early SMC proliferation has been proposed from animal studies to be dependent on an apparent lack of TGF-ß1 in vessels.4 TGF-ß1 is a potent inhibitor of SMC proliferation,5 and escape from its inhibitory effect has been linked to genomic instability of the type II TGF-ß1 receptor gene, a mechanism implicated in atherosclerotic lesions.6 TGF-ß1 also stimulates proteoglycan biosynthesis in human arterial SMCs.7 Proteoglycans interact strongly with lipoproteins, slowing their transit through the vessel wall and thereby increasing the possibility for lipoprotein deposition8 ; biglycan and apolipoprotein deposits colocalize in atherosclerotic plaques.8 Also, modification of lipoproteins by peroxidative processes frequently produces end products, including 4-hydroxy-2,3-nonenal, that can upregulate TGF-ß1 expression in macrophages,9 inducing them to produce urokinase plasminogen activator (uPA) and subsequently plasmin,10 an activator of metalloproteinases,11 systems implicated in plaque instability and rupture.12 TGF-ß1 also suppresses local inflammatory responses13 and is a fibrogenic cytokine.14 In vivo, its effects will depend not only on its own expression but also on the expression of its receptors.
Recently, we demonstrated that vessels in experimental animals contain only small amounts of TGF-ß1 and barely detectable levels of its type I (ALK-5) and type II (TßR-II) signaling receptors; only after activation of SMCs by injury do they express high concentrations of TGF-ß1, ALK-5, and TßR-II and elicit responses attributable to TGF-ß.15 Accordingly, we hypothesized that in human arteries there are likely to be distinct patterns of expression of TGF-ß and its receptors and that their coexpression will define when in the different stages of atherosclerotic lesion progression TGF-ß is likely to exert its effects. We demonstrate in human aortas with atherosclerotic lesions that TGF-ß contributes to specific stages of lesion progression. It is unlikely to be protective, as suggested by animal studies.4 Rather, TGF-ß participates predominantly in the pathogenesis of lipid-rich atherosclerotic lesions.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Tissues
Thoracic and abdominal aortas were collected during autopsy, up to 6 hours after death (see Table
), at the Russian
Cardiology Research Center, from individuals 20 to 73
years old. Ten subjects possessed atherosclerotic lesions of varying
severity (Table). Aortic segments were placed in ice-cold
saline, then frozen in OCT (Miles Inc) and stored at -80°C. Segments
destined for en face analyses of intima/atherosclerotic lesions
were fixed in paraformaldehyde and picric acid
(see below).
|
Immunohistochemistry
Antibodies
Antibodies to detect TGF-ß1,
TGF-ß3, ALK-5, and TßR-II were:
TGF-ß1, 2 polyclonal IgGs, a purified chicken
and turkey IgG raised against human TGF-ß1,
40131 and 40091, from Becton Dickinson; TGF-ß3,
a rabbit polyclonal IgG, sc-83, from Santa Cruz Biotechnology (SCB);
ALK-5, a rabbit polyclonal IgG raised against amino acids 158 to 179 of
human ALK-5, sc-398, from SCB; TßR-II, a rabbit polyclonal IgG raised
against amino acids 246 to 266 of human TßR-II, sc-400, from SCB;
CD-68, a mouse monoclonal antibody against human macrophages,
M0814; and CD-3, a rabbit polyclonal antibody against human T
lymphocytes, A0452, from Dako Corp. Secondary antibodies included
rabbit anti-turkey/chicken, 61-3140, from Zymed Laboratories, and goat
anti-rabbit, PK-6101, and horse anti-mouse, BA-2001, antibodies from
Vector Laboratories.
Immunohistochemical Procedures
TGF-ß and receptors were detected in frozen sections as
previously described.15 Briefly, 6-µm cross sections
were fixed in acetone, washed in 0.1 mol/L PBS, and treated with 3%
H2O2. After washings in 0.1
mol/L PBS, they were incubated in 10% horse or goat serum, followed by
avidin blocking solution ("Elite" Vectastain, Vector Laboratories,
SP-2001). Subsequently, the sections were incubated in 10% serum in
PBS containing the primary antibody or control IgG (dilutions 1:100 to
1:1000). Sections were washed and incubated with the appropriate
biotinylated antibody (1:200 to 1:500), with 10% serum in PBS. The
avidin-biotin-peroxidase complex system (Vector Laboratories) and
3,3'-diaminobenzidine tetrachloride were used for staining; whenever
appropriate, counterstaining was with hematoxylin. Oil red O was used
for neutral lipid staining, which involved rehydrating cryostat
sections in dH2O, rinsing in 60% isopropanol,
staining with aqueous isopropanol (40%) containing 0.25% to 0.5% oil
red O, followed by rinsing in 60% isopropanol and
dH2O.
En face determination of TGF-ß isoforms and cell-specific antigens in unaffected and atherosclerotic intima was carried out by fixing the segments in PBS containing 4% paraformaldehyde and 15% picric acid for 4 to 24 hours at 4°C. After washing, the intimal layer was isolated and dehydrated in a series of graded alcohol solutions before it was embedded in Epon-812 epoxy resin. After polymerization overnight at 60°C, serial en face semithin (1-µm) sections were cut and mounted. The epoxy resin was removed by immersion for 2 minutes into absolute ethanol/saturated sodium ethoxide (2:1). After rehydration, the sections were washed and incubated with either rabbit or goat serum (2% in PBS), and then TGF-ß1 and TGF-ß3 peptides were detected by overnight (4°C) incubation with a TGF-ß1 IgG (1:50) or the TGF-ß3 IgG (1:100). Sections were then incubated with the avidin-biotin-peroxidase complex, as above, and counterstained with methylene blue. Identical procedures were used to detect CD-3 (T lymphocytes) and CD-68 (macrophages). SMCs were CD-68 negative, generally elongated with filamentous processes, stellate in shape, or large and irregularly flattened.16
PCR-SLP Analysis of TGF-ß Type II Receptor
A10 Microsatellite Region
For polymerase chain reaction (PCR)–strand length
polymorphism (SLP) analysis of the
A10 microsatellite region in the TßR-II gene,
DNA was extracted and purified from intimal segments and cells using
proteinase K/SDS, phenol/chloroform extraction, and RNAse digestion.
PCR primers amplifying the specific (157-bp) region were sense,
5'-AACACTAGAGACAGTTTGCC-3' (bases 285 to 304 in the cDNA, GenBank
accession No. M85079), and antisense,
5'-GATGTTGTCATTGCACTCATCAGAGC-3'(bases 416 to 441 in the cDNA). By use
of Pfu DNA polymerase (Stratagene), DNA (
200 ng) was
amplified in a mixture of 20 mmol/L Tris-HCl (pH 8.2), 10
mmol/L KCl, 6 mmol/L
(NH4)2SO4,
2 mmol/L MgCl2, 0.1% Triton X-100, 250 ng
BSA, 0.125 U Pfu, 200 nmol/L dNTPs, 5 µCi
[32P]dCTP, and 0.4 µmol/L
oligonucleotide primers. Amplification conditions were
95°C for 5 minutes, 50°C for 3 minutes, 72°C for 2 minutes, 35
cycles of 95°C for 30 seconds, 50°C for 1 minute, 72°C for 2
minutes, and then 72°C for 8 minutes. Each product was digested
with AluI for 2 hours and mixed with formamide loading dye
(10 mmol/L NaOH, 95% formamide, 0.05% bromphenol blue, 0.05%
xylene xyanol). They were incubated at 90°C for 3 minutes, cooled on
ice, then electrophoresed in prewarmed 6% acrylamide/8
mol/L urea denaturing gels at 80 W for 3 to 4 hours. Dried gels were
exposed to Kodak Biomax MS film for 2 to 15 hours to visualize the
blunt-ended DNA fragments. Control DNA from the human colon carcinoma
cell line HCT 116, containing an A9
microsatellite region,6 allowed the calibration of
fragment sizes.
Automated fluorescent dideoxy cycle sequencing (ABI Prism 100, Perkin-Elmer) using undigested gel-purified PCR products from each sample was carried out to verify the PCR-SLP analysis.
Cell Culture
Human SMCs from internal mammary arteries were cultured as
previously described.17 The effects of 1 to 24 hours of
exposure to TGF-ß1 (1 to 10 ng/mL) on
intracellular TGF-ß1 localization in
serum-deprived SMCs was examined by immunohistochemistry (see above)
after the cells were washed, briefly fixed in 4% formalin, and
incubated in 0.3% Triton X-100. The human colon carcinoma cell line
HCT 116 (Center for Applied Microbiology and Research, Salisbury, UK)
was cultured in McCoy's 5A medium with 10% FCS.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Clinical Status
The clinical status and causes of death are listed in the Table
. No subject died of any serious infectious diseases, and
the entire aorta of each subject was examined grossly for
atherosclerotic lesions. Ten of the 21 aortas possessed atherosclerotic
lesions, mostly in vessels of subjects 
50 years old (Table),
classified either as fatty streaks, fibrofatty lesions, or advanced
fibrous-plaque lesions, according to published
criteria.18 19
TGF-ß and Its Receptors in Nonatherosclerotic Aortas
Nonatherosclerotic aortas were restricted primarily to individuals
<50 years old (Table). There were few, if any,
TGF-ß1 or TGF-ß3
peptides within the aortic media (Figure 1A and 1B). However, significant
concentrations of TGF-ß1 were always
present in the intima, and its diffuse distribution suggested an
extracellular localization. En face sections of the intima confirmed
its extracellular localization and also demonstrated an intracellular
localization; TGF-ß1 was present in 50%
of the intimal stellate-shaped SMC population, and in 80% of these,
it was associated more with nuclei than with cytoplasm (Figure 1C). TGF-ß3 was present in smaller
amounts (Figure 1B), again only in the intima, and mostly
localized intracellularly, in the cytoplasm of the stellate-shaped SMCs
(Figure 1B and 1D).
|
The type I (ALK-5) and type II (TßR-II) receptors were also
differentially expressed between the intima and media. ALK-5 was not
detectable in the media and only very weakly expressed by <50% of the
intimal cells (Figure 1E). In contrast, there were high
concentrations of TßR-II, localized to most cells in the media and
intima (Figure 1F).
TGF-ß and Receptors in Fatty Streaks/Fibrofatty Lesions
Fatty streaks/fibrofatty lesions were defined by oil red O
staining of extracellular and intracellular lipids (not
shown),20 and macrophages differentiated from
smooth muscle expressing TGF-ß on the basis of CD-68 differential
expression (Figure 2). Fatty
streaks/fibrofatty lesions contained high concentrations of
TGF-ß1 and TGF-ß3
(Figure 3A and 3B). Within these lesions,
many macrophages and SMCs contained lipid droplets, seen as
vacuoles in the en face sections (Figure 3C and 3D). Most cell
types contained high concentrations of TGF-ß1
and TGF-ß3; high extracellular concentrations
of TGF-ß1 were also present (Figure 3A and 3C). The cell types expressing
TGF-ß1 included macrophage-derived
foam cells and activated macrophages with an
"amoeboid-like" appearance; T lymphocytes were also present,
identified by their small, rounded appearance, large nuclei (Figure 3C), and CD-3 antigen expression (not shown). These cell types
contained only cytoplasmic TGF-ß1. However,
SMCs, including the lipid-laden SMCs, contained cytoplasmic and
nucleus-associated TGF-ß1 (Figure 3C);
this unexpected distribution was confirmed with a second
TGF-ß1–specific antibody from chickens (not
shown). An identical intracellular distribution was observed in SMC
cultures, in 62% of the quiescent, serum-deprived cells. Incubation of
the SMCs with 10 ng/mL TGF-ß1 for 1 or 25 hours
did not alter the proportion of cells exhibiting this distribution
pattern, suggesting that it was not due to uptake of extracellular
TGF-ß1. Here, <10% of the SMCs possessed
TGF-ß1 exclusively in their cytoplasm.
|
|
Serial en face sections indicated that all cells within the fatty
streaks/fibrofatty lesions that expressed
TGF-ß1 also expressed
TGF-ß3 (see Figure 3C and 3D). These
included the amoeboid-like and lipid-laden macrophages and T
lymphocytes (not shown).
The cells within these lesions also expressed high concentrations of
ALK-5 and TßR-II (Figure 3E and 3F). TßR-II expression was
much greater than in unaffected intima or the adjacent media (compare
Figures 1F and 3F). ALK-5 expression was also high and
colocalized with TßR-II (compare Figures 1E and 3E),
suggesting a highly activated TGF-ß system in these
lesions.
TGF-ß System and Fibrous Plaques
Fibrous plaques, characterized by a fibrous cap overlying a
hypocellular or largely acellular core, possessed significant amounts
of TGF-ß1 (Figure 4A); TGF-ß3
immunoreactivity varied widely, from low to undetectable levels (Figure 4B). Here, the majority of cells containing
TGF-ß1 and TGF-ß3
appeared to be enlarged modified SMCs, surrounded by dense layers of
connective tissue matrix in which their distribution was diffuse, with
no apparent structural organizational pattern (Figure 4C and 4D). Occasional lymphocytes also contained
TGF-ß1 and TGF-ß3
(Figure 4C and 4D). ALK-5 and TßR-II immunoreactivities were
very low in fibrous plaque and the associated media (Figure 4E and 4F).
|
Analysis of TßR-II Microsatellite Region in Aortic
Atherosclerotic Lesions
Although we used an antibody that detects the full-length type II
TGF-ß receptor (TßR-II), the possibility exists of truncated
receptors in the lesions, arising through error-prone replication
within a microsatellite region of 10 adenine residues
(A10) in the third exon of its gene; such
error-prone replication has been reported in SMCs of complex
coronary and carotid artery lesions.6 Accordingly,
we investigated whether such deletions were also a feature of intimal
cells in less severe aortic lesions. In all instances—unaffected
intima, fatty streak, fibrous plaque, lipid fibrous plaque, and complex
fibrous plaque—the normal, nonmutated A10
sequence appeared to be maintained by the cells, as demonstrated by
PCR-SLP analysis and cycle-sequencing of amplified products
(Figures 5 and 6). DNA from HCT 116 cells, containing a
homozygous 1A deletion within the A10
microsatellite, was used as a control; PCR products from these
cells exhibited the expected A9 pattern (Figures 5 and 6).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Although animal models of atherosclerosis have suggested that TGF-ß1 is protective, by preventing intima development,4 21 22 our findings indicate that in human aorta, TGF-ß is unlikely to be similarly protective. In addition, expansion of cell populations with adenine deletions in the A10 microsatellite region of the TßR-II gene does not appear to be necessary for aortic lesion progression. Rather, the restricted coexpression of TGF-ß and its receptors, TßR-II and ALK-5, provides strong, circumstantial evidence for TGF-ß acting predominantly within fatty streaks/fibrofatty lesions. Because TGF-ß is a potent stimulator of proteoglycan biosynthesis in human SMCs,7 its presence in fatty lesions is likely to contribute to the synthesis of lipoprotein-trapping proteoglycans, particularly biglycan.8 Proteoglycan aggregation can lead to reductions in the transmural movement of lipoproteins, their accumulation in the vessel wall,8 and their subsequent chemical modification.1 Also, TGF-ß can stimulate uPA secretion by macrophages,10 a key factor required for the generation of plasmin and activation of proteolytic matrix degradative enzymes.10 11 These properties of TGF-ß, together with its ability to inhibit SMC proliferation, suggest that it is a proatherogenic cytokine.
The evidence that TGF-ß1 normally present in arteries reduces their susceptibility to develop atherosclerotic lesions has been derived mainly from studies that used mice expressing the human apolipoprotein (a) gene.4 In these animals, high concentrations of apolipoprotein (a) in the vessel wall prevent the accumulation of bioactive TGF-ß1 necessary to inhibit SMC proliferation, permitting aortic intima and intimal fatty lesion development.4 21 The ability of tamoxifen to elevate aortic TGF-ß1 and inhibit lesion formation in apolipoprotein E knockout mice is also consistent with these findings.22 In humans, supporting evidence is lacking, although plasma concentrations of TGF-ß1 are greatly reduced in subjects with advanced atherosclerosis.23 Because we could not detect TGF-ß1 or TGF-ß3 in the media of nonatherosclerotic aortas, it is unlikely that TGF-ß1 is responsible for attenuating early intima development by inhibiting medial SMC proliferation; even in the intima, where both TGF-ß isoforms were present, the restricted expression of ALK-5 would significantly limit their potential effects.15 The effects of TGF-ßs are initiated by their binding to TßR-II, followed by recruitment and transphosphorylation of ALK-5.24 In the nonatherosclerotic aortas, TßR-II was present in the media and the intima, but ALK-5 was only very weakly expressed in intima. A similar pattern of TßR-II expression, with no concomitant ALK-5 expression, occurs in SMCs of the developing kidney.25 Also, the TGF-ß system is only marginally active in rat carotid arteries, and only after vessel injury are there large elevations in TGF-ß1, TGF-ß3, ALK-5, and TßR-II and effects attributable to TGF-ß.15 The temporal coexpression of TGF-ß and its signaling receptors in atherosclerotic aortas also suggests that the TGF-ß system is active in fatty streaks/fibrofatty lesions. The increases in TGF-ß1 were due, in part, to increased expression by SMCs, including lipid-laden SMCs, which contain high concentrations of cytoplasmic and nucleus-associated TGF-ß1. We did not determine whether the nuclear localization represented intranuclear accumulation. However, it was also observed in cultured SMCs, and because it was unaffected by exogenous TGF-ß1, it is unlikely to be due to uptake of TGF-ß1 by the SMCs. A similar intracellular distribution of TGF-ß1 has been reported in cardiomyocytes undergoing hypertrophy.26 This intracellular distribution was not observed in monocytes/macrophages or macrophage-derived foam cells. Our findings that macrophages within the fatty/fibrofatty lesions express high concentrations of TGF-ß1 suggest that this cytokine may also be influencing their actions, via local autocrine and paracrine mechanisms. Activated macrophages produce bioactive TGF-ß1,27 and this, in turn, can further activate their TGF-ß system by inducing ALK-5 and TßR-II.28 Analogous mechanisms could elevate ALK-5 and TßR-II expression in SMCs.29 Thus, the marked elevations in ALK-5 and TßR-II concentrations in fatty/fibrofatty lesions would be expected to be attributable to increases in receptors on SMCs and macrophages. An active TGF-ß system in SMCs could promote lipid accumulation through increases in proteoglycan biosynthesis and deposition in the fatty intima.8 Proteoglycans are closely associated with SMCs and TGF-ß1 in experimental atherosclerosis,30 and in human lesions there are close associations of proteoglycans with lipoproteins.8 Macrophages exposed to TGF-ß can also remodel the extracellular matrix and weaken fibrous plaques by secreting uPA.10 These effects of TGF-ß on SMCs and macrophages are likely to be more important for the development and progression of aortic atherosclerotic lesions than expansion of a clonal SMC population resistant to its proliferative inhibitory effects through TßR-II genomic instability.6 We failed to detect adenine deletions in the replication error–prone A10 microsatellite region of the TßR-II gene in cells in the various lesions, suggesting that mutation within this specific sequence of the TßR-II gene in SMCs is not essential for the development or progression of aortic atherosclerotic lesions.
In summary, our findings suggest that the TGF-ß system is most active in fatty atherosclerotic lesions of the human aorta. TGF-ß has the properties to be a proatherogenic cytokine, which can promote the retention of lipoproteins, participate in activating proteolytic systems of macrophages, and also limit SMC proliferation in fatty lesions.
Received October 13, 1998; revision received March 10, 1999; accepted March 26, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–561.
2. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793–795.[Medline] [Order article via Infotrieve]
3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
4. Grainger DJ, Kemp PR, Liu AC, Lawn RM, Metcalfe JC. Activation of transforming growth factor-ß is inhibited in transgenic apolipoprotein(a) mice. Nature. 1994;370:460–462.[Medline] [Order article via Infotrieve]
5. Kirschenlohr HL, Metcalfe JC, Weissberg PL, Grainger DJ. Proliferation of human aortic vascular smooth muscle cells in culture is modulated by active TGFß. Cardiovasc Res. 1995;29:848–855.[Medline] [Order article via Infotrieve]
6. McCaffrey TA, Du B, Consigli S, Szabo P, Bray PJ, Hartner L, Weksler BB, Sanborn TA, Bergman G, Bush HL Jr. Genomic instability in the type II TGF-ß1 receptor gene in atherosclerotic and restenotic vascular cells. J Clin Invest. 1997;100:2182–2188.[Medline] [Order article via Infotrieve]
7.
Chen J-K, Hoshi H, McKeehan WL. Transforming
growth factor type ß specifically stimulates synthesis of
proteoglycan in human adult arterial smooth muscle cells.
Proc Natl Acad Sci U S A. 1987;84:5287–5291.
8.
O'Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M,
Hudkins K, Wight TN, Chait A. Comparison of apolipoprotein and
proteoglycan deposits in human coronary atherosclerotic
plaques: colocalization of biglycan with apolipoproteins.
Circulation. 1998;98:519–527.
9. Leonarduzzi G, Scavazza A, Biasi F, Chiarpotto E, Camandola S, Vogl S, Dargel R, Poli G. The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor ß1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J. 1997;11:851–857.[Abstract]
10. Falcone DJ, McCaffrey TA, Mathew J, McAdam K, Borth W. THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-ß1 via increased expression of urokinase and the urokinase receptor. J Cell Physiol. 1995;164:334–343.[Medline] [Order article via Infotrieve]
11. Farina AR, Coppa A, Tiberio A, Tacconelli A, Turco A, Colletta G, Gulino A, Mackay AR. Transforming growth factor-ß1 enhances the invasiveness of human MDA-MB-231 breast cancer cells by up-regulating urokinase activity. Int J Cancer. 1998;75:721–730.[Medline] [Order article via Infotrieve]
12. Libby P, Clinton SK. The role of macrophages in atherogenesis. Curr Opin Lipidol. 1993;4:355–363.
13. Khanna A, Kapur S, Sharma V, Li B, Suthanthiran M. In vivo hyperexpression of transforming growth factor-ß1 in mice: stimulation by cyclosporine. Transplantation. 1997;63:1037–1039.[Medline] [Order article via Infotrieve]
14.
Border WA, Noble NA. Transforming growth factor
ß in tissue fibrosis. N Engl J Med. 1994;331:1286–1292.
15.
Ward MR, Agrotis A, Kanellakis P, Dilley R, Jennings G,
Bobik A. Inhibition of protein tyrosine kinase attenuates increases in
expression of transforming growth factor-ß isoforms and their
receptors following arterial injury. Arterioscler
Thromb Vasc Biol. 1997;17:2461–2470.
16. Krushinsky AV, Nestaiko GV. Morphology of smooth muscle cells from normal and atherosclerotic human aorta. Soc Med Rev A Cardiol. 1987;1:35–73.
17.
Neylon CB, Little PJ, Cragoe EJ Jr, Bobik A.
Intracellular pH in human arterial smooth muscle:
regulation by Na+/H+
exchange and a novel 5-(N-ethyl-N-isopropyl)
amiloride-sensitive Na+ - and
HCO3- -dependent mechanism.
Circ Res. 1990;67:814–825.
18.
Stary HC, Chandler AB, Glagov S, Guyton JR,
Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler
RW. A definition of initial, fatty streak, and intermediate lesions of
atherosclerosis: a report from the committee on
vascular lesions of the Council on
Arteriosclerosis, American Heart Association.
Circulation. 1994;89:2462–2478.
19.
Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S,
Insull W Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A
definition of advanced types of atherosclerotic lesions and a
histological classification of
atherosclerosis: a report from the committee on
vascular lesions of the Council on
Arteriosclerosis, American Heart Association.
Arterioscler Thromb Vasc Biol. 1995;15:1512–1531.
20.
Chiu DS, Oram JF, LeBoeuf RC, Alpers CE, O'Brien KD.
High-density lipoprotein-binding protein (HBP)/vigilin is expressed in
human atherosclerotic lesions and colocalizes with apolipoprotein E.
Arteriosclerosis Thromb Vasc Biol. 1997;17:2350–2358.
21. Grainger DJ, Metcalfe JC. Transforming growth factor-beta: the key to understanding lipoprotein (a)? Curr Opin Lipidol. 1995;6:81–85.[Medline] [Order article via Infotrieve]
22.
22. Reckless J, Metcalfe JC, Grainger DJ. Tamoxifen
decreases cholesterol sevenfold and abolishes lipid lesion
development in apolipoprotein E knockout mice. Circulation. 1997;95:1542–1548.
23. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-ß is severely depressed in advanced atherosclerosis. Nat Med. 1995;1:74–79.[Medline] [Order article via Infotrieve]
24.
Cárcamo J, Weis FMB, Ventura F, Wieser R, Wrana
JL, Attisano L, Massagué J. Type I receptors specify
growth-inhibitory and transcriptional responses to
transforming growth factor ß and activin. Mol Cell
Biol. 1994;14:3810–3821.
25. Liu A, Ballermann BJ. TGF-ß type II receptor in rat renal vascular development: localization to juxtaglomerular cells. Kidney Int. 1998;53:716–725.[Medline] [Order article via Infotrieve]
26. Li J-M, Brooks G. Differential protein expression and subcellular distribution of TGF ß1, ß2 and ß3 in cardiomyocytes during pressure overload-induced hypertrophy. J Mol Cell Cardiol. 1997;29:2213–2224.[Medline] [Order article via Infotrieve]
27.
Porreca E, Di Febbo C, Mincione G, Reale M, Baccante G,
Guglielmi MD, Cuccurullo F, Colletta G. Increased transforming growth
factor-ß production and gene expression by
peripheral blood monocytes of hypertensive patients.
Hypertension. 1997;30:134–139.
28.
Lastres P, Letamendía A, Zhang H, Rius C,
Almendro N, Raab U, López LA, Langa C, Fabra A, Letarte M,
Bernabéu C. Endoglin modulates cellular responses to
TGF-ß1. J Cell Biol. 1996;133:1109–1121.
29. Ward MR, Agrotis A, Jennings G, Bobik A. Vascular types I and II transforming growth factor-beta receptor expression: differential dependency on tyrosine kinases during induction by TGF-ß. FEBS Lett. 1998;422:197–200.[Medline] [Order article via Infotrieve]
30. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and proximity of platelet-derived growth factor and transforming growth factor-ß. Am J Pathol. 1998;152:533–546.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  P. T.G. Bot, I. E. Hoefer, J. P.G. Sluijter, P. van Vliet, A. M. Smits, F. Lebrin, F. Moll, J.-P. de Vries, P. Doevendans, J. J. Piek, et al. Increased Expression of the Transforming Growth Factor-{beta} Signaling Pathway, Endoglin, and Early Growth Response-1 in Stable Plaques Stroke, February 1, 2009; 40(2): 439 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nakashima, T. N. Wight, and K. Sueishi Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans Cardiovasc Res, July 1, 2008; 79(1): 14 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Dadlani, M. L. Ballinger, N. Osman, R. Getachew, and P. J. Little Smad and p38 MAP Kinase-mediated Signaling of Proteoglycan Synthesis in Vascular Smooth Muscle J. Biol. Chem., March 21, 2008; 283(12): 7844 - 7852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.J. Grande-Allen, N. Osman, M.L. Ballinger, H. Dadlani, S. Marasco, and P.J. Little Glycosaminoglycan synthesis and structure as targets for the prevention of calcific aortic valve disease Cardiovasc Res, October 1, 2007; 76(1): 19 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Grainger TGF-{beta} and atherosclerosis in man Cardiovasc Res, May 1, 2007; 74(2): 213 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yao, A. F. Zebboudj, A. Torres, E. Shao, and K. Bostrom Activin-like kinase receptor 1 (ALK1) in atherosclerotic lesions and vascular mesenchymal cells Cardiovasc Res, May 1, 2007; 74(2): 279 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bobik Transforming Growth Factor-{beta}s and Vascular Disorders Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1712 - 1720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Khurana, M. Simons, J. F. Martin, and I. C. Zachary Role of Angiogenesis in Cardiovascular Disease: A Critical Appraisal Circulation, September 20, 2005; 112(12): 1813 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. D. Norata, E. Callegari, M. Marchesi, G. Chiesa, P. Eriksson, and A. L. Catapano High-Density Lipoproteins Induce Transforming Growth Factor-{beta}2 Expression in Endothelial Cells Circulation, May 31, 2005; 111(21): 2805 - 2811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. Berg, L. J. Myers, M. A. Richardson, G. Sandusky, and B. W. Grinnell Smad6s Regulates Plasminogen Activator Inhibitor-1 through a Protein Kinase C-{beta}-dependent Up-regulation of Transforming Growth Factor-{beta} J. Biol. Chem., April 15, 2005; 280(15): 14943 - 14947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cipollone, M. Fazia, G. Mincione, A. Iezzi, B. Pini, C. Cuccurullo, S. Ucchino, F. Spigonardo, M. Di Nisio, F. Cuccurullo, et al. Increased Expression of Transforming Growth Factor-{beta}1 as a Stabilizing Factor in Human Atherosclerotic Plaques Stroke, October 1, 2004; 35(10): 2253 - 2257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kalinina, A. Agrotis, Y. Antropova, O. Ilyinskaya, V. Smirnov, E. Tararak, and A. Bobik Smad Expression in Human Atherosclerotic Lesions: Evidence for Impaired TGF-{beta}/Smad Signaling in Smooth Muscle Cells of Fibrofatty Lesions Arterioscler Thromb Vasc Biol, August 1, 2004; 24(8): 1391 - 1396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lutgens, R.-J. van Suylen, B. C. Faber, M. J. Gijbels, P. M. Eurlings, A.-P. Bijnens, K. B. Cleutjens, S. Heeneman, and M. J.A.P. Daemen Atherosclerotic Plaque Rupture: Local or Systemic Process? Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2123 - 2130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gojova, V. Brun, B. Esposito, F. Cottrez, P. Gourdy, P. Ardouin, A. Tedgui, Z. Mallat, and H. Groux Specific abrogation of transforming growth factor-{beta} signaling in T cells alters atherosclerotic lesion size and composition in mice Blood, December 1, 2003; 102(12): 4052 - 4058. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Li, L. M. Fan, A. Shah, and G. Brooks Targeting {alpha}v{beta}3 and {alpha}5{beta}1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-{beta}1 Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H1123 - H1131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-H. Oak, M. Chataigneau, T. Keravis, T. Chataigneau, A. Beretz, R. Andriantsitohaina, J.-C. Stoclet, S.-J. Chang, and V. B. Schini-Kerth Red Wine Polyphenolic Compounds Inhibit Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells by Preventing the Activation of the p38 Mitogen-Activated Protein Kinase Pathway Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1001 - 1007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Tyson, J. L. Reynolds, R. McNair, Q. Zhang, P. L. Weissberg, and C. M. Shanahan Osteo/Chondrocytic Transcription Factors and Their Target Genes Exhibit Distinct Patterns of Expression in Human Arterial Calcification Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 489 - 494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sandusky, D. T. Berg, M. A. Richardson, L. Myers, and B. W. Grinnell Modulation of Thrombomodulin-dependent Activation of Human Protein C through Differential Expression of Endothelial Smads J. Biol. Chem., December 13, 2002; 277(51): 49815 - 49819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kalinina, A. Agrotis, E. Tararak, Y. Antropova, P. Kanellakis, O. Ilyinskaya, M. T. Quinn, V. Smirnov, and A. Bobik Cytochrome b558-Dependent NAD(P)H Oxidase-Phox Units in Smooth Muscle and Macrophages of Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2037 - 2043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Kintscher, C. Lyon, S. Wakino, D. Bruemmer, X. Feng, S. Goetze, K. Graf, A. Moustakas, B. Staels, E. Fleck, et al. PPAR{alpha} Inhibits TGF-{beta}-Induced {beta}5 Integrin Transcription in Vascular Smooth Muscle Cells by Interacting With Smad4 Circ. Res., November 29, 2002; 91 (11): e35 - e44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Forrester Prevention of Plaque Rupture: A New Paradigm of Therapy Ann Intern Med, November 19, 2002; 137(10): 823 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Porreca, C. Di Febbo, G. Baccante, M. Di Nisio, and F. Cuccurullo Increased transforming growth factor-beta1 circulating levels and production in human monocytes after 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibition with pravastatin J. Am. Coll. Cardiol., June 5, 2002; 39(11): 1752 - 1757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Ward, A. Agrotis, P. Kanellakis, J. Hall, G. Jennings, and A. Bobik Tranilast Prevents Activation of Transforming Growth Factor-{beta} System, Leukocyte Accumulation, and Neointimal Growth in Porcine Coronary Arteries After Stenting Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 940 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lutgens, M. Gijbels, M. Smook, P. Heeringa, P. Gotwals, V. E. Koteliansky, and M. J.A.P. Daemen Transforming Growth Factor-{beta} Mediates Balance Between Inflammation and Fibrosis During Plaque Progression Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 975 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Little, L. Tannock, K. L. Olin, A. Chait, and T. N. Wight Proteoglycans Synthesized by Arterial Smooth Muscle Cells in the Presence of Transforming Growth Factor-{beta}1 Exhibit Increased Binding to LDLs Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 55 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Taylor, A. Bobik, M. C. Berndt, D. Ramsay, and G. Jennings Experimental Rupture of Atherosclerotic Lesions Increases Distal Vascular Resistance: A Limiting Factor to the Success of Infarct Angioplasty Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 153 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, N. Shiota, M. J. Leskinen, K. A. Lindstedt, and P. T. Kovanen Mast Cell Chymase Inhibits Smooth Muscle Cell Growth and Collagen Expression In Vitro: Transforming Growth Factor-{beta}1-Dependent and -Independent Effects Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1928 - 1933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Argmann, C. H. Van Den Diepstraten, C. G. Sawyez, J. Y. Edwards, R. A. Hegele, B. M. Wolfe, and M. W. Huff Transforming Growth Factor-{beta}1 Inhibits Macrophage Cholesteryl Ester Accumulation Induced by Native and Oxidized VLDL Remnants Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 2011 - 2018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lutgens and M. J.A.P. Daemen Transforming Growth Factor-{beta}: A Local or Systemic Mediator of Plaque Stability? Circ. Res., November 9, 2001; 89(10): 853 - 855. [Full Text] [PDF]
|
|
|
|
 |
|
 C. G. Panousis, G. Evans, and S. H. Zuckerman TGF-{beta} increases cholesterol efflux and ABC-1 expression in macrophage-derived foam cells: opposing the effects of IFN-{{gamma}} J. Lipid Res., May 1, 2001; 42(5): 856 - 863. [Abstract] [Full Text]
|
|
|
|
|
|
K. J. Clark, N. R. Cary, A. A. Grace, and J. C. Metcalfe Microsatellite Mutation of Type II Transforming Growth Factor-{beta} Receptor Is Rare in Atherosclerotic Plaques Arterioscler Thromb Vasc Biol, April 1, 2001; 21(4): 555 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Mohler III, F. Gannon, C. Reynolds, R. Zimmerman, M. G. Keane, and F. S. Kaplan Bone Formation and Inflammation in Cardiac Valves Circulation, March 20, 2001; 103(11): 1522 - 1528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Dubois, F. Blanchette, M.-H. Laprise, R. Leduc, F. Grondin, and N. G. Seidah Evidence that Furin Is an Authentic Transforming Growth Factor-{beta}1-Converting Enzyme Am. J. Pathol., January 1, 2001; 158(1): 305 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Ma, M. Labinaz, J. Goldstein, H. Miller, W. J. Keon, M. Letarte, and E. O'Brien Endoglin Is Overexpressed After Arterial Injury and Is Required for Transforming Growth Factor-{beta}-Induced Inhibition of Smooth Muscle Cell Migration Arterioscler Thromb Vasc Biol, December 1, 2000; 20(12): 2546 - 2552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Neuville, M.-L. Bochaton-Piallat, and G. Gabbiani Retinoids and Arterial Smooth Muscle Cells Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1882 - 1888. [Full Text] [PDF]
|
|
|
|
 |
|
 E. Lutgens, K. B. J. M. Cleutjens, S. Heeneman, V. E. Koteliansky, L. C. Burkly, and M. J. A. P. Daemen Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype PNAS, June 20, 2000; 97(13): 7464 - 7469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Taylor and L. M. Khachigian Induction of Platelet-derived Growth Factor B-chain Expression by Transforming Growth Factor-beta Involves Transactivation by Smads J. Biol. Chem., May 26, 2000; 275(22): 16709 - 16716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Du, C. Fu, K. C. Kent, H. Bush Jr., A. H. Schulick, K. Kreiger, T. Collins, and T. A. McCaffrey Elevated Egr-1 in Human Atherosclerotic Cells Transcriptionally Represses the Transforming Growth Factor-beta Type II Receptor J. Biol. Chem., December 8, 2000; 275(50): 39039 - 39047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Mallat, A. Gojova, C. Marchiol-Fournigault, B. Esposito, C. Kamate, R. Merval, D. Fradelizi, and A. Tedgui Inhibition of Transforming Growth Factor-{beta} Signaling Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Mice Circ. Res., November 9, 2001; 89(10): 930 - 934. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||