Anti-HMGB1 antibody reduces weight gain in mice fed a high-fat diet.

Insulin resistance in obesity is believed to be propagated by adipose tissue and liver inflammation. HMGB1 is a multifunctional protein that is pro-inflammatory when released from cells. It has been previously demonstrated that anti-HMGB1 antibody reduces atherosclerotic lesion pro-inflammatory cells and progression of atherosclerosis in a mouse model. To test the potential beneficial role of blocking HMGB1 in adipose tissue and liver inflammation in mice fed an obesogenic diet, we administered anti-HMGB1 antibody to C57Bl/6 mice fed a high (60%)-fat diet. The mice were treated with weekly injections of an anti-HMGB1 antibody or anti-KLH antibody (isotype control) for 16 weeks. Mice that received the anti-HMGB1 antibody gained less weight than the control-treated animals. Anti-HMGB1 treatment also reduced hepatic expression of TNF-alpha and MCP-1, molecules that promote inflammation. However, adipose tissue inflammation, as measured by gene expression analyses and immunohistochemistry, did not differ between the two groups. There also were no differences in glucose or insulin tolerance between the two groups. When feeding mice a high-fat diet, these data suggest that HMGB1 may have a crucial role in weight gain and liver inflammation.

Deficiency of haematopoietic-cell-derived IL-10 does not exacerbate high-fat-diet-induced inflammation or insulin resistance in mice.

AIMS/HYPOTHESIS: Recent work has identified the important roles of M1 pro-inflammatory and M2 anti-inflammatory macrophages in the regulation of insulin sensitivity. Specifically, increased numbers of M2 macrophages and a decrease in M1 macrophages within the adipose tissue are associated with a state of enhanced insulin sensitivity. IL-10 is an anti-inflammatory cytokine and is a critical effector molecule of M2 macrophages. METHODS: In the present study, we examined the contribution of haematopoietic-cell-derived IL-10 to the development of obesity-induced inflammation and insulin resistance. We hypothesised that haematopoietic-cell-restricted deletion of IL-10 would exacerbate obesity-induced inflammation and insulin resistance. Lethally irradiated wild-type recipient mice receiving bone marrow from either wild-type or Il10-knockout mice were placed on either a chow or a high-fat diet for a period of 12 weeks and assessed for alterations in body composition, tissue inflammation and glucose and insulin tolerance. RESULTS: Contrary to our hypothesis, neither inflammation, as measured by the activation of pro-inflammatory stress kinases and gene expression of several pro-inflammatory cytokines in the adipose tissue and liver, nor diet-induced obesity and insulin resistance were exacerbated by the deletion of haematopoietic-cell-derived IL-10. Interestingly, however, Il10 mRNA expression and IL-10 protein production in liver and/or adipose tissue were markedly elevated in Il10-knockout bone-marrow-transplanted mice relative to wild-type bone marrow-transplanted mice. CONCLUSIONS/INTERPRETATION: These data show that deletion of IL-10 from the haematopoietic system does not potentiate high-fat diet-induced inflammation or insulin resistance.

[Participation of the DNA-binding cytokine amphoterine in triggering the processes of tissue reparation].

Author's studies and literature references indicate that the DNA-binding cytokine: amphoterine, being a non-histone component of chromatin, acts in extracellular milieu as cytokine regulating gene expression in target cells. The amphoterine effects are mediated by its interaction with the cell surface receptors including those of the final glucosiding product (RAGE), which leads to activation of the ERK and p38 MAP-kinases as well as NF-kappaB. Amphoterine prompts inflammatory response by means of activating synthesis of interleukins-1, -6 and -8 as well as tumour necrosis factors (TNF-alpha) and activates tissue regeneration by means of involvement of the precursor cells into the damage foci and induction of the cells' differentiation. These properties of amphoterine suggest that appearance of this protein in extracellular space signals of a tissue damaging.

Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines.

OBJECTIVE: Atherosclerosis is a chronic inflammatory response of the arterial wall to injury. High-mobility group box 1 (HMGB1) is a DNA-binding protein, which on release from cells exhibits potent inflammatory actions. We examined its expression in atherosclerotic lesions and regulation by cytokines. METHODS AND RESULTS: In atherosclerotic lesions, HMGB1 protein is expressed by endothelial cells, some intimal smooth muscle cells, and macrophages. As atherosclerosis develops and progresses from fatty streaks to fibrofatty lesion, the number of HMGB1-producing macrophages increases markedly. Studies using the THP-1 cell line indicated that HMGB1 mRNA expression could be markedly upregulated by inflammatory cytokines, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and also transforming growth factor (TGF)-beta. IFN-gamma, TNF-alpha, TWEAK, and TGF-beta induced an intracellular redistribution of HMGB1 and stimulated secretion by THP-1 cells and human blood monocytes. Inhibitors of MEK1/MEK2, protein kinase C, and PI-3/Akt, which inhibit lysosomal degranulation and mRNA translation, attenuated cytokine-induced HMGB1 secretion. CONCLUSIONS: Macrophage is the major cell type responsible for HMGB1 production in human atherosclerotic lesions. Inflammatory cytokines and TGF-beta increase HMGB1 expression and secretion by monocyte/macrophages. HMGB1 appears to be a common mediator of inflammation induced by inflammatory cytokines and is likely to contribute to lesion progression and chronic inflammation.

[Expression, regulation, and pathophysiological role of NAD(P)H oxidases in atherogenesis]. / ékspressiia, reguliatsiia i rol' NAD(P)H-zavisimykh oksidaz v aterogeneze.

Oxidative stress has been implicated in the development and progression of atherosclerotic lesions. Significant increase of reactive oxygen species production by vascular cells can lead to progression of atherosclerotic lesions and development of unstable plaques due to triggering the apoptosis of endothelial and smooth muscle cells, expression of matrix metalloproteases and inflammatory cytokines. Cytolysis NAD(P)H-dependent oxidases appeared to be involved in reactive oxygen species production in the vascular network. Understanding of functions and regulation of individual NAD(P)H oxidases in atherosclerotic lesions can facilitate the development of novel therapeutic strategy for treating atherosclerosis. This review summarizes current data regarding expression, regulation and pathophysiological significance of these enzymes during development and progression of human atherosclerotic lesions.

[The origin of neointimal cells in the rat carotid artery after balloon angioplasty]. / Proiskhozhdenie kletok neointimy, obrazovannoi v sonnykh arteriiakh krys, posle ballonnoi angioplastiki.

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.

Urokinase plasminogen activator augments cell proliferation and neointima formation in injured arteries via proteolytic mechanisms.

Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor (uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima. Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by tPA, greatly influences SMC proliferation and early neointima formation.

Plasmin-independent gelatinase B (matrix metalloproteinase-9) release by monocytes under the influence of urokinase.

It is shown that the release of matrix metalloproteinase-9 (gelatinase B) by THP-1 and U937 cells into conditioned media is increased under the action of recombinant single-chain urokinase. This effect is not accompanied by proteolytic activation of gelatinase B and is related to release of a pro-form of the enzyme. The action of urokinase on monocytes is time-dependent and becomes significant 12-24 h after the beginning of cell incubation. The dependence of the effect on the concentration of urokinase is characterized by half-maximum at about 20 nM and saturation at about 200 nM. The urokinase-induced gelatinase B release is not dependent on the action of plasmin because plasmin inhibitors aprotinin and alpha2-antiplasmin do not abolish this action. Additionally, tissue type plasminogen activator does not induce gelatinase B release by monocytes as observed under the action of urokinase. Nevertheless, the catalytic activity of urokinase participates in the development of the observed effect because it is significantly depressed by the natural urokinase inhibitor PAI-1. The effect of urokinase is completely abolished by actinomycin D and cycloheximide, indicating the participation of transcription and translation processes in its development.

Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor.

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF.

Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries.

BACKGROUND: Coronary artery angioplasty triggers healing that causes constrictive remodeling. Because collagen accumulation correlates with constrictive remodeling and aldosterone has been implicated in collagen accumulation, we examined how aldosterone and the mineralocorticoid receptor antagonists spironolactone and eplerenone affect remodeling and collagen in porcine coronary and iliac arteries after angioplasty. METHODS AND RESULTS: Twenty-four pigs were allocated into 4 treatment groups: oral eplerenone (100 mg/d), oral spironolactone (200 mg/d), subcutaneous aldosterone (400 microgram/d), or no treatment. Twenty-eight days after angioplasty of the coronary arteries, eplerenone increased total vessel area by 30% (P<0.05) and luminal area by nearly 60% (P<0.05) compared with the no-treatment group, without affecting neointima size. These effects were accompanied by a 65% reduction in neointimal and medial collagen density (both P<0.05). Spironolactone was less effective, and aldosterone tended to exert opposite effects on coronary artery structure after angioplasty. These effects were not observed in angioplastied iliac arteries. CONCLUSIONS: Eplerenone attenuates constrictive remodeling after coronary artery angioplasty by mechanisms involving reduction in collagen accumulation, which thus appears to be an important contributor to constrictive remodeling of angioplastied coronary arteries.

Effects of transforming growth factor-beta(1)on proliferation of smooth muscle cells in human aortic intima and human promonocytic leukemia THP-1 cells.

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.

Low blood flow after angioplasty augments mechanisms of restenosis: inward vessel remodeling, cell migration, and activity of genes regulating migration.

-The predominant cause of restenosis after angioplasty is now thought to be inward remodeling, but the mechanisms responsible are unknown. Remodeling in normal vessels is regulated by the endothelium in response to altered shear stress. Although the endothelium is often damaged by angioplasty, restenosis rates after angioplasty have been correlated with impaired coronary flow. Thus, we examined how increases or decreases in blood flow through balloon catheter-injured rat carotid arteries affect vessel morphometry (4, 10, and 28 days), cell migration (4 days), and levels of promigratory mRNAs (2 and 10 days). After 28 days, the luminal area in vessels with low blood flow was significantly less than in those with normal and high blood flow (0.17+/-0.01 [low] versus 0.24+/-0.06 [normal] versus 0.30+/-0.02 [high] mm(2), P:<0.01), predominantly because of accentuated inward remodeling (or reduced area within the external elastic lamina; 0.42+/-0.02 [low] versus 0.54+/-0.07 [normal] versus 0.53+/-0.04 [high] mm(2), P:<0.05). Low flow also enhanced smooth muscle cell migration 4 days after injury by 90% above normal and high flows (P:<0.01). Two days after injury, low flow significantly increased levels of mRNAs encoding promigratory peptides (integrin alpha(v)ss(3), transforming growth factor-ss(1), CD44v6, MDC9, urokinase plasminogen activator receptor, and ss-inducible gene h3); these changes persisted 10 days after injury and were localized to the neointima. Low blood flow may promote restenosis after angioplasty because of its adverse effect on vessel remodeling, and it is associated with the augmented expression of multiple genes central to cell migration and restenosis.

Urokinase plasminogen activator enhances neointima growth and reduces lumen size in injured carotid arteries.

OBJECTIVES: Increases in urokinase plasminogen activator (uPA) have been reported in tissues undergoing remodelling, but its effects on the vessel intima formation are not known. We investigated its effects on carotid artery intima, media and lumen size, as well as smooth muscle cell (SMC) proliferation and migration in vivo. DESIGN AND METHODS: Carotid arteries of rats were distended with an inflated balloon catheter and uPA, or uPA-neutralizing antibodies were applied perivascularly in pluronic gel; control rats received vehicle. Carotid artery structure, cell migration and proliferation were assessed after 4 days by quantitative morphometry and immunohistochemistry. RESULTS: Four days after increasing vessel uPA, the intima/media ratio was double compared to that in control rats (both P < 0.05). The size of the lumen reduced by 75%, compared to the vehicle-treated vessels (P < 0.05). The elevation in uPA also increased SMC numbers in the intima and media, compared to the vehicle-treated vessels (both P < 0.05). Antibody neutralizing endogenous uPA attenuated the growth responses in the distended arteries, reduced neointimal SMC numbers by approximately 50% and prevented much of the reduction in lumen size. CONCLUSIONS: Thus, local increases in uPA in distended, injured arteries augment SMC migratory and proliferative responses, leading to increases in the thickness of the carotid artery intima and media and a reduction in lumen size; effects at least partially attributable to its proteolytic properties.

Response to balloon injury is vascular bed specific: a consequence of de novo vessel structure?

Relative contributions of remodelling and neointimal hyperplasia to restenosis after coronary angioplasty have been inferred from studies using iliofemoral arteries, despite differences in structure/function and smooth muscle cell lineage. We compared the response to balloon overstretch injury of coronary arteries (C, n = 16) and similar sized branches of the iliac arteries (I, n = 18) using preinjury vessel diameter (P). inflated balloon size in vivo (B) and the manufacturer predicted inflated size (M) to examine arterial compliance, as well as resulting injury and morphology in perfusion fixed vessels. Despite similar degrees of oversizing (M/P) in the coronary and iliac arteries (C, 1.44 +/- 0.04; I, 1.51 +/- 0.02), the compliance to overstretch (B-P/M-P) was significantly greater in the coronary than the iliac arteries (C, 0.71 +/- 0.05; I, 0.51 +/- 0.03) (P <0.05) and was associated with a higher injury score (C, 1.64 +/- 0.31; I, 0.39 +/- 0.18 P < 0.05)--only 5/18 iliac vessels had rupture of the IEL compared with 13/16 in the coronary bed. In a subgroup of animals whose vessels (C:n = 7; I:n = 8) were perfusion fixed 28 days after injury, coronary arteries had greater intimal area (C:1.03 +/- 0.42; I:0.10 +/- 0.03 mm2, P < 0.05) but larger luminal area (C:1.61 +/- 0.71; 1:0.76 +/- 0.51, P < 0.05) due to greater area within EEL (C:3.38 +/- 0.49;1:] .49 +/- 0.54, P < 0.05) or less inward remodelling. The injuries resulting from similar strategies of balloon overstretch in the coronary and the iliac arteries are different and affect healing responses--iliac arteries remodel more while coronary arteries develop more intimal hyperplasia. These results indicate that caution is warranted when extrapolating results from the iliac to the coronary artery when investigating restenosis after angioplasty.

Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthase in hypertension.

Blood flow participates in vascular remodeling during development and growth by regulating cell apoptosis and proliferation. However, its significance in the development of vascular hypertrophy and vascular remodeling in hypertensive patients is not known. We investigated how changing blood flow through the common carotid artery (CA) of young adult rats rendered hypertensive via aortic coarctation affects CA hypertrophy and/or remodeling responses to hypertension. Blood flow was reduced by approximately 50% as a result of ligation of the external CA immediately after hypertension was induced, and the effects of that procedure were compared with those in similarly treated normotensive rats. Reducing blood flow in the hypertensive animals markedly augmented the development of CA hypertrophy over the ensuing 14 days by increasing the vessel wall cross-sectional area. In those animals, CA lumen size was unaltered by reducing blood flow, as was CA structure in normotensive animals. The greater hypertrophy in the hypertensive animals with reduced blood flow was associated with enhanced smooth muscle cell (SMC) proliferation 3 days after the hemodynamic changes were induced. There also appeared to be more extensive remodeling of the endothelium in the hypertensive animals with normal flow; this was indicated by the greater frequency of apoptotic endothelial cells at that time. This reduction in blood flow also attenuated endothelial cell nitric oxide synthase expression in hypertensive animals but not in normotensive animals. Severe reductions in blood flow ( approximately 90%) were required to reduce endothelial cell nitric oxide synthase in the normotensive animals. Increasing CA nitric oxide levels by perivascular application of S-nitroso-N-acetylpenicillamine (SNAP) to the CAs of hypertensive animals with reduced endothelial cell nitric oxide synthase attenuated the greater SMC proliferation. Thus, reduced blood flow in hypertensive animals promotes hypertrophy by enhancing SMC proliferation via mechanisms that reduce the inhibitory effects of nitric oxide on SMC proliferation.

Alternative splicing within the TGF-beta type I receptor gene (ALK-5) generates two major functional isoforms in vascular smooth muscle cells.

We have identified in rat vascular smooth muscle cells (SMCs) the simultaneous expression of two TGF-beta type I receptor (ALK-5) cDNAs, occurring as a consequence of alternate usage of AG splice acceptor motifs separated by 12 nucleotides located at an intron-exon junction. When translated the resultant full length proteins differ from each other only by the in-frame presence or absence of Gly-Pro-Phe-Ser residues adjacent to their transmembrane domain. Stable expression of these alternate ALK-5 isoforms in ALK-5-deficient cells demonstrated that both were competent in signaling TGF-beta-induced growth inhibition and gene transcription, but with an apparently distinct potency. Our data suggest that alternate splicing within the ALK-5 gene is an important mechanism whereby SMCs may regulate their response to TGF-beta.

Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function.

Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions. Colocalization implicates TGF-beta in fibrofatty lesion development.

BACKGROUND: Some animal studies suggest that transforming growth factor-beta (TGF-beta) 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-beta isoforms and the TGF-beta receptors ALK-5 and TbetaR-II in aorta during the various stages of atherosclerotic lesion development. METHODS AND RESULTS: The spatial relationships between TGF-beta1, TGF-beta3, ALK-5, and TbetaR-II expression were compared in aortic segments from 21 subjects. Nonatherosclerotic intima contained predominantly TGF-beta1, low concentrations of TbetaR-II, and barely detectable amounts of ALK-5. In contrast, fatty streaks/fibrofatty lesions contained high concentrations of both TGF-beta 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 TbetaR-II. Despite fibrous plaques containing TGF-beta1, its receptors were at detection limits. We found no evidence for truncated TbetaR-II expression in either normal intima or the various atherosclerotic lesions. CONCLUSIONS: TGF-beta appears to be most active in lipid-rich aortic intimal lesions. The findings support the hypothesis that TGF-beta 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.

GC factor 2 represses platelet-derived growth factor A-chain gene transcription and is itself induced by arterial injury.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for a wide variety of cell types. The genes encoding PDGF A chain (PDGF-A) and PDGF B chain (PDGF-B) reside on separate chromosomes and are independently regulated at the level of transcription. Regulatory events underlying inducible PDGF-A expression have been the focus of much investigation. However, mechanisms that inhibit transcription of this gene are not well understood. In this study, we report the capacity of a newly cloned DNA binding factor, GC factor 2 (GCF2), to repress expression driven by the human PDGF-A promoter. 5' Deletion and transient cotransfection analysis in vascular endothelial cells revealed that GCF2 repression is mediated by a nucleotide region located in the proximal region of the PDGF-A promoter. Electrophoretic mobility shift assays demonstrate that GCF2 binds to this region in a specific and dose-dependent manner. Interestingly, the site bound by GCF2 overlaps those for specificity protein-1 (Sp1) and early growth response factor-1 (Egr-1), zinc finger transcription factors that direct basal and inducible expression of the PDGF-A gene. Gel shift experiments revealed that GCF2 competes with these factors for interaction with the PDGF-A promoter. Overexpression of GCF2 suppressed endogenous PDGF-A expression in vascular endothelial cells and smooth muscle cells. GCF2 was induced on mechanical injury of cells in culture as well as after balloon injury of the rat carotid artery wall. Time course studies revealed the sustained induction of GCF2 after injury while PDGF-A levels sharply returned to baseline. Smooth muscle cell proliferation was inhibited by GCF2, an effect reversed by the addition of exogenous PDGF-AA. These findings demonstrate negative regulation of PDGF-A expression by GCF2. This is the first report of the induction of an endogenous transcriptional repressor in the rat vessel wall.