ACAT inhibition reduces the progression of preexisting, advanced atherosclerotic mouse lesions without plaque or systemic toxicity.

OBJECTIVE: Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice. METHODS AND RESULTS: Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. CONCLUSIONS: Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Prolyl hydroxylase EGLN3 regulates skeletal myoblast differentiation through an NF-kappaB-dependent pathway.

The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the alpha subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1alpha by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-kappaB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IkappaBalpha. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-kappaB activation induced by overexpressed TRAF2 or IkappaB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-kappaB. These data suggest that EGLN3 is a negative regulator of NF-kappaB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-kappaB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.

Vascular smooth muscle-derived tissue factor is critical for arterial thrombosis after ferric chloride-induced injury.

Tissue factor (TF) initiates coagulation, regulates hemostasis, and plays a critical role in mediating arterial thrombosis. TF is up-regulated in vascular smooth muscle cells (VSMCs) in atherosclerosis and arterial injury. To examine the biologic role of VSMC-derived TF, we crossed TF(flox/flox) mice with SM22alphaCre(+/-) mice. TF mRNA and activity were decreased in the aortic media of TF-deficient mice by 96% and 94.8%, respectively. There were no differences in TF activity measured in plasma or concentrated microparticles. TF-deficient mice were generated with the expected frequency, showed no evidence of bleeding or increased mortality, and had similar activated partial thromboplastin and tail vein bleeding times. Thrombus-mediated flow reduction in response to ferric chloride injury of the carotid arteries was significantly attenuated in VSMC-specific TF-deficient. Stable occlusion was seen in 11 of 12 wild-type mice, but in only 6 of 16 VSMC-specific TF-deficient mice (P = .001). These data suggest that VSMC-derived TF is critical in a macrovascular model of arterial thrombosis. This mouse model should be valuable in determining the contribution of VSMC-derived TF in other TF-mediated phenomena, such as restenosis.

Platelets and megakaryocytes contain functional nuclear factor-kappaB.

OBJECTIVE: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules. METHODS AND RESULTS: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels. CONCLUSIONS: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.

15-deoxy-delta12,14-PGJ2 enhances platelet production from megakaryocytes.

Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein, we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly, the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma, but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively, these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients.

Protein kinase Cδ mediates MCP-1 mRNA stabilization in vascular smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF ß and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct "proximal" signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.

Foxp3 regulates megakaryopoiesis and platelet function.

OBJECTIVE: Platelets are crucial for hemostasis and are vital regulators of inflammation. Foxp3 is a key transcription factor for T regulatory cell development. Humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) and the scurfy (Foxp3(sf)) mouse have mutations in the Foxp3 gene that lead to a host of pathologies including autoimmunity and skin diseases. Scurfy mice and some humans with IPEX are also thrombocytopenic. The purpose of this study was to determine whether the absence of functional Foxp3 leads to defects in megakaryocytes and platelets. METHODS AND RESULTS: We discovered that human and mouse megakaryocytes express Foxp3 mRNA and protein. Using shRNA and Foxp3(sf) mice, we demonstrated that Foxp3-deficient mouse and human megakaryocyte progenitors exhibited proliferation defects. Striking platelet abnormalities were observed in both an IPEX patient and Foxp3(sf) mice. Impaired platelet spreading and release of TGF-beta and CD40 ligand (CD40L), and abnormal levels of plasma CD40L were observed in a case of IPEX syndrome. Foxp3(sf) mice were thrombocytopenic and had increased platelet volume and altered serum levels of CD40L, TXB(2), and TGF-beta. CONCLUSIONS: These findings provide compelling new evidence that Foxp3 is needed for proper megakaryopoiesis and plays a role in regulating platelet function including spreading and release.

Tissue factor and VEGF expression in prostate carcinoma: a tissue microarray study.

Tissue factor (TF) is the principal physiologic initiator of coagulation. It also plays an important role in tumor growth and metastasis possibly by contributing to angiogenesis. We evaluated the expression of TF in benign and malignant prostate tissue and correlated it with the expression of the pro-angiogenic protein, vascular endothelial growth factor (VEGF). We used a tissue microarray (TMA) constructed from 80 archival prostatectomy specimens. Core samples were collected from benign prostate tissue (BP) (n= 77), high-grade prostatic intraepithelial neoplasia (PIN) (n= 26), and carcinoma (PCa) (n= 93). TMA sections were stained with an immunopurified polyclonal TF antibody and a rabbit polyclonal VEGF. Two pathologists manually scored staining in epithelial cells using the German Immunoreactive Score. Positive staining for TF was seen predominantly in PCa with rare positive glands in BP and PIN. TF expression was significantly lower in BP versus PCa specimens (p< .001) and in PIN versus PCa specimens (p< .001). Positive staining for VEGF was seen in PCa, BP, and PIN. Rates of VEGF expression were also significantly lower in BP versus PCa specimens (p= .003) but not in PCa versus PIN (p= .430). The majority of PCa samples positive for TF were also positive for VEGF (p< .001). Our findings reinforce the link between angiogenesis and TF expression in PCa. We suggest further exploration of TF-mediated pathways leading to increased tumor aggressiveness in PCa, and the possible use of anti-TF agents in PCa.

Peroxisome proliferator-activated receptor gamma and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles.

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

Inhibition of MCP-1/CCR2 signaling does not inhibit intimal proliferation in a mouse aortic transplant model.

BACKGROUND: Transplant arteriopathy is the leading cause of long term morbidity and mortality following heart transplantation. Animal models have demonstrated that monocyte chemoattractant protein (MCP)-1 is induced early after transplant in cardiac and aortic allografts. We have previously reported that deficiency of MCP-1 or its receptor, CC chemokine receptor 2 (CCR2), is associated with a reduction in intimal proliferation in a mouse femoral artery injury model. Using knockout mice, we have now examined the role of MCP-1 and CCR2 in the development of the intimal proliferation of transplant arteriopathy. METHODS: C57Bl/6 CCR2 and MCP-1 wild-type and knockout mice were used in the studies and aortic transplants were performed between Balb/c mice and C57Bl/6 mice. Aortas from recipient animals were harvested 8 weeks after transplant. RESULTS: Unlike arterial injury, in an aortic transplant model inhibition of MCP-1/CCR2 signaling did not result in reduced intimal proliferation. CONCLUSIONS: Despite a pathology that appears similar, the inflammatory mediators that regulate transplant arteriopathy differ from those regulating intimal proliferation secondary to wire injury. Our results suggest that targeting MCP-1/CCR2 signaling is not sufficient to block transplant arteriopathy across a complete MHC-mismatch barrier.

The role of smooth muscle derived tissue factor in mediating thrombosis and arterial injury.

Tissue factor (TF) is a glycoprotein that initiates coagulation, regulates hemostasis and plays a critical role in arterial thrombosis. Vascular smooth muscle cells (SMC) are the major cellular component of the arterial wall. Under normal conditions, SMC express minimal levels of TF; however TF is rapidly induced in SMC by growth factors and cytokines and is expressed in abundance in arterial SMC in response to injury and during atherogenesis. Recent studies have suggested that SMC-derived TF plays an important role in promoting arterial thrombosis and in mediating intimal hyperplasia in response to arterial injury. This latter role may be related to non-procoagulant properties of TF.

Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer.

PURPOSE: Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer. EXPERIMENTAL DESIGN: Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n=10), intraductal papillary mucinous neoplasms (n=70), pancreatic intraepithelial neoplasia (n=40), and resected or metastatic pancreatic adenocarcinomas (n=130). RESULTS: TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade >or=2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P<0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P=0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P=0.04). CONCLUSIONS: TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

The platelet as a therapeutic target for treating vascular diseases and the role of eicosanoid and synthetic PPARgamma ligands.

The platelet was traditionally thought only to serve as the instigator of thrombus formation, but now is emerging as a pivotal player in cardiovascular disease and diabetes by inciting and maintaining inflammation. Upon activation, platelets synthesize eicosanoids such as thromboxane A2 (TXA2) and PGE2 and release pro-inflammatory mediators including CD40 ligand (CD40L). These mediators activate not only platelets, but also stimulate vascular endothelial cells and leukocytes. These autocrine and paracrine activation processes make platelets an important target for attenuating inflammation. The growing interest and recent discoveries in platelet biology has lead to the search for therapeutic platelet targets. Recently, platelets, although anucleate, were discovered to possess the transcription factor PPARgamma. Treatment with eicosanoid and synthetic PPARgamma ligands blunts platelet release of the bioactive mediators, soluble (s) CD40L and TXA2, in thrombin-activated platelets. PPARgamma ligand treatment may prove useful for dampening unwanted platelet activation and chronic inflammatory diseases such as cardiovascular disease.

Chemokines in the pathogenesis of vascular disease.

Our increasing appreciation of the importance of inflammation in vascular disease has focused attention on the molecules that direct the migration of leukocytes from the blood stream to the vessel wall. In this review, we summarize roles of the chemokines, a family of small secreted proteins that selectively recruit monocytes, neutrophils, and lymphocytes to sites of vascular injury, inflammation, and developing atherosclerosis. Chemokines induce chemotaxis through the activation of G-protein-coupled receptors, and the receptors that a given leukocyte expresses determines the chemokines to which it will respond. Monocyte chemoattractant protein 1 (MCP-1), acting through its receptor CCR2, appears to play an early and important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury. Acute thrombosis is an often fatal complication of atherosclerotic plaque rupture, and recent evidence suggests that MCP-1 contributes to thrombin generation and thrombus formation by generating tissue factor. Because of their critical roles in monocyte recruitment in vascular and nonvascular diseases, MCP-1 and CCR2 have become important therapeutic targets, and efforts are underway to develop potent and specific antagonists of these and related chemokines.

Lysophosphatidylcholine stimulates monocyte chemoattractant protein-1 gene expression in rat aortic smooth muscle cells.

OBJECTIVE: Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for approximately 60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells. METHODS AND RESULTS: LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs -146 and -261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal-regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor. CONCLUSIONS: LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein.

CCL11 (Eotaxin) induces CCR3-dependent smooth muscle cell migration.

OBJECTIVE: CCL11 (Eotaxin) is a potent eosinophil chemoattractant that is abundant in atheromatous plaques. The major receptor for CCL11 is CCR3, which is found on leukocytes and on some nonleukocytic cells. We sought to determine whether vascular smooth muscle cells (SMCs) possessed functional CCR3. METHODS AND RESULTS: CCR3 mRNA (by RT-PCR) and protein (by Western blot analysis and flow cytometry) were present in mouse aortic SMCs. CCL11 induced concentration-dependent SMC chemotaxis in a modified Boyden chamber, with maximum effect seen at 100 ng/mL. SMC migration was markedly inhibited by antibody to CCR3, but not to CCR2. CCL11 also induced CCR3-dependent SMC migration in a scrape-wound assay. CCL11 had no effect on SMC proliferation. CCR3 and CCL11 staining were minimal in the normal arterial wall, but were abundant in medial SMC and intimal SMC 5 days and 28 days after mouse femoral arterial injury, respectively, times at which SMCs possess a more migratory phenotype. CONCLUSIONS: These data demonstrate that SMCs possess CCR3 under conditions associated with migration and that CCL11 is a potent chemotactic factor for SMCs. Because CCL11 is expressed abundantly in SMC-rich areas of the atherosclerotic plaque and in injured arteries, it may play an important role in regulating SMC migration.

CCR2 deficiency decreases intimal hyperplasia after arterial injury.

Monocyte chemoattractant protein (MCP)-1 is upregulated in atherosclerotic plaques and in the media and intima of injured arteries. CC chemokine receptor 2 (CCR2) is the only known functional receptor for MCP-1. Mice deficient in MCP-1 or CCR2 have marked reductions in atherosclerosis. This study examines the effect of CCR2 deficiency in a murine model of femoral arterial injury. Four weeks after injury, arteries from CCR2(-/-) mice showed a 61.4% reduction (P<0.01) in intimal area and a 62% reduction (P<0.05) in intima/media ratio when compared with CCR2(+/+) littermates. The response of CCR2(+/-) mice was not significantly different from that of CCR2(+/+) mice. Five days after injury, the medial proliferation index, determined by bromodeoxyuridine incorporation, was decreased by 59.8% in CCR2(-/-) mice when compared with CCR2(+/+) littermates (P<0.05). Although leukocytes rapidly adhered to the injured arterial surface, there was no significant macrophage infiltration in the arterial wall of either CCR2(-/-) or CCR2(+/+) mice 5 and 28 days after injury. These results demonstrate that CCR2 plays an important role in mediating smooth muscle cell proliferation and intimal hyperplasia in a non-hyperlipidemic model of acute arterial injury. CCR2 may thus be an important target for inhibiting the response to acute arterial injury.

MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.

Mice deficient in tissue factor demonstrate attenuated intimal hyperplasia in response to vascular injury and decreased smooth muscle cell migration.

Tissue factor (TF) is the primary initiator of the coagulation cascade and is thought to play a key role in the generation of arterial thrombosis. Recent studies have suggested that TF mediates inflammatory processes in the arterial wall and may be an important regulator of intimal hyperplasia. We have employed genetically engineered mice (mTF(-/-) /hTF(+)) with markedly diminished TF activity ( approximately 1% normal levels) o examine the role of TF in mediating the response to arterial injury. mTF(-/-)/hTF(+) displayed a marked reduction in intimal hyperplasia (46% decrease in intimal area, 60% decrease in intimal/medial ratio) in response to femoral artery injury when compared to wild type controls. The decreased intimal hyperplasia seen in low TF mice was noted in a model of vascular injury not associated with significant thrombosis, suggesting that it may be mediated by non-procoagulant properties of TF. Smooth muscle cells from mTF(-/-)/hTF(+) mice grew normally in response to serum, but exhibited a marked defect in cell migration in a modified Boyden chamber assay. In contrast, there was no difference in platelet derived growth factor- induced migration, suggesting that the effect of TF on smooth muscle cell migration is agonist dependent. These data suggest that TF may mediate intimal hyperplasia by regulating smooth muscle cell migration.