mTOR kinase inhibition reduces tissue factor expression and growth of pancreatic neuroendocrine tumors.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

Tissue factor variants induce monocyte transformation and transdifferentiation into endothelial cell-like cells.

Essentials Monocytes (Mo) transdifferentiate into endothelial cell-like (ECL) cells. Mo induce tissue factor (TF) expression and secretion in microvascular endothelial cells (mECs). TF interacts with Mo in a paracrine fashion, inducing their transdifferentiation into ECL cells. TF generates a positive feedback crosstalk between Mo and mECs that promotes angiogenesis. SUMMARY: Background Monocytes (Mo) increase neovascularization by releasing proangiogenic mediators and/or transdifferentiating into endothelial cell-like (ECL) cells. Recently, we have reported that Mo-microvascular endothelial cells (mECs) crosstalk induces mEC-tissue factor (TF) expression and promotes angiogenesis. However, the effect of TF on Mo remains unknown. Objective Here, we analyzed whether TF might exert angiogenic effects by inducing transdifferentiation of Mo. Methods Full-length TF (flTF) and alternatively spliced TF (asTF) were overexpressed in mECs, and their supernatants were added to Mo cultures. CD16 positivity and expression of vascular endothelial cell (VEC) markers in Mo were analyzed by fluorescence activated cell sorting. The capacity to form tube-like structures were visualized in three-dimensional cultures. Results In mECs flTF and asTF expression and release were increased in cultures with Mo-conditioned media. TF variants induced expansion of a CD16+ Mo subset and Mo transdifferentiation into ECL-cells expressing VEC markers that can form new microvessels. CD16+ Mo exposed to TF showed an increased expression of VE-cadherin, von Willebrand factor (VWF) and eNOS. Mo cultured with supernatants obtained from TF-silenced mECs did not transdifferentiate to ECL-cells or expressed VEC markers. Blocking ß1-integrin in Mo significantly blocked the effects of the TF variants. Conclusions Mo induce mECs to express and release TF, which drives CD16- Mo to transform into CD16+ Mo and to transdifferentiate into ECL-cells that can form new microvessels. Our results reveal a TF-mediated positive feedback between mECs and Mo that stimulates Mo differentiation and induces angiogenesis.

Interplay between alternatively spliced Tissue Factor and full length Tissue Factor in modulating coagulant activity of endothelial cells.

BACKGROUND: Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system. OBJECTIVE: To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface. METHODS: Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels. RESULTS: Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF - but not flTF - protein expression. CONCLUSION: At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Procoagulant and proinflammatory effects of red blood cells on lipopolysaccharide-stimulated monocytes.

BACKGROUND: We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. METHODS: Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. RESULTS: In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. CONCLUSIONS: Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Alternatively spliced tissue factor synergizes with the estrogen receptor pathway in promoting breast cancer progression.

BACKGROUND: Procoagulant full-length tissue factor (flTF) and its minimally coagulant alternatively spliced isoform (asTF), promote breast cancer (BrCa) progression via different mechanisms. We previously showed that flTF and asTF are expressed by BrCa cells, resulting in autoregulation in a cancer milieu. BrCa cells often express hormone receptors such as the estrogen receptor (ER), leading to the formation of hormone-regulated cell populations. OBJECTIVE: To investigate whether TF isoform-specific and ER-dependent pathways interact in BrCa. METHODS: Tissue factor isoform-regulated gene sets were assessed using ingenuity pathway analysis. Tissues from a cohort of BrCa patients were divided into ER-positive and ER-negative groups. Associations between TF isoform levels and tumor characteristics were analyzed in these groups. BrCa cells expressing TF isoforms were assessed for proliferation, migration and in vivo growth in the presence or absence of estradiol. RESULTS: Ingenuity pathway analysis pointed to similarities between ER- and TF-induced gene expression profiles. In BrCa tissue specimens, asTF expression was associated with grade and stage in ER-positive but not in ER-negative tumors. flTF was only associated with grade in ER-positive tumors. In MCF-7 cells, asTF accelerated proliferation in the presence of estradiol in a ß1 integrin-dependent manner. No synergy between asTF and the ER pathway was observed in a migration assay. Estradiol accelerated the growth of asTF-expressing tumors but not control tumors in vivo in an orthotopic setting. CONCLUSION: Tissue factor isoform and estrogen signaling share downstream targets in BrCa; the concomitant presence of asTF and estrogen signaling is required to promote BrCa cell proliferation.

BMP-7 induces TF expression in human monocytes by increasing F3 transcriptional activity.

BACKGROUND: Bone morphogenetic protein (BMP)-7, a major regulator of bone metabolism, inhibits ectopic calcification in atherosclerotic plaques. We have recently reported that BMP-7 is also a potent inducer of tissue factor (TF) in human mononuclear cells (MNCs). While nuclear factor kappa beta (NF-kB) and activation protein-1 (AP-1) are the transcription factors essential for inducible expression of human TF gene (F3), the mechanisms responsible for TF induction by BMP-7 are not known. OBJECTIVE: To elucidate the molecular mechanisms governing BMP-7-triggered TF expression in human MNCs. METHODS: Human blood monocytes were stimulated with BMP-7 and western blotting, qRT-PCR, and flow cytometry studies were carried out to assess F3 expression; promoter studies were also performed using a panel of reporter constructs. Procoagulant TF activity was measured using a validated FXa generation assay. The significance of NF-kB transcriptional activity was verified via pharmacological inhibition. RESULTS: BMP-7 increased TF protein levels, procoagulant activity, surface presentation, and TF mRNA expression. This increase was accompanied by activation of NF-kB as evidenced by reduced IkB-α levels and elevated transcriptional activity of an NF-kB-sensitive reporter in transfected MNCs. Although treatment with BMP-7 also led to a strong phosphorylation of c-Jun, activation of AP-1 alone was not sufficient to induce TF expression: JSH-23, a potent and specific NF-kB inhibitor, completely blocked BMP-7-induced TF expression. CONCLUSIONS: We report that BMP-7-dependent activation of TF in human MNCs is mediated via increased activity of NF-kB, leading to enhanced F3 transcription in human MNCs.

Contributions of thrombin targets to tissue factor-dependent metastasis in hyperthrombotic mice.

BACKGROUND: Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. OBJECTIVE: Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TM(Pro) mice). METHODS: Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. RESULTS: TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TM(Pro) mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TM(Pro) mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TM(Pro) mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TM(Pro) mice. CONCLUSIONS: Metastasis in the hyperthrombotic TM(Pro) mouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells.

Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?

BACKGROUND: Tumor-derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor-derived TF in this model. METHODS: Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin-antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. RESULTS: Particles in both CM and plasma were < 0.4 µm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 µm. Injection of < 0.1 µm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor-bearing mice plasmas was non-sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 µm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. CONCLUSIONS: Tumor-derived particles of < 0.1 µm and non-sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.

Splice variants of tissue factor promote monocyte-endothelial interactions by triggering the expression of cell adhesion molecules via integrin-mediated signaling.

BACKGROUND: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. OBJECTIVE: To examine the role of integrin signaling in TF-dependent recruitment of monocytes by endothelial cells. METHODS: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ-TF) and recombinant asTF were assessed. RESULTS: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid-rich plaques. Tumor lesions, as well as stromal CD68(+) monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ-TF, and this was completely blocked by anti-ß1 integrin antibody. asTF- and LZ-TF-treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF-elicited adhesion was more pronounced than that elicited by LZ-TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E-selectin, ICAM-1 and VCAM-1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3-fold under MCP-1 gradient. CONCLUSIONS: TF splice variants ligate ß1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non-proteolytic, integrin-mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Bone morphogenetic protein -7 increases thrombogenicity of lipid-rich atherosclerotic plaques via activation of tissue factor.

Thrombogenicity of atherosclerotic plaques largely depends on plaque morphology. Tissue factor (TF) expression is higher in lipid-rich than in calcified lesions. Although bone morphogenetic protein (BMP) -7 is a known inhibitor of vascular calcification, the role of BMP-7 in the development of plaque thrombogenicity is uncertain. We hypothesized that increased thrombogenic potential of lipid-rich plaques is attributed to activation of TF by BMP-7. We measured levels of BMP-7 and TF proteins in lipid-rich and calcified carotid plaques, and tested the effects of BMP-7 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-7 revealed that lipid-rich plaques contained more TF antigen than calcified ones (158.6±25.3 vs 37.4±8.8 AU, p<0.008). Lipid-rich plaques also expressed higher levels of BMP-7 (60.7±5.2 AU) than calcified lesions (31.8±8.6 AU, p<0.021). In vitro treatment of whole blood with BMP-7 markedly increased the population of TF-positive monocytes from 1.5±0.6 % to 31.0±7.6 % (p<0.001). Stimulation of blood with BMP-7 was accompanied by elevated surface presentation of TF antigen in monocytes as TF-dependent fluorescence intensity increased from 5.0±2.6 AU in unstimulated conditions to 15.8±1.9 AU after incubation with BMP-7 (p<0.002). Our data suggest that BMP-7 contributes to increased thrombogenicity of lipid-rich plaques via enhancement of TF expression.

Alternatively spliced tissue factor induces angiogenesis through integrin ligation.

The initiator of coagulation, full-length tissue factor (flTF), in complex with factor VIIa, influences angiogenesis through PAR-2. Recently, an alternatively spliced variant of TF (asTF) was discovered, in which part of the TF extracellular domain, the transmembrane, and cytoplasmic domains are replaced by a unique C terminus. Subcutaneous tumors produced by asTF-secreting cells revealed increased angiogenesis, but it remained unclear if and how angiogenesis is regulated by asTF. Here, we show that asTF enhances angiogenesis in matrigel plugs in mice, whereas a soluble form of flTF only modestly enhances angiogenesis. asTF dose-dependently upregulates angiogenesis ex vivo independent of either PAR-2 or VIIa. Rather, asTF was found to ligate integrins, resulting in downstream signaling. asTF-alphaVbeta3 integrin interaction induces endothelial cell migration, whereas asTF-dependent formation of capillaries in vitro is dependent on alpha6beta1 integrin. Finally, asTF-dependent aortic sprouting is sensitive to beta1 and beta3 integrin blockade and a TF-antibody that disrupts asTF-integrin interaction. We conclude that asTF, unlike flTF, does not affect angiogenesis via PAR-dependent pathways but relies on integrin ligation. These findings indicate that asTF may serve as a target to prevent pathological angiogenesis.

SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells.

BACKGROUND: Human monocytes express two naturally occurring forms of circulating tissue factor (TF) - full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. OBJECTIVE: To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. METHODS AND RESULTS: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87-117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. CONCLUSIONS: SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87-117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.

Identification and characterization of murine alternatively spliced tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that initiates coagulation and plays a critical role in regulating hemostasis and thrombosis. We have recently reported a naturally occurring, soluble form of human tissue factor (asTF) generated by alternative splicing. This splice variant has a novel C-terminus with no homology to that of the full-length TF (flTF), lacks a transmembrane domain, and is active in the presence of phospholipids. Mouse models offer unique opportunities to examine the relative importance of flTF and asTF in mediating thrombosis, the response to arterial injury, and ischemic damage. To that end, we have identified and characterized murine asTF (masTF). Like the human splice variant, masTF lacks a transmembrane domain and has a unique C-terminus. We have generated antibodies specific to masTF and murine flTF (mflTF) to examine the expression of both forms of TF. masTF antigen is widely and abundantly expressed, with a pattern similar to that of mflTF, in adult tissues, in experimentally induced thrombi, and during development. These studies demonstrate that masTF contributes to the pool of total TF and may thus play an important role in mediating TF-dependent processes.

Platelet-derived growth factor-specific regulation of the JE promoter in rat aortic smooth muscle cells.

JE is a member of the family of "immediate early" genes induced by growth factors and cytokines. JE encodes a low molecular weight secretory glycoprotein analogous to the human monocyte chemoattractant protein, MCP-1. JE and MCP-1 proteins are thought to play an important role in inflammation and in the recruitment of monocyte/macrophages to the vessel wall during the development of atherosclerosis. We have previously reported that the induction of JE in rat aortic smooth muscle cells (SMC) was specific to platelet-derived growth factor (PDGF) and was not seen with other growth agonists. Using a luciferase reporter system and transient transfection assays of rat aortic SMC, we now report the identification of a region in the proximal rat JE promoter that is responsive to PDGF but not to other growth factors (angiotensin II and alpha-thrombin) or cytokines (interleukin 1-beta and tumor necrosis factor-alpha). The full response to PDGF (approximately 6-fold) requires the cooperative activity of two potentially novel cis-acting elements, at positions -146 to -128 and -84 to -59. While each element produces a different pattern in electrophoretic mobility shift assays, they appear to bind the same PDGF-responsive species. Further analysis of these regions should provide important insights into PDGF-specific responses in vascular SMC.

Secretion of monocyte chemotactic activity by cultured rat aortic smooth muscle cells in response to PDGF is due predominantly to the induction of JE/MCP-1.

Inflammation is a critical feature of atherosclerosis and is characterized in part by the migration of circulating monocytes to the atherosclerotic plaque. These monocytes, together with macrophages, are a source of cytokines, growth factors, proteases, and procoagulants, which contribute to the progression of the atherosclerosis lesion. This study employed a modified Boyden chamber to examine the secretion of monocyte chemotactic activity by cultured rat aortic vascular smooth muscle cells in response to growth factors and cytokines. The induction of monocyte chemotactic activity showed a surprising specificity for platelet-derived growth factor-BB. This activity was blocked by actinomycin D and cycloheximide and thus required de novo transcription and protein synthesis. The ability to stimulate monocyte migration appeared to be solely due to the secretion of the monocyte chemoattractant protein JE/MCP-1 and was completely blocked by antisense oligonucleotides and antibodies to JE/MCP-1. The induction of chemotactic activity was also blocked by dexamethasone, an inhibitor of JE mRNA accumulation. This study suggests that the secretion of monocyte chemotactic activity by vascular smooth muscle cells is a highly regulatable and specific event and underscores the importance of JE/MCP-1 in the inflammatory response of the vessel wall.