Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT 1-R͞cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT 2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD 4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT 2-R-and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at Ϸ60 min, were unaffected by a cysLT 1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD 4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT 2-R activation results in a proinflammatory EC phenotype. Because LTD 4 and thrombin are likely to be formed concomitantly in vivo, cysLT 2-R and PAR-1 may cooperate to augment vascular injury.cysteinyl leukotriene 2 receptor gene signature ͉ protease-activated receptor 1 gene signature ͉ vascular inflammation L eukotrienes (LTs), i.e., LTB 4 and the cysteinyl LTs (cysLT) LTC 4 , LTD 4 , and LTE 4 constitute a group of lipid mediators derived from the 5-lipoxygenase (5-LO) pathway (1, 2). LTs are either produced by leukocytes at sites of inflammation or formed through transcellular metabolism after uptake and metabolism of leukocyte-derived LTA 4 by downstream enzymes of the 5-LO pathway (LTA 4 hydrolase and LTC 4 synthase) in cells that normally do not express 5-LO, such as endothelial cells (ECs) (3, 4). LTs act through G protein-coupled surface receptors (GPCRs), i.e., the LTB 4 receptors and the cysLT receptors (LT-Rs) (cysLT 1 -R and cysLT 2 -R) (5-10). LT-Rs are expressed on multiple target cells, including leukocytes, smooth muscle cells, and ECs (1). Recent studies implicate the 5-LO pathway in cardiovascular disease (11)(12)(13)(14)(15)(16)(17).Considerable information is available on cysLT 1 -R, whereas little is known about cysLT 2 -R. We have used human umibilical vein (HUV)ECs as a model of vascular cells to study cysLT 2 -R activation by demonstrating that cysLTs exclusively signal through cysLT 2 -R in this cell type (18): In fact, HUVECs are the first primary cell type that s...

Objective-Inflammatory infiltrates and atherosclerotic lesions emerge when monocytes adhere to endothelial cells (ECs), migrate into the subendothelial space, and become macrophages (M⌽s). Leukotrienes (LTs), products of 5-lipoxygenase, are powerful inflammatory mediators. 5-lipoxygenase ϩ M⌽s have been shown to increase during atherogenesis, and LT receptor (LT-R) transcripts were identified in diseased arteries. To investigate LT-Rs in cells involved in inflammation and atherogenesis, we used the in vitro models of human umbilical vein ECs (HUVECs) and monocyte-derived M⌽s. Methods and Results-HUVECs primarily expressed transcripts of the cysteinyl (cys) LT 2 -R, which was strongly upregulated by interleukin-4. By contrast, M⌽s predominantly expressed transcripts of the cysLT 1 -R. Calcium responses toward LTs revealed differential cysLT-R utilization by both cell types: HUVECs responded to both cysLTs, whereas M⌽s preferentially responded to LTD 4 ; HUVECs, but not M⌽s, were resistant toward a cysLT 1 -R antagonist, montelukast; cysLTs generated regular calcium oscillations in HUVECs that lasted Ͼ60 minutes, resulting in Ͼ500 oscillations per cell. By contrast, calcium elevations in M⌽s returned to baseline within seconds and were nonoscillatory. Conclusions-Our data raise the possibility that M⌽-derived LTs differentially activate cysLT 2 -Rs via paracrine stimulation and cysLT 1 -Rs via autocrine and paracrine stimulation during inflammation and atherogenesis. 4 and LTC 4 , namely, cysLT-Rs. 5,6 Whereas LTB 4 mediates chemotaxis, 3 cysLTs trigger a variety of tissue responses, including increases in vascular permeability. 7 CysLT 1 -R is primarily present in the spleen, blood leukocytes, and lung macrophages (M⌽s), 5 whereas cysLT 2 -R is expressed in the heart and brain. 6 However, the precise cell lineage-specific expression of LT-Rs, their mode of regulation, and the signaling pathways that they trigger remain to be determined.We observed transcripts of all 4 LT-Rs in human atherosclerotic lesions. 8 These data raised the possibility that the 5-lipoxygenase (5-LO) pathway forms foci of inflammation within the arterial wall. Because human umbilical vein endothelial cells (HUVECs) and M⌽s have been used to study inflammation and recent studies have associated the 5-LO pathway with experimental atherosclerosis, 9 -15 we examined their LT-R expression patterns and characterized their calcium responses. http://atvb.ahajournals.org/ Downloaded from fura 2-AM, from Calbiochem; fluo 4-AM, from Molecular Probes; transforming growth factor-␤1 (TGF ␤-1), interleukin-1␤ (IL-1␤), tumor necrosis factor-␣ (TNF-␣), and IL-4, from R&D Systems; LTs, from Cayman Chemicals; CD14 MicroBeads, from Miltenyi Biotech; and other reagents, from Sigma. Monocytes were purified from peripheral blood mononuclear cells by adherence or CD14 microbead adsorption and maintained at 1ϫ10 6 cells/mL for 5 to 9 days in RPMI-1640 medium plus 20% autologous human serum. HUVECs were cultured as described 16 and used at passages 1 to 2. Carotid ...

Abstract-There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI 2 ) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI 2 .Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smooth muscle cells by prostaglandins, endogenously formed via the COX-2 pathway. Because TM is an important inhibitor of blood coagulation, these findings provide a novel platelet-independent mechanism to explain the prothrombotic effects of COX-2 inhibitors.

This study investigates, whether in addition to the protease‐activated receptor‐1 (PAR‐1), PAR‐4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR‐1 and PAR‐4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. mRNAs for both, PAR‐1 and PAR‐4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT – PCR). Treatment of the SMC with GYPGQV (200 μM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. Stimulation of the SMC with 10 nM thrombin or 200 μM SFLLRN caused a time‐dependent activation of the extracellular signal‐regulated kinases‐1/2 (ERK‐1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 μM of GYPGQV induced a prolonged phosphorylation of ERK‐1/2 with a maximum at 60 min. These data suggest that PAR‐1 and PAR‐4 are activated by thrombin at distinct concentrations and with distinct kinetics. GYPGQV stimulated [3H]‐thymidine incorporation in SMC. At 500 μM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 μM SFLLRN, respectively. These data indicate that a functionally active PAR‐4 is present in SMC and, in addition to PAR‐1, might contribute to thrombin‐induced mitogenesis. British Journal of Pharmacology (2001) 132, 1441–1446; doi:

Abstract-Pro-matrix metalloproteinase-2 (pro-MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro-MMP-2 (72 kDa) into MMP-2. Pro-MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro-MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). Key Words: matrix metalloproteinase-2 Ⅲ factor Xa Ⅲ vascular smooth muscle cells Ⅲ extracellular matrix invasion Ⅲ mitogenesis M atrix metalloproteinases (MMPs) are a family of structurally related zinc-endopeptidases. MMPs are thought to play an important role in the physiological turnover of extracellular matrix (ECM) components. This includes embryonic tissue morphogenesis, tissue repair, and angiogenesis. In pathological conditions such as atherosclerosis, arthritis, glomerulonephritis, gastric ulcer, tumor invasion, and metastasis, MMPs are also involved in ECM degradation.

Objective-Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors. Methods and Results-In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4 -activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor ␣ gene expression. This enhancement of thrombinstimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown. PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-␤ and -␦ isoforms or nuclear factor (NF)B. Nuclear translocation of NFB in high glucose-stimulated SMCs led to PKC-dependent NFB binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels. Key Words: diabetes mellitus Ⅲ thrombin Ⅲ vascular muscle Ⅲ protease-activated receptors C hronically elevated plasma glucose levels in diabetes are associated with cardiovascular complications such as vascular remodeling and poor outcome after revascularization. 1,2 At the cellular level, hyperglycemia is a potent stimulus for the proliferation and migration of vascular smooth muscle cells (SMCs), 3 which are major factors contributing to vascular remodeling and accelerated atherosclerosis. 4 In addition, diabetes represents a hypercoagulable state associated with enhanced thrombin generation and increased risk of thrombotic complications. 5 Thrombin is the central component of the coagulation cascade that becomes activated when vascular injury allows contact between blood-borne components and tissue factor-expressing cells such as fibroblasts and SMCs. 6 However, the overwhelming majority of total thrombin generated is released by the thrombus after clotting is completed, 6,7 indicating functions beyond coagulation. Conclusion-HighThrombin can exert direct coagulation-independent actions such as SMC migration and proliferation through activation of a unique family of G protein-coupled receptors, the protease-activated receptors (PARs). 8 PARs are activated through proteolytic cleavage of the extracellular N terminus, which unmasks a new N terminus that acts as a tethered ligand to autoactivate the receptor. 9 Synthetic hexapeptides mimicking this tethered ligand can elicit most of the biological actions of thrombin independently of receptor cleavage. Of the 4 PARs identified to date, PAR-1, PAR-3, and PAR-4 are activated by thrombin, whereas another receptor, PAR-2, is activated by other proteases such as activa...

1 This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinaseactivated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2 Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar e ect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic e ect. 3 The speci®c PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4 When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca 2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5 Trypsin (2 nM) as well as SLIGRL (100 mM) activated the nuclear factor kB (NFkB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkB by thrombin (10 nM) was detected after 4 ± 5 h. 6 These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkB via either PAR-1 or PAR-2 does not predict mitogenesis.

Abstract-The endothelial cell protein C receptor (EPCR) is expressed on endothelial cells and regulates the protein C anticoagulant pathway via the thrombin-thrombomodulin complex. Independent of its anticoagulant activity, activated protein C (APC) can directly signal to endothelial cells and upregulate antiapoptotic and antiinflammatory genes. Here we show that vascular smooth muscle cells (SMCs) also express EPCR. EPCR protein on SMCs was detected by flow cytometry and Western blotting. EPCR mRNA was identified by quantitative RT-PCR. To examine the functionality of EPCR, intracellular signaling in APC-stimulated SMCs was analyzed by determination of intracellular free calcium transients using confocal laser scanning microscopy. Phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) was detected by immunoblotting. APC-induced ERK-1/2 phosphorylation was inhibited by an anti-EPCR antibody and by a cleavage site blocking anti-PAR-1 antibody, indicating that binding of APC to EPCR and cleavage of protease-activated receptor-1 (PAR-1) were involved. APC elicited an increase in