Protease activated receptor 2 and the cardiovascular system

Protease activated receptors (PARs) represent a novel class of seven transmembrane domain G-protein coupled receptors activated by proteolytic cleavage. Up to now, four members of this class of receptors have been identified, PAR1 to PAR4 (Hollenberg, 1999). The mechanism for activation of PARs involves a proteolytic unmasking of an N-terminal sequence that acts as a tethered ligand. In the absence of proteolysis, PAR1, PAR2 and PAR4, but not PAR3, can be activated by synthetic peptides (PAR-activated peptides, PAR-APs) ranging between five and 14 aminoacids, whose sequence mimics the tethered ligand (Dery et al., 1998). The thrombin receptor PAR1 was the first PAR to be discovered and cloned (Vu et al., 1991). Successively, a second member of PAR family was identified by Nystedt (1994) who cloned a mouse genomic DNA sequence encoding a proteolitically activated receptor, related to but with a different sequence from the thrombin receptor PAR1. The receptor was termed PAR2 and it was found to be activated by trypsin that cleaves the receptor within the sequence N34SKGR/SLIGRLETQP48 of its extracellular -NH2 terminus, exposing a tethered ligand, SLIGRLETQP, that binds the cleaved receptor (Figure 1). Synthetic peptides mimicking the tethered ligand sequence, SLIGRLETQ., can activate the receptor even in the absence of proteolysis. The site for the receptor proteolytic cleavage contains the sequence SLIGRL and SLIGKV, murine and human respectively (Nystedt et al., 1994). Schematic representation of PAR2 activation by trypsin. PAR2 is activated upon trypsin cleavage within the sequence N34SKGR/SLIGRLETQP48 of its extracellular -NH2 terminus. This event unmasks a tethered ligand, SLIGRLETQP, that binds the cleaved receptor activating it. Synthetic peptides mimicking the tethered ligand sequence, SLIGRLETQ …, can activate the receptor even in absence of proteolysis. The physiological ligand of PAR2 has not as yet been identified. However, it has been shown that besides trypsin, the receptor may be activated by tryptase (Molino et al., 1997) and factor Xa (Fox et al., 1997), but it is resistant to activation by thrombin, even at concentrations as high as 100 nM. PAR2 mRNA has been found in several human and animal tissues, including kidney, stomach, pancreas, liver, colon and small intestine (Nystedt et al., 1994). Furthermore, by using immunohistochemical techniques, PAR2 has been localized in almost all human organs; in particular, a strong immunoreactivity has been detected in endothelial and epithelial cells, and in smooth muscle of both vascular and non vascular origin (D'andrea et al., 1998). To date, the physiological and/or pathological role of PAR2 is unknown. The presence of PAR2 on highly vascularized organs strongly suggests a role for the receptor in the control of vascular reactivity (D'andrea et al., 1998). This hypothesis is supported by a large amount of work that has been produced, since its discovery and cloning, giving evidence for the ability of PAR2 in regulating vascular function. On the basis of the present knowledge, the aim of this review is to recapitulate the cardiovascular functions of PAR2. The finding that PAR2 is highly expressed on endothelial cells is not surprising, considering that a large amount of proteases circulate in the blood. Most of them circulate in their proteolitically inactive form of zymogens, becoming activated following injury. Trypsin, tryptase, factor Xa can all activate PAR2 on endothelial cells and induce cellular effects involving either tissue degradation or regeneration processes. Evidence that PAR2 is expressed on both endothelium and vascular smooth muscle has suggested a role for this receptor in the control of vascular reactivity. A functional role for PAR2 on vascular endothelial cells was first described by Al-Ani et al. (1995), who found that the peptide mimicking the murine tethered ligand sequence, SLIGRL-NH2, and trypsin caused an endothelium-dependent relaxation of rat thoracic aorta. The effect of PAR2-activating peptide (PAR2-AP) was inhibited by L-NAME, implying the involvement of nitric oxide. At the same time, by RT – PCR analysis, receptor expression in the rat aortic tissue was also demonstrated. Subsequently, several studies have been performed to investigate: (i) whether PAR2 on vascular tissues was functional; (ii) whether vascular effects triggered by peptides activating PAR2 were due to activation of another receptor alone or in addition to PAR2 and (iii) whether thrombin could also activate the receptor. All these questions have now been addressed and it has been clearly shown that peptides activating PAR2, as well as trypsin, cause endothelium-dependent relaxation of rat thoracic aorta, as previously demonstrated (Al-Ani et al., 1995; Hollenberg et al., 1996; Magazine et al., 1996; Saiffedine et al., 1996). The effect is likely due to the activation of PAR2 per se, since several antagonists, such as atropine, phenoxybenzamine, ritanserine, propranolol, do not modify the response. In addition, structure activity relationship studies have shown that among peptides of different length, the hexapeptide SLIGRL is the most active in causing the endothelium dependent relaxation of rat aorta, and this activity is linked to the presence of the leucine and arginine residue at positions 2 and 5 respectively (Hollenberg et al., 1996). Although PAR2 is not activated by thrombin, a striking similarity has been observed between the two receptors. PAR1-APs, such as PAR2-APs, causes endothelium dependent relaxation of rat aorta that involves nitric oxide release (Muramutsu et al., 1992; Hollenberg et al., 1996; Magazine et al., 1996). Structure activity relationship studies have shown that for both receptors, PAR1 and PAR2, activating peptides with a free carboxyl group are less active than their carboxyamide derivatives (Hollenberg et al., 1996). However, in contrast to PAR1-AP, PAR2-AP fails to cause contraction of endothelium denuded aorta, although it is expressed on vascular smooth muscle (Magazine et al., 1996; Saiffedine et al., 1996). Moreover, there is no cross-desensitization between the two receptors; however, peptides activating PAR1 also activate PAR2 (Blackhart et al., 1996). This implicates that PAR2 might be activated by the sequence of the cleaved receptor PAR1. Although Blackhart et al. (1996) considered physiologically irrelevant this cross-reactivity, since the tethered ligand would be insufficient for an intramolecular interaction, Chen et al. (1994) have shown that intramolecular signalling by the thrombin receptor tethered ligand can occur. It is feasible that the two receptors are very closely located on endothelial cell plasma membrane. In this respect, a cross-talk between the two receptors, PAR1 and PAR2, on HUVEC has been demonstrated; it has been observed that a trans-activation of PAR2 by cleaved PAR1 contributes to the endothelial response to thrombin (O'brien et al., 2000). This result agrees with previous evidence for a functionally coupling between PAR1 and PAR2 receptors on HUVEC, given by Mirza et al. (1996), and it is not surprising considering that a cross-talk among G proteins coupled receptors has already been demonstrated (Djellas et al., 1998). Thus, it remains to be established whether PAR2 contributes to modulate thrombin vascular effects. This hypothesis could justify the increased response to thrombin when compared to the response obtained with agonist peptides, both in vitro and in vivo assays, as probably due to PAR1 activation and PAR2 trans-activation by the cleaved PAR1, that cannot occur with the use of the agonist peptides. It will be possible to discriminate between these two receptors activation only when PAR2 selective antagonists will be available. What is now clearly ascertained is that PAR2 is present on both endothelial and vascular smooth muscle cells. On endothelium, its activation causes a relaxation that involves nitric oxide. The release of nitric oxide from rat aortic rings stimulated with PAR2-AP (SLIGRL) has been demonstrated and also a role for endothelin-1 through Etb receptor activation has been shown (Magazine et al., 1996). Both trypsin and the peptide activating PAR2 (SLIGRL) also cause an endothelium-dependent relaxation of porcine coronary artery (Table 1). The effect of trypsin is clearly dependent upon its proteolytic activity since it is inhibited by soybean trypsin inhibitor (Hwa et al., 1996). However, relaxation induced by PAR2 activation is only partially inhibited by L-NAME, suggesting that it is dependent upon a NO-dependent and NO-independent mechanism (Hwa et al., 1996). More recently it has been shown that the NO-independent component of vasorelaxation induced by SLIGRL on porcine isolated coronary artery might be due to a hyperpolarizing factor, likely endothelium derived hyperpolarizing factor (EDHF) (Hamilton & Cocks, 2000). Besides large conduit vessels, such as thoracic aorta, PAR2 activation has also shown an endothelium-dependent vasorelaxant effect on small arteries, such as rat femoral artery and vein (Emilsson et al., 1997; Roy et al., 1998); rat renal, mesenteric and pulmonary arteries, (Roy et al., 1998), and on mouse renal arteries (Moffatt & Cocks, 1998) suggesting a role in controlling peripheral resistance. In any vascular district examined, the relaxant effect is dependent upon the presence of a functional endothelium, and it is abolished by inhibition of nitric oxide synthase. In the absence of endothelium, neither contraction nor relaxation has been observed, apart from a small contractile effect on femoral arteries without endothelium achieved only by a high concentration (100 μM) of PAR2-agonist peptide (Emilsson et al., 1997), and a contractile effect on mouse isolated renal artery (Moffatt & Cocks, 1998) described in the next section. Conversely, an endothelium-dependent vasoconstriction has been observed following PAR2 activation on renal, mesenteric, pulmonary arteries and on the aorta, that is independent of endothelin, angiotensin, nor-adrenaline or arachidonic acid metabolites (Roy et al., 1998). However, this contractile effect of PAR2-AP has been postulated to be dependent upon the activation of another receptor for several reasons. First, the peptide SLIGRL-NH2 caused contraction even in a preparation desensitized to the contractile effect of trypsin; second, peptides SLIGRL-NH2 and SLIGKV-NH2, rat and human sequence respectively, were equipotent to cause contraction, while showed different potencies in the relaxation assay, being SLIGKV-NH2 1/5 as potent as SLIGRL-NH2. Finally, the PAR2-AP derivative, transcynnamoil-LIGRLO-NH2 (Tc-LIGRLO-NH2), that is known to be a very selective PAR2 agonist, did not cause contraction, while it was equally active with SLIGRL-NH2 in the relaxation assay (Roy et al., 1998). However, to date, no PAR2 subtypes have been described. Functional PAR2 has also been demonstrated on cerebral arteries. Indeed, a nitric oxide-dependent relaxation of rat basilar artery has been demonstrated in vivo (Sobey & Cocks, 1998). Interestingly, the effect of PAR2 activation on cerebral vasculature is preserved in spontaneous hypertensive rats (SHR), despite the impaired nitric oxide production in this model of hypertension. This has suggested a protective role for the receptor in the cerebral circulation during chronic hypertension (Sobey & Cocks, 1998; Sobey et al., 1999). This finding further supports the hypothesis that besides nitric oxide other mechanisms might mediate PAR2 effects. While on animal vascular tissues the relaxant effect of PAR2 has been extensively characterized, even though still much work is needed to identify the signal transduction mechanism involved, the effect of PAR2 on human tissues is still controversial. A functional PAR2 on human vascular endothelial cells (HUVEC) has been demonstrated, and its activation causes cell proliferation and elevation of intracellular calcium, a signal known to activate NOS (Mirza et al., 1996). A study performed by using a ‘sandwich’ assay between aorta and human umbilical vein (HUV) has shown that while both trypsin and PAR2-AP cause nitric oxide dependent vasorelaxation, only PAR2-AP stimulates the release of an endothelium-derived contracturing factor from the same tissue, that is not identifiable with nor-adrenaline, angiotensin nor with endothelin (Saiffedine et al., 1998). This result agrees with data obtained on rat tissues (Roy et al., 1998) and both imply the existence of a novel PAR or of a PAR2 subtype, whose activation is responsible for the release of an endothelium derived contracturing factor (EDCF). Human coronary arteries with endothelium have been shown to relax in response to thrombin and trypsin, but not to the human sequence SLIGKV-NH2 or the mouse SLIGRL-NH2, in contrast to porcine coronary arteries that respond to all stimuli (Hamilton & Cocks, 2000). On the basis of this finding, it has been postulated that human coronary arteries present only PAR1 that can be activated by both thrombin and trypsin, but not PAR2. Inflammatory stimuli, such as TNFα, IL-1 or LPS, cause an over-expression of PAR2 on cultured HUVEC (Nystedt et al., 1996). Similarly, we have demonstrated that animals challenged with bacterial endotoxin present an increased expression of PAR2 on endothelium and smooth muscle cells, from both jugular vein and carotid artery (Cicala et al., 1999). Recently, it has been shown an increased PAR2 expression on human coronary artery following exposure to inflammatory stimuli (Hamilton et al., 2001). This finding, together with the finding of a proliferative effect of PAR2 on HUVEC, given previously (Mirza et al., 1996), suggests that this receptor could play a role in inflammation, in particular in the proliferative/reparative processes. The role of PAR2 on endothelial cells has not yet been clearly established. However, all data accumulated up to now suggest that it modulates vascular reactivity (Table 1). It remains to be clarified whether its main role is under physiological or pathological conditions. In order to address this question, it is necessary to identify the physiological ligand of PAR2. Previously described studies have shown a vasorelaxant endothelium-dependent effect following PAR2 activation, but no increase in vascular tension has been observed on tissues without endothelium, or in L-NAME pre-treated tissues, as described for PAR1 activation (Al-Ani et al., 1995; Magazine et al., 1996; Saiffedine et al., 1996). However, trypsin, that activates the receptor, has been shown to contract endothelium-denuded rabbit aorta (Komuro et al., 1997). In some cases, this discrepancy between PAR2 and trypsin effect has led to suggest the involvement of other PARs or of a PAR2 subtype. Among human vascular tissues, PAR2 expression has been found predominantly on arterial smooth muscles and less on venous smooth muscles. Conversely, myocardial smooth muscles are negative for PAR2 expression (D'andrea et al., 1998). By measuring changes in cytosolic calcium following stimulation with the agonist peptides (SLIGKV and SLIGRL), Molino et al. (1998) have shown that PAR2 normally expressed on smooth muscle cells from human aorta and coronary arteries is functional, while smooth muscles from saphenous vein do not show any functional receptor. These data demonstrate that there are differences in PAR2 expression between venous and arterial vascular beds, that might implicate a specific role for PAR2 in the modulation of peripheral resistance (Molino et al., 1998). Despite previous a vascular smooth muscle contraction in response to both trypsin and PAR2 ligand has been described on mouse isolated renal arteries (Moffatt & Cocks, 1998). The that the effect is due to PAR1 activation has been by the finding that tissue to PAR1 agonist, not induced by PAR2 agonist, SLIGRL-NH2. This the first evidence for a contractile effect of PAR2 on vascular smooth apart from a small contractile effect triggered by a high concentration of on endothelium-denuded rat femoral artery (Emilsson et al., 1997; The that the increase in intracellular calcium following PAR2 activation on aortic smooth muscles is than that caused by PAR1 activation suggests an expression of PAR2 than PAR1 on smooth on endothelial cells there is no evidence for in the expression between these two receptors (Molino et al., 1998). PAR2 is expressed on smooth muscles from large and small arteries, both in and in however, on endothelium-denuded there is neither relaxation nor contraction following its activation, with the only of the receptor on mouse renal arteries, as described (Moffatt & Cocks, 1998). This suggests that the presence of the receptor is not a to a response by in tissue the that vascular to PAR2 might be due to activation of another receptor alone or in addition to PAR2 (Saiffedine et al., 1996). To address this question, selective PAR2 antagonists be available. PAR2 on vascular smooth muscle might mediate different on the vascular and/or animal This hypothesis is with a proliferative effect of PAR2 agonist and trypsin on human aortic smooth muscle cells et al., 1997) and also on human endothelial cells (Mirza et al., 1996). increased expression of PAR2 has been found in rat carotid artery in with smooth muscle and smooth muscle cells of the et al., Thus, hypothesis could be that during vascular PAR2 on smooth muscles could be the of cell This would also be with the presence of cells at the site of vascular and likely high concentration et al., 1997). In with this hypothesis is the well ability of proteases to induce proliferation of several cell in some cases, it has been demonstrated that the effect is through PAR2 activation et al., et al., 2000). possible to mediate smooth muscle proliferation through PAR2 activation is factor Indeed, it has been shown that factor Xa stimulates rat and human vascular smooth muscle cell proliferation and the effect to be by the cell et al., 1992; et al., 1996; et al., 1998). However, evidence that only proteolitically active factor Xa cellular suggests that another receptor, than is involved, since not Interestingly, an between and PAR2 has recently been demonstrated in factor Xa effects on endothelial cells et al., 2000). effects due to PAR2 activation have also been shown in by using (Table of PAR2 agonist, rats causes in and independent of since it is not by functional and it is likely due to a peripheral The is partially inhibited by L-NAME, suggesting as well as in nitric oxide is (Emilsson et al., 1997). A following of PAR2 agonist (SLIGRL) has also been demonstrated in et al., 1998). in this the effect of the peptide activating PAR2 is likely due to a peripheral effect of the than to an effect on as demonstrated by the absence of to et al., 1998). have recently shown that induced by SLIGRL in rats is due to an NO-dependent and NO-independent the when the vascular the hypothesis of a effect on (Cicala et al., 2001). Similarly, in vitro data have shown that PAR2 activation on porcine coronary artery causes NO-dependent and relaxation (Hamilton & Cocks, 2000). In previous performed either in or PAR2 activation has been shown to induce only a without causing any hypertensive effect (Emilsson et al., 1997; et al., 1998; et al., 1999). However, we have recently shown that is by a hypertensive due to a and by nitric oxide. Indeed, hypertension induced by PAR2 agonist (SLIGRL) is abolished in animals and with L-NAME (Cicala et al., 2001). Most this hypertensive response to peptide was not in previous since the of the was not A hypertension is with the hypothesis of a peripheral of PAR2-AP and also with a increase in observed in rats following of PAR2-AP (Emilsson et al., 1997), although differences among different have been induced by PAR2 activation is 2 – The effect of L-NAME, only the of induced by has been by all the studies performed in vivo (Emilsson et al., 1997; et al., 1998; et al., 2001). This has suggested that other mechanisms besides nitric oxide release for the vasorelaxant effect of peptides activating PAR2. The of vascular response to PAR2 receptor activation has been by performed on and response to is in Conversely, in the same response to PAR1 activation is This suggests that PAR2 might modulate thrombin vascular effects et al., and further supports the hypothesis of PAR2 trans-activation by cleaved PAR1, described (O'brien et al., 2000). The physiological ligand of PAR2 is not The receptor is activated by trypsin, tryptase and factor and it could mediate and/or vascular effects of these proteases at the site of an injury. A effect of factor Xa has been demonstrated et al., 1998) and it has been postulated the involvement of However, recently it has been shown that activation of endothelial cells by factor Xa is related to activation of and PAR2 et al., and that PAR2 is in factor vasorelaxation of rat thoracic aorta et al., 2001). Thus, PAR2 might be the of induced by factor Furthermore, PAR2 can be activated by factor in the presence of tissue factor and factor et al., 2000). activates factor the proteolitically active form in activates PAR2. A previous work by the same et al., showed that only proteolitically active factor and factor a calcium response in cells. Thus, it is feasible that a receptor is involved, most likely PAR2. recently it has been demonstrated that factor Xa in the with activates PAR2 & 2001). All these a role for PAR2 in effects. effect of PAR2 agonist, SLIGKV-NH2, and trypsin is increased in rats with bacterial this effect is coupled to an increased PAR2 expression on endothelium and smooth muscles from either jugular vein and carotid artery (Cicala et al., 1999). This finding is in with in vitro data an increased expression of PAR2 on endothelial cells due to inflammatory stimuli (Nystedt et al., 1996; et al., 2001). increased expression of PAR2 has been also demonstrated in the rat following et al., The physiological role of PAR2 has not been as well as it has not been established whether the receptor is for the control of and changes in under physiological or only in pathological Evidence that PAR2 is physiologically expressed on the vascular tissues suggests that it normally in the control of vascular However, its ligand is not known and possible such as trypsin, tryptase or factor do not normally circulate in the at concentrations to activate the receptor and it is that are to activation of such an receptor. On the other the finding that the receptor is on tissues stimulated with inflammatory either in vitro and in vivo (Nystedt et al., 1996; et al., et al., strongly suggests that PAR2 might in the well established between and as of proteases (Cicala & 1998; et al., 2000). On the basis of evidence accumulated since its and cloning, it is possible to a cardiovascular for PAR2. The receptor is highly expressed on vascular tissues and both vascular and suggesting that it to the physiological control of vascular On the other PAR2 is also in pathological suggesting a role in the mechanisms activated following vascular injury. In this it might be considered as a of proteases during On the endothelial cells, PAR2 relaxation both in large and in small arteries, suggesting a role in the control of and It remains to be the mechanism at the basis of this since nitric oxide to be only partially the role of PAR2 on vascular smooth muscles. Indeed, although its activation not cause in tension in any vascular tissue examined, but in mouse renal arteries, as described by & on vascular smooth muscle PAR2 has been to be functional; it remains to be established whether PAR2 on smooth muscle is functionally to another receptor, likely another G-protein coupled receptor, or whether its main effect is to induce smooth muscle under pathological conditions. are in a of represent a between and and their strongly increase following vascular either or at site of an injury. is much evidence that such as et al., and et al., et al., have a effect on inflammatory such as The discovery of and in particular of PAR2, has a on proteases and on their cellular effects. Evidence that PAR2 is also activated by that even proteases mediate cellular effects. To the role of PAR2 in the cardiovascular still much work is This review was to that all data obtained since its discovery to PAR2 in the vascular modulates vascular and vascular cell both under physiological and pathological conditions. in a it will be to study mechanisms in cardiovascular without in PAR2.