Published online before print September 1, 2005, doi: 10.1161/01.ATV.0000184770.01494.2e
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© 2005 American Heart Association, Inc.
Vascular Biology |
Proteinase-Activated Receptor-2 Mediates Arterial Vasodilation in Diabetes
From Dipartimento di Farmacologia Sperimentale (F.R., M.B., V.B., A.D.L., G.C.), Università di Napoli Federico II; Dipartimento di Scienze Farmaceutiche (L.P.), Università di Salerno; Dipartimento di Aria Critica Medico Chirurgica (P.G.), Università di Firenze; Dipartimento di Medicina Sperimentale (S.F., S.Fiorucci), Università di Perugia; Dipartimento di Fisiopatologia e Medicina Sperimentale (G.L.), Università di Siena.
Correspondence to Giuseppe Cirino, PhD, Dipartimento di Farmacologia Sperimentale, via Domenico Montesano 49 80131 Napoli, Italy. E-mail cirino{at}unina.it
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Abstract |
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Objective— Proteinase-activated receptor-2 is widely expressed in vascular tissue and in highly vascularized organs in humans and other species. Its activation mainly causes endothelium-dependent vasorelaxation in vitro and hypotension in vivo. Here, using nonobese diabetic (NOD) mice at different disease stages, we have evaluated the role of PAR2 in the arterial vascular response during diabetes progression.
Methods and Results— High (NOD-II; 20 to 500 mg/dL) or severe glycosuria (NOD-III; 500 to 1000 mg/dL) provokes a progressive reduction in the response to acetylcholine paralleled by an increase in the vasodilatory response to PAR2 stimulation. Western blot and quantitative real-time polymerase chain reaction (RT-PCR) studies showed that this effect is tied to an increased expression of PAR2 coupled to cyclooxygenase-2 expression. Pharmacological dissection performed with specific inhibitors confirmed the functional involvement of cyclooxygenase-2 in PAR2 vasodilatory effect. This vasodilatory response was confirmed to be dependent on expression of PAR2 in the smooth muscle component by immunohistochemistry studies performed on aorta isolated by both NOD-III and transgenic PAR2 mice.
Conclusions— Our data demonstrate an important role for PAR2 in modulating vascular arterial response in diabetes and suggest that this receptor could represent an useful therapeutic target.
On diabetes development in NOD mice there is a diminished vasodilatory response to acetylcholine that is counterbalanced by an increased expression of PAR-2 both as protein and message. The expression is mainly localized on the smooth muscle cell component as demonstrated by the immunohistochemistry and functional studies
Key Words: cyclooxygenase-2 • diabetes, type I • proteinase-activated receptor-2 • smooth muscle
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Introduction |
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Proteinase-activated receptors (PARs) are a recently described novel family of 7-transmembrane G-protein-coupled receptors.1–3 PARs are activated enzymatically through proteolysis of the receptor by a well-characterized family of enzymes that require serine within active site, the serine proteases. On cleavage at these specific sites, the new N-terminal for each receptor functions as a tethered ligand.2 At the present stage, 4 subtypes PARs have been characterized: PAR1,4 PAR3,5 and PAR4,6 selectively activated by thrombin and PAR2 that is activated by trypsin, tryptase, tissue factor, factor VII, and factor X.1
PAR2 has been shown to be involved in cardiovascular function.7,8 Functional studies have shown that activation of the receptor by using a small agonist peptide derived from the tethered ligand sequence (PAR2AP) causes an endothelium-dependent vasodilatation.9–13 Subsequent studies have clearly shown that PAR2 is expressed in the human endothelial14 and smooth muscle cells.15 Systemic administration of PAR2AP in vivo in rats and mice causes hypotension.10,11,16,17 Interestingly, also in humans in vivo PAR2 AP causes vasodilatation that is dependent on nitric oxide (NO) release and prostanoids.18 As opposed to PAR1, PAR2 expression is upregulated by inflammatory stimuli such as tumor necrosis factor-
, bacterial endotoxin (lipopolysaccharide [LPS]) and IL-1 in endothelial cells.16,19,20 Likewise, LPS administration to rats increases PAR2 expression on endothelium and smooth muscle cells from both jugular vein and carotid artery, which translates in an increased vasodilatory response to PAR2 AP.16 Consistent with these findings, the dose necessary to activate the receptor in several inflammatory conditions is reduced to 1 or 2 orders of magnitude in comparison to normal conditions. Whereas all these data suggest a role for PAR2 in cardiovascular inflammation, it is unclear whether the activation of this receptor plays a pathological or compensatory role.20,21
One example of endothelial dysfunction coupled to cardiovascular inflammation is represented by diabetes. Duration of diabetes and poor glycemic control are the main predictor factors for peripheral arterial disease, indicating a close link between metabolic impairment and arterial injury.22,23 Although peripheral arterial disease results from atherosclerotic narrowing of the blood vessel lumen, endothelial dysfunction has been shown to play a role in the progression of this disease. Further, these processes are enhanced by the metabolic disturbances associated with diabetes and denervation of the smooth muscle of the tunica media of arteries caused by the diabetic neuropathy. Whether PAR2 plays a role in the pathogenesis of arterial dysfunction in diabetes and if it is upregulated in this condition are unknown.
In the present study, by using a genetic mouse model of type I diabetes and PAR2 transgenic mice, we have investigated the role of PAR2 in vessel function during the disease progression. The results of our studies indicate that arterial expression of PAR2 increases in response to diseases progression and exerts a potent vasorelaxant effect, suggesting that development of PAR2 agonist might be helpful in treating patients with diabetic arthropathy.
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Materials and Methods |
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Animals
Female nonobese diabetic mice (NOD/Ltj) and CD-1 mice were purchased from Charles River (Italy). CD-1 mice were used as control animal because they display a similar pattern of contraction and relaxation to NOD-I (Figure I, available online at http://atvb.ahajournals.org). Transgenic PAR2 (tg-PAR2) mice were bred at University of Siena and have been previously described.24 NOD mice were divided according to the glycosuria value (Figure II, available online at http://atvb.ahajournals.org) in the following groups: NOD- I: low or null glycosuria (5±2 weeks; 0 to 20 mg/dL); NOD-II: high glycosuria (13±3; 20 to 500 mg/dL); and NOD-III severe glycosuria (22±3; 500 to 1000 mg/dL). For further details, see http://atvb.ahajournals.org.
In Vitro Experimental Protocols
In each experiment, rings were standardized using phenylephrine (1 µmol/L) until the responses were reproducible. To evaluate tissue vasorelaxation, cumulative concentration response curve to Ach (10 nmol to 30 µmol/L), and to the PAR2 tethered ligand peptide (10 nmol to 30 µmol/L) were performed on 5HT (3x10–7 mol) precontracted rings. Curves to PAR2AP were constructed in the absence and presence of L-NAME (100 µmol/L, 20 minutes), 1400 W (10 µmol/L), ibuprofen (10 µmol/L), DFP (10 µmol/L), FR-122047 (20 µmol/L), and SQ-22,536 (100 µmol/L). When L-NAME was added, a submaximal dose (EC80) of 5-HT (3x10–8 mol) was used.
Western Blotting
Western blotting studies were performed on aortic tissue samples homogenized in lysis buffer using a Talon homogenizer and were processed identically. The immunoblots were developed with 1:500 dilutions for PAR2 and 1:1000 for COX-2 and the signal was detected with the ECL System according to the manufacturer’s instructions (Amersham Pharmacia Biotech).
Quantitative Real-Time Polymerase Chain Reaction Studies
After the mice had been killed, aortas were removed and immediately snap-frozen on liquid nitrogen (LN2) and stored at –80°C until used. Total RNA was isolated using TRIzol reagent (Life Technologies, Milan, Italy) as previously described.26,27 Quantification of the expression mouse genes was performed by quantitative real-time polymerase chain reaction (RT-PCR) by using specific primers.
Immunohistochemistry
The aortas from the different groups of mice were fixed with buffered formalin (5%) for 24 hours. Tissue sections (6 µm) were stained for PAR2 receptors by an immunoperoxidase method. The primary polyclonal antibody (Ab) used was a goat polyclonal Ab raised against the carboxyl terminus of PAR2 of human origin, and the specificity of the antibody was tested using the blocking peptide (sc-8205P). The Ab was used at dilution of 1:200.
Statistical Analysis
Data are expressed as mean±SEM. The level of statistical significance was determined by 1-way analysis of variance (ANOVA) followed by Bonferroni’s t-test for multiple comparisons, using the GraphPad Prism software.
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Results |
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Diabetes Progression Increases PAR-2AP–Induced Vasodilatation and Upregulates PAR-2 and COX-2 Expression
In NODI and CD1 mice, that have normal glycosuria, the relaxant response induced by PAR2AP on isolated aorta is similar (Figure 1a). The disease progression leads to a significant decrease in the relaxant response to acetylcholine (Figure 1d) with a concomitant increase in PAR2AP-induced vasodilatation of NOD-II (Figure 1b) and NOD-III (Figure 1c) aortas. There is a significant shift of PAR2AP-induced vasorelaxant effect of the EC50 from 1x10–6 mol of NOD-I aorta to 6.5x10–7 for NOD-II aorta and to 4x10–7 mol/L for NOD-III aorta. The scramble peptide LSIGRL-NH2 was inactive on both NOD-I and NOD-III mice aorta (Figure III, available online at http://atvb.ahajournals.org). Because it is known that inflammatory stimuli can upregulate both PAR2 and COX-2 expression, we have evaluated expression of PAR2 in aortas obtained from normal and NOD mice at different stages of illness (NOD-I, NOD-II, and NOD-III). Quantitative RT-PCR showed an increase in mRNA expression of PAR2 (Figure 2A, panel f) and COX-2 (Figure 2A, panel b), whereas PAR1 (Figure 2A panel e), COX-1 (Figure 2A, panel a), inducible nitric oxide synthase (iNOS) (Figure 2A, panel d), and endothelial nitric oxide synthase (eNOS) (Figure 2A, panel c) mRNA levels were unchanged. Western blot analysis showed that there is a concomitant significant increase of PAR2 and COX-2 protein expression in NOD-II and NOD-III aortas when compared with either NOD-I or CD-1 aortas (Figure 2B), whereas iNOS was unchanged (data not shown).
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Role of NO and Prostanoids in PAR2AP-Induced Vasodilatation in NOD Mice
Next, to investigate on the role played by NO and prostanoids, we tested the effect of l-NAME and ibuprofen. Each drug was used at concentrations known to inhibit NO-dependent vasorelaxation and COX-1/COX-2 activity, respectively. Incubation of aorta rings of CD-1 (Figure 3a) or NODI (Figure 3b) with L-NAME abrogated the vasodilatory effect of PAR2AP. Conversely, ibuprofen did not affect PAR2AP-induced vasodilatation of either CD-1(Figure 3a) or NODI (Figure 3b) mouse aorta.
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Aorta isolated from NOD-II mice show a reduced inhibitory effect of L-NAME that was removed at lower doses with a significant shift of Emax (maximal relaxation achievable) from 90% to 25% (Figure 3d). Similarly, there is a significant inhibition by ibuprofen (Figure 3d) particularly marked at lower doses of PAR2AP. NOD-III isolated aortas displayed a similar inhibitory pattern with a more marked inhibitory effect displayed by ibuprofen (Figure 3e). When aortic rings were incubated with both ibuprofen and L-NAME, there was a further significant inhibition of the relaxant response to PAR2AP. Prostaglandins produce their effect through cAMP. To further confirm cAMP involvement we used SQ-22,536 an inhibitor of adenylate cyclase. SQ-22,536 significantly reduced PAR2AP-induced vasorelaxation of NOD-III aorta (Figure 3f), but it was ineffective in NOD-I aortas (Figure 3c). Functional studies performed on aortas isolated by CD-1 (Figure 4a), NOD-I (Figure 4b), and NOD-III (Figure 4c) by using specific inhibitors of COX-1 (FR-122047), COX-2 (DFP), and iNOS (1400W) showed that the major contribute to the relaxation observed in NOD-III mice is given by prostanoids mainly derived by COX-2. These data fit well with the increased COX-2 expression observed in NOD-II and NOD-III mouse aortas (Figure 2). Aorta harvested form CD-1 mice age-matched with NOD mice did not show any change in relaxation induced by acetylcholine or PAR2AP; similarly, glycosuria was unchanged (Figure I).
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Tg-PAR2 Mice
To confirm these data, we used transgenic mice overexpressing PAR2. The vasorelaxant response of NOD-III aorta to PAR2AP (Figure 4a) is similar to that of tg-PAR2 mouse isolated aorta (Figure 4c). Furthermore, similarly to what happens in NOD-III aortas (Figure 4a), PAR2AP-induced vasorelaxation is still present in endothelium-denuded aortic rings obtained by tg-PAR2 mice (Figure 4c). These data clearly suggest an active role of smooth muscle cells in PAR2-mediated vasorelaxation in tg-PAR2 mice, suggesting that a similar increase in PAR2 expression in the smooth muscle component of NOD-II and NOD-III mice. We therefore evaluated the expression of PAR2 in situ by using immunohistochemical staining of aortas obtained from wild-type and NOD-III mice, as well as from transgenic overexpressing PAR2 (Figure 5). As shown in Figure 5, PAR2 expression was minimal but nonetheless detectable in a patchy distribution in aorta and peri-aortal tissue of wild-type mice with very low positive reaction on the smooth cell component (Figure 5, panel A). By contrast, expression of PAR2 was diffusely greater in tissues of NOD mice (Figure 5, panel B). The specific staining for PAR2 we observed on the smooth muscle cell component in aortas from NOD-III mice (Figure 5, panel B) equaled, or exceeded, in strength that of aortas from mice overexpressing PAR2 (Figure 5, panel C). The positive staining was removed by addition of the blocking peptide confirming the specificity of the primary antibody (Figure 5, panel D). Thus, the immunohistochemistry data support the functional data. Next, to further address this similarity between NOD-III and tg-PAR2 mice, we performed a comparative study using ibuprofen, L-NAME, SQ-22,536, DFP, and FR-122047. Similar to what happens in NOD-III mice, ibuprofen (Figure 4e) and SQ-22,536 (Figure 4d) both inhibited PAR2AP-induced vasorelaxation in intact rings harvested from tg-PAR2 mice. As in NOD-III mice, L-NAME inhibitory effect on tg-PAR2 mouse aorta was reduced as Emax (Figure 4d). DFP, the selective COX-2, inhibitor significantly reduced PAR-2AP–induced relaxation in NOD- III aorta (Figure 4b). Conversely, the selective COX-1 inhibitor (FR-122047) that was ineffective in NOD-III mice significantly inhibited PAR2AP-induced vasorelaxation in tg-PAR2 mice whether DFP had no effect (Figure 4f).
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Discussion |
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Cardiovascular diseases are currently the principal causes of morbidity and mortality in patients with type I diabetes.22,23 The loss of modulator tone by the endothelium is considered one of the critical factor in the development of diabetic vascular diseases. There is evidence that an impaired response to endothelium-dependent agonists in different vascular beds develops in response to both chemically induced and genetic models of type I diabetes or in vivo in experimental animals.28 A constant feature of these studies is an impaired response to acetylcholine. We have recently shown that NOD mice vessel reactivity follows the diabetes progression and, in particular, there is an impairment of the vasorelaxant effect to acetylcholine and isoprenaline.25,29
Here, we show that response to PAR2 selective stimulation is increased in diabetic mice with a clear shift of the EC50. This increased response is strictly linked to the disease progression being maximal in NOD-III mice in which vasodilatation to acetylcholine is strongly impaired through an alteration of the post-translational mechanisms involved in eNOS regulation.25 The vasorelaxant effect of PAR2 on vessels is linked to the presence of an intact endothelium and only in part through the NO release as demonstrated both in vitro and in vivo.7,10,11,30 Recently it has been shown that PAR2 activation increases COX-2 protein and mRNA expression and promotes PGI2 release from human umbilical vein endothelial cell. Thus, COX-2 induction takes part in the functional response of endothelial cells to PAR2 activation,31 suggesting that PAR2 promotes a sustained upregulation of prostanoid production in endothelium. To gain further insights into this mechanism we have analyzed the effect of L-NAME, an NO synthase inhibitor, and ibuprofen, a COX-1/2 inhibitor. In control and NOD-I mice (low or null glycosuria), L-NAME virtually abrogates the PAR2AP induced vasorelaxation whereas ibuprofen was ineffective. Conversely, when diabetes is clearly established, such as in NOD-II mice, there is a reduction in the inhibitory effect of L-NAME and the appearance of ibuprofen inhibitory effect on PAR2AP-induced relaxation, which is particularly evident at the lower doses of PAR2AP. In NOD-III mice, that have a more severe glycosuria, there is equally a loss in efficacy of L-NAME and the inhibitory effect of ibuprofen is significantly more pronounced. The simultaneous administration of ibuprofen and L-NAME further inhibited the relaxant response to PAR-2AP. These results imply that, as the disease progresses, there is a gradual switch of the vessel relaxant mechanism toward the PAR2 signaling pathway with an increased contribute of the cyclooxygenase pathway. Functional studies performed using selective inhibitors of COX-1 (FR-122047) and COX-2 (DFP) confirmed that PAR-2AP induced vasorelaxation occurs with a COX-independent mechanism in control condition, eg, in NOD-I and CD1 mouse aortas. Conversely, in pathological condition, such as in NOD-III mice, there is a clear involvement of prostanoids, mainly driven by COX-2 as suggested by the lack of inhibitory effect of the selective COX-1 inhibitor FR-122047 and by the marked effect of SQ22,536, a selective cAMP inhibitor. This interpretation is supported by the molecular studies that clearly show by quantitative RT-PCR an increase in PAR2 and COX-2 mRNAs expression that correlates well with protein expression in NOD-III mice. Interestingly, the immunohistochemistry study clearly demonstrated that diabetes development causes an increased expression of PAR2 on the smooth muscle cell component and in peri-aortic areas. Thus, it appears that there is a linkage among NO, PAR2, and COX-2 that becomes evident in pathological condition. A similar linkage among PAR2AP vasodilatory effect, NO, and COX products has been recently shown also in human volunteers18 and in experimental animals.30 Using rat aorta has been shown that basal NO modulates the vascular effects linked to PAR2 activation and that both cGMP and cAMP are involved.30 In human healthy volunteers it has been shown that vasodilatation of the dorsal hand vein induced by local administration of PAR2AP is inhibited by both L-NAME and aspirin.16 In this context it is important to note that PAR2, as opposed to PAR1, can be upregulated by inflammatory stimuli such as tumor necrosis factor-, IL-1ß, and LPS.16 This upregulation is also present in vivo after administration of LPS in the arterial and venous tissue of rats19 and in vitro in human coronary vessels.32 Similar results that further support the hypothesis that PAR2 can be "unmasked" by an inflammatory cardiovascular event were obtained by using an animal model of balloon vascular injury.33
Because in our vessel preparation there is still a residual activity to Ach, and to better-understand the role of endothelium in our experimental condition, we tested the effect of PAR2AP in rings where the endothelium was mechanically removed. Endothelium removal in NOD-I mice comported a complete loss of the relaxing activity similar to what can be observed in CD-1 mice as it has been demonstrated also by others. Conversely, no significant changes were observed in NOD-III mice in relaxation in aorta after endothelium removal, furthermore the pattern of relaxation operated by PAR-2AP in vitro was very close to that displayed by aorta isolated by tg-PAR-2 mice. In a more complete analysis of the response to PAR2AP of tg- PAR2 aorta, the similarity with the response to NOD-III mice was even more striking. Similar to NOD-III isolated aortas, tg-PAR2 mice isolated aortas displayed a reduced vasodilatory response to PAR2AP in presence of ibuprofen and SQ-22,546 as well as reduced inhibition by L-NAME. Interestingly, the selective COX-1 inhibitor was ineffective on aortas isolated from NOD-III, whereas it significantly inhibited tg-PAR2 mouse aorta, further supporting that the disease development causes a selective induction of the COX-2 isoform. Thus, these data support the hypothesis that after endothelial injury there is an increased expression of PAR2 on the smooth muscle cells to counterbalance the loss in vasodilatory component. In NOD- III mice, the prostanoid effect is mainly driven by COX-2 as opposed to tg-PAR-2 mice, in which it is mainly driven by COX-1. This is not surprising because in NOD mice, the slow development of diabetes can cause the COX-2 induction, whereas in tg-PAR-2 mice the increased expression of PAR-2 is obtained by genetic manipulation and, for this reason, couples to the constitutive form of the enzyme, ie, COX-1.
In conclusion, our data show that in diabetes development there is a gradual switch of the vessel relaxant function toward PAR2 and COX-2. This most likely represents a functional response to the injury to the endothelium that in this condition displays a reduced functionality. These data also suggest that PAR2AP peptide may be useful vasodilator in diabetes or in other pathology in which an endothelial damage on inflammatory basis is present. In this context it has been recently demonstrated a beneficial effect of PAR2AP in a murine model of hind-limb ischemia in which PAR2AP administration increases capillarity resulting in an accelerated hemodynamic recovery and enhanced limb rescue.34
Received May 31, 2005; accepted August 8, 2005.
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