Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.     

Regulation of vascular smooth muscle cell apoptosis. Modulation of bad by a phosphatidylinositol 3-kinase-dependent pathway.

Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal-induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I-induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Deltap85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase-dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.     

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage

Fas and its ligand (FasL), are a receptor-ligand pair identified as promoting cell death in several tissues. Apoptosis of vascular smooth muscle cells (VSMCs) in human atherosclerotic plaque may contribute to weakening of the fibrous cap, ultimately resulting in plaque rupture. We investigated the ability of monocytes to induce apoptosis of cultured VSMCs through Fas/FasL pathway. In addition, we examined the association of FasL with apoptosis in human coronary plaques. Both activated monocytes and the supernatant obtained from activated monocytes were able to kill cultured VSMCs. The apoptotic response of VSMCs was almost completely blocked by the caspase inhibitor z-VAD-fmk and was partially blocked by incubation with antagonistic anti-Fas IgG1 which suggests that Fas/FasL system was involved in the induction of cell death. An approximate 30 kDa protein, which represents a cleaved, soluble form of FasL, was identified in culture medium from activated monocytes, but not in culture medium from control, unactivated monocytes. Immunohistochemical analysis of human atherosclerotic coronary lesions showed that FasL is expressed by macrophages, and microvessels in the adventitia as well as in the plaque. Finally, double-staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and FasL antibody showed that FasL enriched lesions always included a number of TUNEL-positive cells. These data suggest that Fas/FasL pathway can be employed by monocytes/macrophages to induce VSMC apoptosis in the atherosclerotic lesions.     

Chemokine receptor-8 (CCR8) mediates human vascular smooth muscle cell chemotaxis and metalloproteinase-2 secretion

The response of the arterial vascular wall to injury is characterized by vascular smooth muscle cell (VSMC) migration, a process requiring metalloproteinase production. This migration is induced by cytokines, however the agonists involved are not fully defined. The CC chemokine receptor 8 (CCR8) is expressed on monocytes and T lymphocytes and is the sole receptor for the human CC chemokine 1 (CCL1, I-309) and for the viral chemokine, vCCL1 (viral macrophage inflammatory protein 1 [vMIP-1]). We have reported that CCR8 is expressed on human umbilical vein endothelial cells (HUVECs) and mediates chemotaxis induced by CCL1. The conditioned medium from incubation mixtures of lipoprotein(a) (Lp(a)) and HUVECs (LCM) contained CCL1 and stimulated both monocyte and HUVEC chemotaxis, providing novel mechanisms for the atherogenicity of Lp(a). We now report that CCL1, vCCL1, and LCM stimulate chemotaxis of human VSMCs that is blocked by murine monoclonal antibody against CCR8 and by the G-protein inhibitor pertussis toxin. The effect of anti-CCR8 was specific, as this antibody failed to effect the chemotaxis of VSMCs in response to CCL3 or by platelet-derived growth factor BB (PDGF-BB). VSMCs contained CCR8 mRNA and CCR8 antigen coprecipitated with VSMC membranes. Antibodies against metalloproteinase-2 (MMP-2) inhibited the CCL1-induced chemotaxis of VSMCs, whereas anti-MMP-9 was less effective. CCL1 induced VSMC pro-MMP-2 mRNA and protein secretion. Poxvirus MC148 inhibited the increase in MMP-2 induced by CCL1, documenting that CCR8 was the receptor responsible. In mouse femoral arteries, CCR8 and TCA3 antigen colocalized with VSMCs and were up-regulated after injury. The induction of CCR8 and CCL1/TCA3 under conditions associated with VSMC proliferation and migration raises the possibility that CCR8 may play an important role in vessel wall pathology.     

Regulation of smooth muscle cell proliferation by beta-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of beta-catenin. In this study we demonstrate directly that growth factors stimulate beta-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether beta-catenin/TCF signaling regulates VSMC proliferation via modulation of the beta-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited beta-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of beta-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by beta-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting beta-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and beta-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 beta-catenin/TCF responsive genes.     

Resistin is secreted from macrophages in atheromas and promotes atherosclerosis

OBJECTIVE: Resistin belongs to a family of cysteine-rich secreted polypeptides that are mainly produced by monocytes/macrophages in humans. Recently, high concentrations of resistin were shown to induce vascular endothelial dysfunction and vascular smooth muscle cell proliferation. We examined if resistin was secreted from macrophages locally in atheromas and if it affected vascular cell function in human. METHODS AND RESULTS: Immunohistochemical staining of human vessels showed that aortic aneurysms exhibited resistin-positive staining areas along macrophage infiltration, while normal arteries and veins did not. Co-localization of resistin and CD68 (a marker for macrophages) was observed in immunofluorescent double staining of aneurysms. Resistin mRNA was expressed much higher in cultured monocytes/macrophages than in human vascular smooth muscle cells (VSMCs) and human umbilical venous endothelial cells (HUVECs). This suggested that the resistin in aneurysms originates from macrophages within the vessels. To determine the effects of resistin on atherosclerosis, HUVECs and human VSMCs were incubated with resistin (10-100 ng/mL for 4 approximately 24 h). In HUVECs, plasminogen activator inhibitor (PAI)-1 release was assayed by ELISA, while the PAI-1 and endothelin (ET)-1 mRNA levels were analyzed by Northern blotting. Both were increased significantly with resistin treatment by factors of 1.3-2.5 (p<0.05). Migratory activity of VSMCs measured by scratched wound assay also increased significantly (1.6 times, p<0.05). In summary, macrophages infiltrating atherosclerotic aneurysms secrete resistin, and resistin affects endothelial function and VSMC migration. CONCLUSIONS: Resistin secreted from macrophages may contribute to atherogenesis by virtue of its effects on vascular endothelial cells and smooth muscle cells in humans.     

Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human vascular smooth muscle cells

OBJECTIVE: Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II. METHODS: Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration ([Ca2+]i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries. RESULTS: Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic [Ca2+]i response (Emax = 636 +/- 123 nmol/l). The initial [Ca2+]i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+]i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src-/-mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax = 40 +/- 6.5%). These responses were attenuated by PP2 (Emax = 7.8 +/- 0.08%) but not by PP3 (Emax = 35 +/- 4.5%). CONCLUSIONS: Our findings identify c-Src as an important regulator of VSMC [Ca2+]i signaling and implicate a novel contractile role for this non-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.     

AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration

Development of vascular restenosis is a multifaceted process characterized by migration and proliferation of vascular smooth muscle cells (VSMCs), resulting in loss of lumen diameter. Characterization of proteins that mediate this process is essential in our understanding of the pathogenesis of arterial injury. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding protein that is expressed in VSMCs by allograft and balloon angioplasty injury. AIF-1 is not present in cultured human VSMCs but is induced by cytokines, and overexpression of AIF-1 results in increased VSMC growth and cell-cycle gene expression. To characterize AIF-1 modulatory effects in primary human VSMCs, AIF-1-interacting proteins were identified by an AIF-1/glutathione S transferase fusion protein affinity assay. MALDI-TOF mass spectrophotometric amino analysis identified actin as an AIF-1 interacting protein. This interaction was verified by coimmunoprecipitation. This is a functional interaction, because AIF-1 binds to and polymerizes F-actin in vitro. In unstimulated VSMCs, AIF-1 colocalizes with F-actin but translocates to lamellipodia on stimulation with platelet-derived growth factor. VSMCs stably transduced with AIF-1 retrovirus migrate 2.6-fold more rapidly (85.1+/-2.9 versus 225.5+/-16.6; P<0.001) in response to platelet-derived growth factor versus control cells. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. These data indicate that AIF-1 binds and polymerizes F actin and also regulates Rac1 activity and VSMC migration. Considering the AIF-1 expression pattern in injured arteries, this suggests that AIF-1 may be involved in the cytoskeletal signaling network leading to vascular remodeling.     

Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.     

Interaction between monocytes and vascular smooth muscle cells induces expression of hepatocyte growth factor

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF)--a multifunctional factor implicated in tissue regeneration, wound healing and angiogenesis--that is induced by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs), using coculture of human VSMCs and cells of the human monocytoid cell line, THP-1. METHODS: We collected supernatant from the coculture medium and measured HGF concentrations with an enzyme-linked immunosorbent assay. Northern blot analysis of HGF mRNA was performed using a specific cDNA. To explore which types of cells produce HGF, we performed immunohistochemistry. RESULTS: Coculture of VSMCs with THP-1 cells for 24 h caused a fivefold increase in HGF concentrations over that in control VSMC culture. Northern blot analysis showed an induction of HGF mRNA in the coculture with a peak at 3 h. Separated cocultures demonstrated that both direct contact and soluble factors contribute to the production of HGF. Immunohistochemistry demonstrated that both types of cells in the coculture produce HGF. Neutralizing antibodies against tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 inhibited the HGF production in THP-1 cells and VSMCs that was induced by the coculture conditioned medium. The protein kinase C inhibitors H-7, calphostin C and K252b, and the tyrosine kinase inhibitor, genistein, significantly inhibited the production of HGF in the coculture. CONCLUSIONS: Cell-to-cell interactions between monocytes and VSMCs induced HGF synthesis in both types of cells, suggesting that local HGF production induced by this cell-to-cell interaction has an important role in the pathogenesis of hypertension, atherosclerosis or vascular remodelling.     

Proliferative effect of lipoprotein lipase on human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.     

Dismantling of cadherin-mediated cell-cell contacts modulates smooth muscle cell proliferation

Proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal thickening during atherosclerosis and restenosis. The cadherins are transmembrane proteins, which form cell-cell contacts and may regulate VSMC proliferation. In this study, N-cadherin protein concentration was significantly reduced by stimulation of proliferation with fetal calf serum (FCS) and platelet-derived growth factor-BB (PDGF-BB) in human saphenous vein VSMCs. Furthermore, overexpression of a truncated N-cadherin, which acts as a dominant-negative increased VSMC proliferation. The amount of an extracellular fragment of N-cadherin (approximately 90 kDa) in the media after 24 hours was increased by 12-fold by FCS and 11-fold by PDGF-BB, suggesting that N-cadherin levels are regulated by proteolytic shedding. Incubation with a synthetic metalloproteinase inhibitor or adenoviral overexpression of the endogenous tissue inhibitors of metalloproteinases (TIMPs) demonstrated that metalloproteinase activity was responsible in part for this proteolysis. Although total levels of beta-catenin protein were not affected, beta-catenin was translocated to the nucleus after stimulation with FCS and PDGF-BB. Our data indicates cadherin-mediated cell-cell contacts modulate proliferation in VSMCs. Furthermore, disruption of N-cadherin cell-cell contacts mediated in part by metalloproteinase activity occurs during VSMC proliferation, releasing beta-catenin and possibly inducing beta-catenin-mediated intracellular signaling.     

信号转导子和激活子3通路调控大鼠血管平滑肌细胞增殖迁移机制研究

目的研究转录信号转导子和激活子3（stat3）通路与大鼠血管平滑肌细胞（VSMCs）增殖迁移的关系,明确VSMCs增殖的信号转导过程。方法应用脂质体转染Stat3反义寡核苷酸作用于大鼠VSMCs,应用酶联免疫法（ELISA）检测Stat3水平变化及MTF法检测细胞增殖状态,蛋白免疫印迹法（Western blot）检测stat3、磷酸化stat3及其靶基因产物Cyclin D1、Bcl—XL的表达。结果转染Stat3反义寡核苷酸后,大鼠VSMCs中Stat3水平明显下降（P〈0．01）,同时其增殖水平降低,而相应空白对照组、脂质体组、转染正义寡核苷酸组变化不明显。转染Stat3反义寡核苷酸的VSMCs中stat3、p-Stat 3、Cyclin D1蛋白表达水平随作用时间延长而下降（P〈0．01）,而Bcl—xL水平无明显变化。结论癌基因stat3信号通路与大鼠VSMCs增殖高度相关,可能通过其下游靶基因Cyclin D1影响其增殖,而阻断此通路则可抑制VSMCs的增殖。     

Aquaporin-1 is expressed by vascular smooth muscle cells and mediates rapid water transport across vascular cell membranes

The aquaporins are a rapidly expanding family of highly conserved proteins which function as transmembrane water channels. We have previously shown that the gene for aquaporin-1 (AQP-1) is expressed in rat, aortic vascular smooth muscle cells (VSMCs) implying a specific role for AQP-1 in vascular function. In this study we set out to document the expression of AQP-1 in human arteries and found mRNA and protein in normal endothelial and VSMCs of human arteries and capillaries and in a subset of VSMCs in human atherosclerotic plaques. Secondly, we examined the regulation of AQP-1 gene expression during vascular development and following vascular injury. Studies in the rat demonstrated that AQP-1 mRNA is induced in the neonatal aorta at week 2 of postnatal development and that the protein is present in neointimal VSMCs following balloon injury. Finally, by measuring the rate of change in cell size induced by changes in external osmolarity and demonstrating that water transport can be inhibited with mercuric chloride, we show that AQP-1 is responsible for water transport across human VSMC membranes. Thus, this study provides evidence for a hitherto unrecognised role for aquaporins in mediating rapid water transport across human VSMC membranes. By analogy with other tissues, these data argue for an important role for AQP-1 in regulating transcellular fluid flow and tissue hydration.     

Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)-1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I-induced increase in IRS-I-associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I-stimulated VSMC proliferation and reduced IGF-I-directed VSMC migration by approximately 60%. These results indicate that activation of PI3 kinase is required for both IGF-I-induced VSMC proliferation and migration.     

Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation

An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of Chlamydia pneumoniae in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study, C pneumoniae elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to C pneumoniae EBs (3x10(7) inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12+/-3-fold. UV- and heat-inactivated C pneumoniae EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of C pneumoniae was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 microg/mL) replicated the effect of C pneumoniae, stimulating BrdU incorporation 7+/-3-fold. Exposure to C pneumoniae or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of C pneumoniae and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60-induced VSMC proliferation and attenuated C pneumoniae-induced MAPK activation and VSMC proliferation. Together these results indicate that C pneumoniae and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.