The vasodilator effects of thrombin depend on activation of proteinase-activated receptor (PAR)-1 and the subsequent release of endothelin (ET)-1, which stimulates the generation of nitric oxide and PGs. We recently showed that thrombin released matrix metalloproteinase-2 (MMP-2) from rat arteries. We have now studied the significance of this release for the vasodilator effects of thrombin. Thrombin (≥100 pmol), but not a PAR-1-activating peptide (TFLLR-NH2), produced a long-lasting (>10 min) vasorelaxation of rat mesenteric arteries, as detected by a microperfusion bioassay. Thrombin induced a simultaneous release of vascular MMP-2 into arterial perfusates, as revealed by zymography. Interestingly, the vasodilator effects of thrombin were inhibited by a tissue inhibitor of MMP-2 (TIMP-2, 10 pmol). Moreover, infusion of exogenous MMP-2 (5 pmol) resulted in vasorelaxation. These vasodilatory effects of thrombin and MMP-2 were significantly (P < 0.05) inhibited by endothelium denudation and by PD-142893 (2 nmol), an antagonist of ET receptors. Furthermore, both thrombin and MMP-2 constricted endothelium-denuded arteries. These results show that the vasodilator effects of thrombin may depend, in part, on a release of vascular MMP-2 and downstream activation of ETs. Thus MMP-2-dependent signaling may complement the PAR-1-dependent pathway of vasodilator action of thrombin.
- nitric oxide
the serine protease thrombin, when generated at sites of vascular injury, modulates both hemostasis and vessel wall contractility (7). An exaggerated activation of platelets or the vessel wall by thrombin can result in thrombosis, inflammation, and vasospasm (7, 12, 25, 26). Therefore, characterization of thrombin signaling is crucial to the understanding and treatment of the vascular conditions associated with increased generation of this enzyme.
Recent investigations have implicated G protein-coupled protease-activated receptors (PARs) in a first stage of thrombin signaling (1, 12, 25, 26). Three PAR-type receptors (PAR-1, -3, and -4) have been found to mediate actions of thrombin in various systems (1,12, 25, 26). Signaling via PAR-1 has received the most attention (25). PAR-1 is activated when thrombin cleaves its NH2-terminus (25). This new NH2-terminus then serves as a tethered peptide ligand that binds to the remainder of the receptor molecule, allowing transmembrane signaling. Synthetic peptides that mimic this tethered ligand (e.g., TFLLR-NH2) can trigger PAR-1-mediated signaling and can reproduce many biological actions of thrombin (1, 12, 25). Indeed, PAR-1-activating peptides mimic the endothelium-dependent effects of thrombin on vascular reactivity (2,16, 17, 22). Activation of PAR-1 on the vascular smooth muscle results in vasoconstriction, whereas stimulation of PAR-1 on the endothelium relaxes precontracted arteries (16, 17). This vasorelaxation is modulated by a concomitant release of small quantities of endothelin (ET)-1-like peptide(s) (16, 17). Subsequent binding of ET-1 peptide(s) to ETB receptors on the surface of endothelial cells presumably triggers nitric oxide (NO) and prostacyclin (PGI2) production, thus modulating vasodilator effects of thrombin (16, 17, 23). However, it has not been clarified how PAR-1 activation results in the release of biologically active ET-1-like peptide(s) (i.e., capable of binding to ETB). Indeed, ET peptides are biosynthesized as essentially inactive precursors (prepro-ET-1, pro-ET-1, and Big ET-1) requiring exocytosis and activation by specific proteases (5, 23, 27, 28).
Recently, we found that Big ET-1 can also be activated by matrix metalloproteinase-2 (MMP-2, gelatinase A; see Ref. 8), a protease expressed by the smooth muscle and the endothelium for the (patho)physiological breakdown of specific extracellular matrix proteins (collagens, proteoglycan, laminin-5, and elastin; see Ref.21). Cleavage of big ET-1 by MMP-2 rendered a novel vasoactive peptide, ET-1-(1—32), that binds to ETB receptors to express its vasoactivity (8). Moreover, we have observed that thrombin induces a rapid release of MMP-2 from arteries (9). Thus we have hypothesized that thrombin-induced vasodilation is mediated, at least in part, by vascular MMP-2, suggesting a mechanism by which bioactive ETs [particularly, ET-1-(1—32)] could be released in response to thrombin.
MATERIALS AND METHODS
Human recombinant pro-MMP-2, a tissue inhibitor of MMP-2 (TIMP-2), and affinity-purified polyclonal MMP-2 antibody (immunoreactive against the hinge region of MMP-2) were obtained from Chemicon International (Missisauga, ON, Canada). Control rabbit IgG,l-phenylephrine, PD-142893, and ET-1 fragment (Cys11-Trp21) were obtained from Sigma (Oakville, ON, Canada). TFLLR-NH2 was prepared by solid-phase synthesis, purified by HPLC, and characterized by mass spectrometry and amino acid analysis (Peptide Synthesis Facility, University of Calgary). Dr. Morley Hollenberg (Departments of Pharmacology and Therapeutics, University of Calgary, Canada) kindly provided this peptide.
Microperfusion bioassay of the rat mesenteric artery.
The vasodilator effects of thrombin were studied using rat mesenteric arteries mounted on a microperfusion bioassay (14) and preconstricted 40–60% using phenylephrine (100 nM). Animal protocols were conducted in accordance with institutional guidelines issued by the Canada Council on Animal Care. Male Sprague-Dawley rats (350–450 g) were anesthetized with methohexital sodium (50 mg/kg) and exsanguinated. A section of mesentery (5–10 cm) distal to the pylorus was rapidly removed and placed in ice-cold HEPES-phosphate saline solution [PSS, pH 7.4; composition (in mM) 142 NaCl, 4.7 KCl, 1.17 MgSO4, 1.56 CaCl2, 10 HEPES, 1.18 KH2PO4, and 5.5 glucose]. Arteries were dissected from fat tissue and adventitia and were transferred to a dual-chamber arteriograph (Living Systems Instrumentation, Burlington, VT). Arteries (209 ± 12-μm ID, 1- to 1.5-mm length) were mounted on pulled glass cannulas (1-mm OD, 0.5-mm ID) and were tied on both ends. All experiments were conducted on arteries cannulated and perfused at constant temperature (37°C) and flow rate (10 μl/min) with standard HEPES-PSS, pH 7.4, supplemented with glucose (5.5 mM). In some experiments, the arteries were denuded of endothelium. Endothelium removal was performed mechanically using a human hair threaded through the lumen of the artery and rubbed back and forth (20). To confirm the absence of endothelial function, the artery was first preconstricted by superfusion with phenylephrine (1–5 μM) to reduce the artery inner diameter to 50 ± 5% of its resting value. Next, the preconstricted artery was tested for the absence of relaxation to methacholine chloride (1 μM) added to the superfusion buffer. This concentration of methacholine relaxed arteries with an intact endothelium. Phenylephrine-preconstricted arteries maintained the tone over a period of at least 1 h.
Small volumes (1–5 μl) of thrombin, TFLLR-NH2, recombinant MMP-2, TIMP-2, MMP-2 antibody, control IgG, or specified drugs were injected in the perfusion line toward the preconstricted artery (see above). In studies of dose dependence to thrombin, increasing doses of thrombin (1–400 pmol or 0.05–20 units) were injected toward the arteries. The different inhibitors used showed no significant effect on the diameter of phenylephrine-constricted arteries. TIMP-2 (up to 1 μM) did not inhibit the proteolytic activity of thrombin, as tested by the conversion of fibrinogen (0.8 mg/ml) to insoluble fibrin by thrombin (0.2 U/ml). The injection of the drugs, without introducing flow rate change-related artifacts, was facilitated by using an HPLC injection valve (Rheodyne model 9725I; Mandel Scientific, ON, Canada) provided with a 20-μl loop. All tubings in contact with the perfusion buffer or drugs were 0.010 in. (ID) and were made of inert polymer (PEEK; Mandel Scientific).
To determine whether thrombin-induced vasodilation was mediated by vascular MMP-2, thrombin (1–400 pmol) was infused in the arteries together with either TIMP-2 (10 pmol), an MMP-2 specific antibody (Chemicon), or control rabbit IgG (50 pmol each). TIMP-2 binds strongly to the COOH-terminus of MMP-2 to block the catalytic activity, not the release, of vascular MMP-2 that is expressed on the cell membrane (8,21, 24). The MMP-2 antibody binds to the hinge region of MMP-2 and would inhibit vascular MMP-2 by affecting its native folded structure (21).
To test the role of PAR-1 receptor, in other similar experiments, thrombin was replaced by a synthetic PAR-1-activating peptide (TFLLR-NH2, 2–200 pmol).
To assess the involvement of ET receptors in the vasodilator effects of thrombin and MMP-2 on mesenteric arteries, an ETB receptor antagonist [the ET-1 fragment (Cys11-Trp21)] or the ETA/Bantagonist PD-142893 was injected (2 nmol each) in the line. The inhibitors were allowed to perfuse the artery during 10 min at a flow rate of 1 μl/min (14). The flow rate was then restored to 10 μl/min. Five minutes later, thrombin (100 pmol) or MMP-2 (5 pmol) was coinfused with 2 nmol of the specified antagonist.
To determine the contribution of nitric oxide synthase (NOS) to the vasodilator effects of thrombin, the arteries were preincubated withN ω-nitro-l-arginine methyl ester (l-NAME, 100 μM) during 15 min before thrombin was infused through the arteries. The combined contribution of NOS and PGH synthase (PGHS) was assessed by preincubating the arteries inl-NAME (100 μM) and meclofenamate (1 μM) for 15 min before thrombin infusion.
For measuring gelatinolytic activity, the perfusate of the mesenteric artery was collected over a period of 10 min. Perfusates were immediately subjected to electrophoresis on 7.5% SDS-PAGE copolymerized with gelatin (2.5 mg/ml). After separation, the gel was washed with Triton X-100 (0.1%; 3 times, 20 min). The gelatinolytic activity was developed by first incubating the gel for 48 h (37°C) in enzyme assay buffer containing (in mM) 25 Tris, 5 CaCl2, 142 NaCl, and 0.5 Na3N and subsequently staining the gel with Coomassie blue. Gelatinases were identified as transparent bands against the background of Coomassie blue-stained gelatin.
Results are means ± SE of at least three independent experiments. They were analyzed using one-way ANOVA. When significant differences were found, the Tukey multiple-comparison's test was used (Jandel SigmaStat statistical software). Statistical significance was considered at P < 0.05.
Evidence for MMP-2 involvement in the vasodilator effects of thrombin on rat mesenteric arteries with intact endothelium.
Thrombin (1–400 pmol), when infused in rat mesenteric arteries, produced a dose-dependent vasorelaxation (Fig.1). The vasodilator effects of thrombin (≥100 pmol) persisted for at least 10 min after thrombin infusion was discontinued (Fig. 2 A). Thrombin effects were associated with a release of vascular MMP-2 (Fig.2 A, top; see Ref. 9).
To determine whether MMP-2 contributes to vasodilator effects of thrombin, the effects of coinfusing a TIMP-2 with thrombin were tested. TIMP-2 (10 pmol) inhibited the vasorelaxation response to thrombin (100 pmol; Fig. 2 B). In addition, MMP-2 antibody (50 pmol), but not control IgG, inhibited thrombin-induced vasorelaxation (by 51.1 ± 7.8%).
The contribution of PAR-1 was investigated next. Similar to thrombin, dose-dependent vasorelaxation was observed when a PAR-1-activating peptide (TFLLR-NH2, 20–200 pmol) was infused (Fig.3 A). In contrast to thrombin, this vasorelaxation was short (<3 min) lasting (Fig. 3 B) and was not accompanied by MMP-2 release neither on mesenteric arteries (Fig.3 B, top) nor on the rat aorta (9). Moreover, TIMP-2 did not inhibit TFLLR-NH2-induced vasorelaxation (Fig.3 B). These data indicated that the vasoactive effects of TFLLR-NH2 are not dependent on vascular MMP-2 activity.
ET receptor mediates thrombin-induced vasodilation.
Because MMP-2 can cleave big ET-1, yielding ET-1-(1—32) that acts on ETB receptor to express its vasoactivity (8), the contribution of this receptor to vasodilator effects of thrombin on mesenteric arteries was studied. In the presence of a selective antagonist of ETB receptor, the ET-1 fragment (Cys11-Trp21, 2 nmol), thrombin-induced vasodilation was abolished, and vasoconstrictor effects of thrombin were observed (Fig. 4). Similar results were obtained with PD-142893, a structurally unrelated potent antagonist of ETA and ETB receptors (data not shown).
Vasodilator PGs and NO.
Vasodilator effects of thrombin were inhibited by l-NAME (100 μM), a cell-permeable inhibitor of NOS (Fig.5 A). Moreover, in the presence of l-NAME and meclofenamate (1 μM), an inhibitor of PGHS, thrombin-induced vasodilation was abolished, and vasoconstrictor effects were apparent (Fig.5 A).
Removal of the endothelium abrogated the vasodilator effects of thrombin, unmasking the vasoconstrictor effects of thrombin (Fig. 5,A and B). These effects were small regardless of the artery tone (data not shown) and were associated with a release of vascular MMP-2 (Fig. 5 B, top); this release of MMP-2 was similar to that seen on arteries with intact endothelium (9).
Vasoactive effects of MMP-2.
To determine whether or not a release of vascular MMP-2 by thrombin could modulate vascular reactivity, recombinant human MMP-2 was infused in arteries. MMP-2 induced a significant and long (>5 min)-lasting vasodilation (Fig. 6 A). This effect was significantly reduced in the presence of an ET receptor antagonist (Fig. 6 B) and was abolished when the endothelium was removed. Indeed, MMP-2 constricted endothelium-denuded arteries (Fig.6 C).
The most significant biological effects of MMP-2 have been associated with its ability to cleave proteins of the extracellular matrix (collagens, proteoglycans, laminin-5, elastin; see Ref. 21). This investigation has revealed that vascular MMP-2 may also mediate effects of thrombin on the reactivity of rat mesenteric arteries, which are transduced in parallel to the classical thrombin receptor, PAR-1. First, we have studied a contribution of MMP-2 to the effects of thrombin in arteries with intact endothelium. Under these conditions, thrombin induced long-lasting vasodilator effects and a dose-dependent release of vascular MMP-2. The vasodilator effects were mimicked by an infusion of recombinant MMP-2 but were inhibited by TIMP-2 (an inhibitor of MMPs with preferred effects on MMP-2; see Ref. 21) and by MMP-2 specific antibody. The pharmacological profile of TFLLR-NH2 was, however, different from that of thrombin. The vasorelaxation evoked by this peptide was short lasting, not associated with a release of MMP-2, and not inhibited with TIMP-2. These results clearly show that, although the vasodilator effects of TFLLR-NH2 are MMP-2 independent, those of thrombin require the activity of vascular MMP-2.
MMP-2 is released from arteries mainly as a latent form (pro-MMP-2) that is activated during a cell membrane event (21). Recently, we have studied the downstream events after the generation of MMP-2 in rat mesenteric arteries. MMP-2 was shown to cleave big ET-1, yielding a novel vasoactive form, ET-1-(1—32), whose effects are mediated by ET receptors (8). Interestingly, pharmacological antagonists of ET receptors inhibited the vasodilator effects of thrombin and significantly reduced MMP-2-induced vasodilatation. Moreover, endothelium removal abolished the vasodilator effects of MMP-2 as well as those of thrombin. These results suggest the involvement of endothelial ETB receptors in the vasodilator effects of both thrombin and MMP-2, which is released by thrombin from the vessel wall. Therefore, we suggest that vascular MMP-2, once released by thrombin, may contribute to thrombin-induced vasodilatation by generating ET-1-(1—32). Binding of this ET-1-(1—32) to endothelial ETB receptors would transduce vasodilator effects of thrombin, as shown in Fig. 7. These results are in agreement with recent reports (16, 17) showing that thrombin stimulates the generation and release of vasoactive ET-1-like peptide(s) capable of activating ETB receptors, one of which could be ET-1-(1—32). Importantly, when thrombin was incubated with Big ET-1, no cleavage of Big ET-1 was detected (data not shown). This result excludes a possibility that thrombin could directly cleave Big ET-1 to yield a vasoactive peptide. Whether or not MMP-2 cleaves other peptides/proteins distinct of big ET-1 to contribute to the vasodilator effects of thrombin is under current investigation in our laboratory.
Taken together, our data suggest that thrombin-induced vasorelaxation is transduced through MMP-2-dependent and MMP-2-independent (e.g., PAR-1-dependent) pathways that merge at the level of ETBreceptors. Downstream of these receptors, NO and vasodilator PG(s) are produced, since inhibitors of NOS and PGHS abolished thrombin-induced vasorelaxation (Fig. 7). This is consistent with previously reported data whereby thrombin stimulation of the vascular endothelium triggers a production of endothelium-derived vasodilators (mainly NO and PGI2; see Refs. 1, 2, 7, 14, 16, 17, 22).
Vascular MMP-2 was released by thrombin from arteries denuded of, as well as with intact, endothelium (present data and Ref. 9). Hence, it is likely that thrombin releases vascular MMP-2 mainly, although not solely, from smooth muscle cells (9). Indeed, on mesenteric arteries, MMP-2-like immunoreactivity and net gelatinolytic activity colocalize mainly to medial and intimal smooth muscle cells (8). However, the exact mechanism by which thrombin induces a release of MMP-2 is not completely clear. There is evidence indicating that PAR-1 receptor does not mediate the MMP-2-releasing action of thrombin (9, 21, 29). This is in agreement with the data obtained with TFLLR-NH2 and suggests the involvement of an as yet undetermined thrombin receptor(s) distinct of PAR-1 (perhaps, PAR-3 or PAR-4; see Refs. 1, 3, 9, 12, 29and references therein). Thrombin itself does not activate pro-MMP-2 (9, 29). In turn, it is likely that thrombin induces a release of pro-MMP-2 (9) that is subsequently and rapidly activated by a different protease(s) from the vessel wall (e.g., membrane-type MMP; see Refs. 10and 21). Consistent with a contribution of vascular MMP-2 to short-term vasoactive effects of thrombin is the fact that the basal and thrombin-induced release of MMP-2 is differentially and rapidly modulated by vascular protein tyrosine kinase/phosphatase activity (9). Interestingly, an MMP-2-releasing action of thrombin has been demonstrated for macrovascular and microvascular endothelial and smooth muscle cells in culture (4, 18, 29). Yet, thrombin may not induce a release of MMP-2 from some cell types such as human mesangial cells (13).
MMP-2 was found to constrict arteries denuded of endothelium. This observation suggests that a release of MMP-2 by thrombin at sites of endothelial injury may contribute to vasoconstriction. Previous research suggested that thrombin-induced vasoconstriction is partly dependent on the activity of ET-1-like peptide(s), which act on ETA and ETB receptors in the smooth muscle to produce vasoconstriction (16, 17). Accordingly, an MMP-2-dependent conversion of smooth muscle Big ET-1 to ET-1-(1—32) could contribute to vasoconstrictor effects of thrombin, since ET-1-(1—32) can act as a potent vasoconstrictor where the endothelium is damaged (8). Therefore, at sites of inflammation where the smooth muscle represents an important source of MMP-2 and Big ET-1, MMP-2-dependent vasoconstriction could be important (8, 21, 23). However, in addition to ET-1-like peptides (16, 17), other mediators likely contribute to the vasoconstrictor effects of thrombin (14). Importantly, endothelium-derived vasodilators (NO and PGs) were found to counteract the vasoconstrictor effects of thrombin. Therefore, the endothelial dysfunction associated with decreased production of NO and vasodilator prostaglandins (e.g., at sites of tissue injury) is likely to lead to vasospasm and is likely to contribute to the ischemic injury (14-17, 26).
Recent studies have shown that thrombin and ET-1 can induce angiogenesis, cell migration, and proliferation (3, 15, 19). These processes are important in the response to vascular injury and in the remodeling of the vasculature during embryogenesis. Interestingly, these processes are also associated with an upregulation of MMP-2-dependent degradation of the extracellular matrix (21). This association suggests that the thrombin-MMP-2-ET-(1—32) pathway of signal transduction plays a role during these events. Vasculopathies such as preeclampsia, atherosclerosis plaque rupture, and restenosis are associated with the generation of growth factors, thrombin, MMP-2, and ET-1-like peptides (3, 15, 19, 21, 23). The combined activity of these mediators in the vessel wall-platelet microenvironment is likely to alter vascular reactivity and promote platelet activation, smooth muscle cell migration and proliferation, and exaggerated expansion of the extracellular matrix (3, 15, 19, 21, 24).
We conclude that the vasodilator effects of thrombin involve at least two pathways of signaling that proceed in an MMP-2-dependent and MMP-2-independent (PAR-1-dependent) manner. Signaling via MMP-2 may be an important component of the (patho)physiological effects of thrombin.
C. Fernandez-Patron is a postdoctoral fellow of the Canadian Hypertension Society, Medical Research Council of Canada (MRC), and Alberta Heritage Foundation for Medical Research (AHFMR). M. W. Radomski is a scholar of the AHFMR. S. T. Davidge is a scholar of the AHFMR and the Heart and Stroke Foundation of Canada. This work was supported by a grant from the MRC to M. W. Radomski and grants from the MRC and the Heart and Stroke Foundation of Canada to S. T. Davidge.
Address for reprint requests and other correspondence: S. T. Davidge, Perinatal Research Centre, 232 HMRC, Depts. of Obstetrics/Gynaecology and Physiology, Univ. of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail:).
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