Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin.

Addition of platelet-derived growth factor (PDGF) to growth-arrested cultured smooth muscle cells (SMC) induces the synthesis and secretion of thrombospondin (TS), a glycoprotein component of the SMC extracellular matrix in vitro. This induction occurs at PDGF concentrations that are suboptimal for a mitogenic response. In this study we examined the effect of TS on the proliferation of SMC, using a serum-free mitogenesis assay. Addition of either epidermal growth factor (EGF) or purified human platelet TS to quiescent rat vascular SMC did not substantially stimulate mitogenesis; the 30-hr nuclear labeling index increased from a mean of 7% in control cells to 20% for EGF-treated SMC and 17% for cells exposed to TS alone. However, TS and EGF acted synergistically to stimulate DNA synthesis by SMC, increasing the labeling index to 47%. The facilitative effect of TS on EGF-mediated mitogenesis was inhibited by heparin, a known inhibitor of SMC growth and migration that also blocks incorporation of TS into the SMC extracellular matrix. The effect was specific for EGF; TS did not augment the response of cells to insulin or insulin-like growth factor 1. These data establish a functional role for cell-derived TS and provide evidence for the presence of an autocrine, growth-supportive mechanism involving the extracellular matrix. In addition, our experiments support the existence of a novel, heparin-sensitive SMC mitogenic pathway and suggest a mechanism whereby heparin-like molecules may inhibit SMC proliferation.     

Thrombin- and factor Xa-induced DNA synthesis is mediated by transactivation of fibroblast growth factor receptor-1 in human vascular smooth muscle cells

Thrombin and factor Xa (FXa) are agonists for G protein-coupled receptors (GPRCs) and may contribute to vascular lesion formation by stimulating proliferation of vascular smooth muscle cells (SMCs). Mitogenic signaling of GPCRs requires transactivation of receptor tyrosine kinases (RTKs). In rat SMCs, thrombin transactivates the epidermal growth factor receptor (EGFR) via a pathway that involves heparin-binding EGF-like growth factor (HB-EGF) as ligand for EGFR. The purpose of this study was to investigate in human SMCs the role of receptor transactivation in the mitogenic response to thrombin and FXa. Thrombin (10 nmol/L) and FXa (100 nmol/L) cause a 3.3- and 2.6-fold increase in DNA synthesis, respectively. In human SMCs, neither thrombin nor FXa causes EGFR phosphorylation, and blockade of EGFR kinase does not inhibit DNA synthesis. However, DNA synthesis and phosphorylation of fibroblast growth factor receptor-1 (FGFR-1) induced by thrombin or FXa are inhibited by antibodies neutralizing basic fibroblast growth factor (bFGF) or by heparin. Hirudin inhibits thrombin-, but not FXa-induced mitogenesis, indicating that FXa acts independently of thrombin. We further demonstrate by ELISA that upon thrombin and FXa stimulation, bFGF is released and binds to the extracellular matrix. Our data suggest that in human vascular SMCs, both thrombin and FXa rapidly release bFGF into the pericellular matrix. This is followed by transactivation of the FGFR-1 and increased proliferation. Heparin may inhibit the mitogenic effects of thrombin and FXa in human SMCs by preventing bFGF binding to FGFR-1.     

Cellular effects of factor Xa on vascular smooth muscle cells--inhibition by heparins?

In addition to its key function as a clotting enzyme, factor Xa (FXa) also elicits cellular effects. In cultured human venous smooth muscle cells (SMCs), FXa induced a mitogenic response that was independent of thrombin and platelet-derived growth factor (PDGF). Unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) (enoxaparin) inhibited the mitogenic effects of FXa, thrombin and fetal calf serum (FCS), but did not reduce mitogenesis induced by PDGF. Similarly, both UFH and LMWH inhibited the activation of extracellular signal-regulated kinase (ERK-1/2) by FXa, thrombin and FCS, but not by PDGF. This indicates that heparins can influence cellular signaling in SMC via an antithrombin-II (AT-III)-independent mechanism. The inhibition of ERK-1/2 correlated with the inhibition of mitogenesis by the heparins. Thus, the inhibition of ERK-1/2 phosphorylation by heparins might predict an antimitogenai response in this system.     

Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury.

The migration and proliferation of smooth muscle cells (SMCs) within the intima of arteries following mechanical injury is thought to be initiated by vessel wall injury and release of growth factors, in particular the platelet-derived growth factor (PDGF). However, the mechanism by which SMC proliferation is regulated after platelet interaction with the vessel wall has ceased is unknown. Here we show that SMCs derived from the intima of injured rat arteries (intimal SMCs) are phenotypically distinct from SMCs from unmanipulated vessels (medial SMCs). Intimal SMCs secrete 5-fold greater amounts of PDGF-like activity into conditioned medium in culture, have fewer receptors for 125I-labeled PDGF, and are not mitogenically stimulated by exogenous purified PDGF. This study demonstrates that two SMC phenotypes can develop in the adult rat artery and suggests that SMC proliferation in vivo may be controlled, in part, by SMCs that produce PDGF-like molecules.     

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

AIM: We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. METHODS AND RESULTS: In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. CONCLUSION: These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.     

Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury

Phenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markers in cultured SMCs. Here, we derive mice deficient for Klf4 by conditional gene ablation and analyze their vascular phenotype following carotid injury. Klf4 expression was rapidly induced in SMCs of control mice after vascular injury but not in Klf4-deficient mice. Injury-induced repression of SMC differentiation markers was transiently delayed in Klf4-deficient mice. Klf4 mutant mice exhibited enhanced neointimal formation in response to vascular injury caused by increased cellular proliferation in the media but not an altered apoptotic rate. Consistent with these findings, cultured SMCs overexpressing Klf4 showed reduced cellular proliferation, in part, through the induction of the cell cycle inhibitor, p21(WAF1/Cip1) via increased binding of Klf4 and p53 to the p21(WAF1/Cip1) promoter/enhancer. In vivo chromatin immunoprecipitation assays also showed increased Klf4 binding to the promoter/enhancer regions of the p21(WAF1/Cip1) gene and SMC differentiation marker genes following vascular injury. Taken together, we have demonstrated that Klf4 plays a critical role in regulating expression of SMC differentiation markers and proliferation of SMCs in vivo in response to vascular injury.     

D-erythro-N,N-dimethylsphingosine inhibits bFGF-induced proliferation of cerebral,aortic and coronary smooth muscle cells

The role of sphingosine kinase (SphK) on basic fibroblast growth factor (bFGF)-induced proliferation of cerebral, aortic and coronary smooth muscle cells (SMC) was addressed using D-erythro-N,N-dimethylsphingosine (DMS), an inhibitor of SphK which blocks conversion of sphingosine to sphingosine-1-phosphate (S1P). DMS concentration-dependently reduced the bFGF-induced proliferation of rat cerebral and aortic, and human coronary SMC. This suggests that SphK is one of the key enzymes in the mitogenic response to bFGF in vascular SMC as supported by the finding that S1P stimulated proliferation of SMC. Fumonisin B1, a dihydroceramidesynthase inhibitor which blocks the conversion of dihydrosphingosine to seramide, did not affect SMC proliferation induced by bFGF. Staurosporine, an inhibitor of protein kinase C (PKC), inhibited proliferation of SMC induced by bFGF, and both bFGF- and S1P-induced proliferation of SMC was sensitive to pertussis toxin (PTX), an inhibitor of Gi-protein activity. The present study thus demonstrates that SphK, PKC and Gi-protein activities are required for bFGF-mitogenic signaling in SMC. The bFGF mitogenic effect in vascular SMC might at least in part act via the SphK pathway and a Gi-protein.     

Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor.

Proliferation of smooth muscle cells (SMCs) represents an important event in vascular lesion formation. Despite the common belief that growth factors contribute to the development of the atherosclerotic plaque, until now there has been no direct evidence for a role of mitogens in the development of arterial lesions. Balloon catheter injury of the rat carotid artery is accompanied by death of medial SMCs and is typically followed by proliferation of SMCs with subsequent formation of an intimal lesion. Our hypothesis is that injury causes mitogens to be released from dead cells, which then stimulate cell proliferation. One such mitogen that may be important in this process is basic fibroblast growth factor (bFGF), which can be detected immunocytochemically in SMCs and endothelial cells of adult rat carotid arteries. Systemic injection of a neutralizing antibody against bFGF prior to balloon catheterization significantly decreased the induced SMC proliferation by approximately 80%. The intimal lesion that developed within 8 days after injury, however, was not significantly reduced. The results of this study support the concept that endogenous bFGF is the major mitogen controlling the growth of vascular smooth muscle cells following injury. These data may have implications for the observed failure of endarterectomy and angioplasty procedures.     

Rapamycin reduces neointima formation during vascular injury

BACKGROUND: Proliferation and migration of vascular smooth muscle cells (SMCs) mark the key processes in the development of bypass graft disease and during neointima formation in restenosis after angioplasty. Growth factors are potent SMC mitogens as they are involved in SMC proliferation and in extracellular matrix (ECM) synthesis. Based on these premises, we examined the effect of the proliferation inhibitor rapamycin in human SMC culture and in a rabbit vascular injury model. MATERIALS AND METHODS: Injection of rapamycin or its vehicle was performed with an infusion-balloon catheter directly into the vessel wall during vascular injury. The intima/media ratio was determined histologically whereas the protein expression was analysed using the powerful two-dimensional gel electrophoresis (2D page) technique. Inhibition of proliferation after rapamycin application was estimated in a human SMC culture for time and dose dependent effects. RESULTS: Rapamycin treatment resulted in a significant reduction of intima media ratio compared to vehicle treated animals after three weeks (0.65 +/- 0.1 vs. 1.2 +/- 0.2 intima-media-ratio, p < 0.05). 2D electrophoresis analysis proved increased ECM synthesis following angioplasty (i.e., lamin, vimentin) in vehicle treated animals. Local rapamycin administration resulted in profound reduction of ECM synthesis after vascular injury. In in-vitro experiments exposure of cultured human SMCs to rapamycin resulted in a significant and dose-dependent (1 nm-100 nm) reduction of human smooth muscle cell proliferation measured by cell counting. CONCLUSION: These above mentioned results suggest that protein synthesis in addition to reduction of cellular proliferation plays an important role following vascular injury, since application of rapamycin resulted in the reduction of SMC proliferation and ECM-synthesis.     

Regulation of platelet-derived growth factor ligand and receptor gene expression by alpha-thrombin in vascular smooth muscle cells.

Since the expression of genes for platelet-derived growth factor (PDGF)-A and PDGF beta-receptor are reciprocally regulated in vascular wall cells after balloon injury, we have investigated the ability of specific vasoactive molecules or growth factors to reproduce the injury pattern of gene expression in cultured rat smooth muscle cells (SMCs) and assessed the effect of inactivating alpha-thrombin on injury-induced expression of PDGF-A mRNA by vascular wall cells in vivo. The molecules investigated, to which vascular SMCs may be locally exposed after mechanical injury, included vasoactive factors (alpha- and beta-adrenergic agonists, serotonin, histamine, angiotensin II, and endothelin) and growth factors (PDGF-AA, PDGF-BB, basic fibroblast growth factor, insulin-like growth factor, epidermal growth factor, and alpha-thrombin). In cultured rat SMCs, only alpha-thrombin (0.1-100 nM), among these compounds, produced the pattern of transiently increased PDGF-A and decreased PDGF beta-receptor mRNA. PDGF-B chain mRNA levels remained undetectable in these cultured SMCs. The dependence of these changes in gene expression on the proteolytic activity of alpha-thrombin was shown by the interruption of altered gene expression or DNA synthesis after incubating the cultured SMCs with covalently inactivated alpha-thrombin using D-Phe-Pro-Arg chloromethyl ketone, a synthetic direct active-site irreversible inhibitor of alpha-thrombin. Continuous intravenous infusion of this synthetic antithrombin into baboons for 6 hours (100 nmol/kg per minute maintaining constant plasma levels of 3.0 +/- 0.5 microns/ml) after inducing balloon-catheter arterial injury also prevented the threefold increase in expression of PDGF-A mRNA characteristically exhibited by untreated mechanically injured vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia

The activation of vascular smooth muscle cells (SMCs) in neointimal hyperplasia involves signaling through receptor tyrosine kinases as well as G protein-coupled receptors. Overexpression of G protein-coupled receptor kinase-2 (GRK2) in SMCs can attenuate mitogenic signaling and proliferation in response to not only several G protein-coupled receptor agonists, but also platelet-derived growth factor (PDGF). To test whether overexpression of GRK2 could inhibit other SMC responses implicated in neointimal hyperplasia, we assessed SMC chemotaxis and mitogenic signaling evoked by PDGF and G(q)-coupled receptor agonists. To test the effects of GRK2 overexpression on neointimal hyperplasia in vivo, we employed a rabbit autologous vein graft model system. GRK2 overexpression reduced PDGF-promoted SMC chemotaxis by 85% (P<0.01), but had no effect on chemotaxis promoted by epidermal growth factor (EGF). Congruently, GRK2 overexpression reduced by approximately 50% (P<0.05) the [(3)H]thymidine incorporation induced by combinations of PDGF and Gq-coupled receptor agonists, but had no effect on that induced by PDGF plus EGF. PDGF-, but not EGF-promoted phosphoinositide 3-kinase activity in SMCs was also inhibited by GRK2 overexpression. In rabbit vein grafts, we achieved GRK2 overexpression in medial SMCs, reduced cell proliferation during the first week after graft implantation, and reduced steady state neointimal thickness by 29% (P<0.01), without affecting medial thickness or potentiating SMC apoptosis. Because of its ability to dampen chemotactic and mitogenic signaling through PDGF and Gq-coupled receptors, GRK2 overexpression in SMCs may be a useful therapeutic approach for neointimal hyperplasia.     

Ras/Rac-Dependent activation of p38 mitogen-activated protein kinases in smooth muscle cells stimulated by cyclic strain stress

p38, a subfamily of the mitogen-activated protein kinases (MAPKs), is a crucial signal transducer between a variety of extracellular stimuli and gene expression in mammalian cells. This kinase is activated in cultured cells stimulated by heat shock, osmotic stress, and proinflammatory cytokines, but a similar activation of p38 MAPKs in vascular smooth muscle cells (SMCs) stimulated by mechanical stress has yet to be studied. We studied signal pathways leading to time- and strength-dependent p38 activation in rat SMCs in response to cyclic strain stress. p38 phosphorylation in stressed SMCs showed maximal activation at 10 minutes. This activation was significantly inhibited by pretreatment of the SMCs with pertussis toxin, a G-protein antagonist, and enhanced by treatment with suramin, a growth factor receptor antagonist, but opposite effects in the activation of extracellular signal-regulated kinases stimulated by mechanical forces were found. p38 activation was markedly reduced in stressed SMCs after protein kinase C depletion. Interestingly, SMC lines stably expressing dominant-negative ras (ras N17) or rac1 (rac1 N17) almost abolished p38 phosphorylation induced by cyclic strain stress. When p38 activation was inhibited by the specific inhibitor SB 202190, SMC migration, determined in a Boyden chamber in response to stimulation with platelet-derived growth factor-BB, and SMC proliferation, stimulated by cyclic strain stress, were abrogated. Thus, we provide the first evidence that cyclic strain stress rapidly activates p38 MAPKs via activation of protein kinase C ras/rac signal pathways, suggesting that p38 MAPKs are important signal transducers mediating the mechanical stress-induced cell responses essential for SMC migration and proliferation.     

Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids

Vascular smooth muscle cell (SMC) migration and proliferation contribute to neointimal hyperplasia and restenosis after vascular injury. The epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess vasodilatory, antiinflammatory, and fibrinolytic properties. To determine whether these compounds also possess antimigratory and antiproliferative properties, we stimulated rat aortic SMCs with either 20% serum or platelet-derived growth factor (PDGF-BB, 20 ng/mL). In a concentration-dependent manner, treatment with EETs, particularly 11,12-EET, inhibited SMC migration through a modified transwell filter by 53% to 60%. EETs, however, have no inhibitory effects on PDGF-stimulated SMC proliferation. Adenoviral-mediated overexpression of the CYP isoform, CYP2J2, in SMCs also inhibited serum- and PDGF-induced SMC migration by 32% and 26%, respectively; both effects of which were reversed by the CYP inhibitors SKF525A or clotrimazole, but not by the K(Ca) channel blocker, charybdotoxin, or the cyclooxygenase inhibitor, diclofenac. The effect of EETs correlated with increases in intracellular cAMP levels. Indeed, forskolin and 8-bromo-cAMP exert similar inhibitory effects on SMC migration as 11,12-EET and the effects of 11,12-EET were blocked by cAMP and protein kinase A (PKA) inhibitors. These findings indicate that EETs possess antimigratory effects on SMCs through the cAMP-PKA pathway and suggest that CYP epoxygenase-derived eicosanoids may play important roles in vascular disease and remodeling.     

Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery

Although the level of plasminogen activator (PA) expression has been correlated with cellular proliferation and migration in vitro, this relation has not been established in tissue undergoing repair. In a rat model of arterial injury, we have measured the expression of PAs by vascular smooth muscle cells (SMCs) during entry into the growth cycle (0-24 hours) and subsequent migration from the media to the intima (starting at approximately 4 days). In normal rat carotid, low levels of urokinase-type PA (uPA) and tissue-type PA (tPA) are present; after removal of the endothelium, only uPA is detected in the media. uPA activity in extracts of carotid arteries increases and reaches a maximum between 16 and 24 hours after injury; uPA mRNA increases steadily and is maximal at 7 days. tPA activity appears at 3 days and is maximal at 7 days; tPA mRNA is present in normal vessels and reaches a maximum by 7 days. Most of the tPA in the media is associated with SMC and not with regenerating endothelium. Furthermore, tPA is present in the media before the SMCs migrate into the intima. These results demonstrate that PA expression by vascular SMCs is differentially regulated, with uPA present during mitogenesis and tPA during migration.     

Removal of heparan sulfate by heparinase treatment inhibits FGF-2-dependent smooth muscle cell proliferation in injured rat carotid arteries

Smooth muscle cells (SMC) of the rat carotid arterial media proliferate and migrate in response to injury during the formation of a neointima. The interaction of fibroblast growth factor (FGF-2), which is released at the site of injury, with heparan sulfate proteoglycans (HSPGs) is necessary to induce signaling, which elicits an FGF-dependent mitogenic response by arterial smooth muscle cells, and also serves as a mechanism for storage of the growth factor within the extracellular matrix. However, whether these interactions are critical during neointimal formation has not been directly tested. In this study, a model of FGF-2-dependent medial SMC mitogenic response in balloon-injured rat carotid artery was used to test the effect of degradation of vessel wall heparan sulfate on subsequent SMC proliferation. Treatment of balloon-catheterized rat carotid arteries with chondroitin ABC lyase and/or heparin lyases eliminated heparan sulfates in the vessel wall, as determined by immunoperoxidase staining. In contrast, the distribution in the carotid vessel wall of the large core protein of perlecan, a major vessel wall HSPG that binds FGF-2, is not decreased. The effect of glycosaminoglycan digestion in situ on medial SMC proliferation in response to a bolus injection of FGF-2 after injury was determined by measuring the percentage of SMC nuclei that incorporated 5-bromo-2'-deoxyuridine (BrdU) 48 h after injury. Enzymatic removal of heparan sulfate reduced BrdU incorporation into medial SMC by 60-70% (P < 0.001) at 48 h after injury. Moreover, pre-incubation of FGF-2 with heparin prior to injection restored SMC replication to the levels present in injured vessels treated with buffer alone (P < 0.01). These experiments indicate that endogenous HSPGs are essential to promote FGF-2-driven medial SMC proliferation following injury, and that heparinase treatment can abrogate FGF-2-dependent responses in vivo.     

Periostin mediates vascular smooth muscle cell migration through the integrins ανβ3 and ανβ5 and focal adhesion kinase (FAK) pathway

Smooth muscle cell (SMC) migration involves interactions of integrin receptors with extracellular matrix (ECM) and is an important process of neointimal formation in atherosclerosis and restenosis after vascular interventions. Previous studies have shown that periostin (PN), a novel ECM protein, is upregulated in rat carotid artery after balloon injury, and growth factor-stimulated expression of PN promotes SMC migration in vitro. Here, we address the mechanism by which PN–integrin interaction mediates SMC migration in vitro. Aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate in vitro. Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN?/? and wildtype SMCs, respectively. In both types of SMCs, adenovirus-mediated overexpression of HA-tagged PN to a similar extent, which induced a robust cell migration concomitantly with an increase in β3-integrin expression and phosphorylation of FAK (Tyr397). Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-ανβ3 and PN-ανβ5, but not PN-β1 integrins, are required for SMC migration. Inhibition of FAK signaling by overexpression of an endogenous FAK inhibitor termed FRNK (FAK-related nonkinase) significantly attenuated FAK (Tyr397) phosphorylation and the SMC migration induced by PN. These results reveal a mechanism whereby PN mediates vascular SMC migration through an interaction with alphaV-integrins (mainly ανβ3) and subsequent activation of FAK pathway.     

Galectin 1 is involved in vascular smooth muscle cell proliferation

OBJECTIVE: Smooth muscle cell (SMC) migration and proliferation are the key steps in the development of atherosclerosis and restenosis. Matricellular proteins have been implicated in cell adhesion, migration and proliferation. Here we investigated the role of the matricellular protein galectin-1 (Gal-1), a beta-galactoside-binding lectin, in SMC proliferation in atheroma and DNA synthesis in cell culture. METHODS: Protein expression was visualised by tissue section immunostaining. RNA expression was analysed using Northern blot analysis. DNA synthesis of human vascular SMCs was determined by 3H-thymidine incorporation. Recombinant glutathione S-transferase-galectin-1 fusion protein (Gal FP) binding to extracellular matrix (ECM) proteins was measured by ELISA. Gal-1 binding to cells and ECM was estimated using 125I-labelled Gal FP. RESULTS: Prominent Gal-1 staining coincided with SMC proliferation in human coronary endarterectomy samples in organoid culture. In cell culture, Gal-1 mRNA was upregulated in growing SMCs. Gal FP increased serum-induced DNA synthesis of human SMCs on plastic or endogenous ECM, but not of a rat PAC1 SM cell line. Also, Gal FP slightly increased SMC adhesion to ECM. SMCs exhibited a complex pattern of receptor-ligand interactions with Gal FP. The Gal-1 binding to SMCs was much stronger than to ECM, produced by these SMCs. We identified new ECM proteins: thrombospondin, vitronectin and osteopontin, which bound to Gal FP in a dose- and beta-galactoside-dependent manner in ELISA. CONCLUSIONS: Gal-1 binding to SMCs was stronger than to ECM, although ECM of atherosclerotic blood vessels contained additional ECM proteins which bound to Gal-1. Gal-1 was upregulated during SMC growth and Gal FP enhanced serum-induced DNA synthesis in SMCs. Overall, Gal-1 upregulation is likely to provide a reinforcement of serum-induced events during vascular injury.