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Background: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease., Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3 , and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression., Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P =0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression., Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

Proteinase-activated receptors (PAR-2) are expressed by the cardiovascular system and mediate vasodilation, plasma protein extravasation, and endothelial cell proliferation, all regarded as essential steps for neovascularization. We investigated the angiogenic action of PAR-2 signaling in vivo. The effect of the PAR-2 activating peptide (PAR-2AP, SLIGRL-NH2) was assessed in the absence of ischemia, and the therapeutic potential of PAR-2AP and the PAR-2 agonist trypsin (at 300 and 1.5 nmol IM daily for 21 days, respectively) was also tested in mice subjected to unilateral limb ischemia. PAR-2AP increased capillarity in normoperfused adductor skeletal muscles, whereas neither the vehicle of the PAR2-AP nor the PAR-2 reverse peptide (PAR-2RP, LRGILS-NH2) did produce any effect. In addition, both PAR-2AP and trypsin enhanced reparative angiogenic response to limb ischemia, an effect that was not produced by PAR-2RP or the vehicle of PAR-2 agonists. Potentiation of reparative angiogenesis by PAR-2AP or trypsin resulted in an accelerated hemodynamic recovery and enhanced limb salvage. In conclusions, our study is the first to demonstrate the angiogenic potential of PAR-2 stimulation in vivo. If similar effects occur in humans, PAR-2AP agonists could have some therapeutic potential for the treatment of tissue ischemia.

Protease-activated receptor (PAR)1 and PAR2 are expressed on vascular endothelial cells and mediate endothelium-dependent relaxation in several species, and PAR4 agonists cause similar responses in rat aortas. To date, only PAR1 has been reported to mediate relaxation of human arteries despite endothelial cell expression of both PAR1 and PAR2 in these tissues. Because inflammatory stimuli increase PAR2 expression in human endothelial cells in culture, the present study investigated the effect of similar stimuli on PARs in human isolated coronary arteries (HCAs). In HCA ring segments suspended for isometric tension measurements, the selective PAR1-activating peptide, TFLLR (0.01 to 10 micromol/L), caused endothelium-dependent relaxation of precontracted preparations. Little or no change in vascular tension was elicited by either the PAR2- or PAR4-activating peptides, SLIGKV and GYPGQV, respectively (up to 100 micromol/L). Exposure of HCAs to interleukin (IL)-1alpha (1 ng/mL, 12 hours) or tumor necrosis factor-alpha (3 nmol/L, 12 hours) did not affect PAR1 expression but increased PAR2 and PAR4 mRNA levels by approximately 5- and 4-fold, respectively, as determined by quantitative polymerase chain reaction. Similar IL-1alpha treatment did not affect TFLLR-induced relaxations but revealed significant endothelium-dependent relaxations to SLIGKV (100 micromol/L, 61.4+/-6.7%) and GYPGQV (100 micromol/L, 34.8+/-6.4%). These studies are the first to demonstrate functional PAR2 and PAR4 in human arteries in situ. The selective upregulation of PAR2 and PAR4 expression and the increased vascular response in HCAs after exposure to inflammatory stimuli suggest a role for these endothelial receptors during inflammation.

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Thrombin has been implicated as an important mediator of vascular lesion formation in atherosclerosis and restenosis. To investigate a potential role for thrombin signaling in the vascular response to hypertension, we have studied thrombin receptor (TR) expression and regulation in hypertensive rats. Aortic TR mRNA was upregulated by angiotensin II (Ang II)-induced hypertension (10.7 +/- 2.5 times control, P < .02), which correlated with a 4-fold increase in thrombin-induced constriction in isolated endothelium-denuded aortic rings. The AT1 receptor antagonist losartan normalized blood pressure and TR mRNA. Conversely, lowering blood pressure to the same degree with hydralazine did not abolish the upregulation of TR mRNA expression. When low-renin low-Ang II hypertension was induced in Dahl salt-sensitive rats, there was no detectable increase in the expression of aortic thrombin receptor mRNA. Finally, treatment with a chimeric heparin-binding form of the recombinant human Cu/Zn superoxide dismutase caused complete inhibition of TR mRNA upregulation, suggesting that an increased rate of superoxide anion production is an important signaling mechanism. Thus, increased TR expression via a redox-sensitive mechanism in the aortic smooth muscle of rats treated with Ang II represents a novel in vivo mechanism through which the hypertensive effects of Ang II are mediated.

Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

Blood vessels respond to injury by initiating cell proliferation and migration that result in vascular lesion formation. To determine the roles of thrombin and the thrombin receptor in this process, we characterized thrombin receptor expression in normal and injured arteries, thrombin receptor-mediated smooth muscle cell mitogenesis, and the regulation of thrombin receptor mRNA expression in vitro. Thrombin receptor mRNA was not detected in normal rat or baboon arteries by in situ hybridization. Immunohistochemistry using an antithrombin receptor antibody (TR-R9), directed against the thrombin cleavage site of the rat aortic smooth muscle cell thrombin receptor, revealed low-level staining for thrombin receptor protein in endothelial cells and smooth muscle cells of normal arteries. In contrast, balloon catheter injury increased thrombin mRNA expression in medial smooth muscle cells within 6 hours. This increased thrombin receptor expression continued within the media and in neointimal cells throughout vascular lesion formation, predominantly in areas of active cell proliferation. In vitro, alpha-thrombin stimulates rat aortic smooth muscle cell proliferation in a concentration-dependent manner. That thrombin receptor activation is required for the mitogenic response was confirmed by demonstrating that the polyclonal antibody TR-R9 inhibits thrombin-induced cell proliferation. Thrombin receptor mRNA synthesis was induced by both basic fibroblast growth factor (maximal stimulation of 1.8-fold at 1 hour) and platelet-derived growth factor (maximal stimulation of 2.4-fold at 8 and 24 hours) in quiesced cultured rat aortic smooth muscle cells. In summary, upregulation of smooth muscle cell thrombin receptor expression occurs very early after vascular injury and continues throughout neointimal development.(ABSTRACT TRUNCATED AT 250 WORDS)