Upregulation of Proteinase-Activated Receptor-2 and Increased Response to Trypsin in Endothelial Cells After Exposure to Oxidative Stress in Rat Aortas

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  Fax +41 61 306 12 34E-Mail karger@karger.chwww.karger.com  Research Paper J Vasc Res 2010;47:494–506DOI: 10.1159/000313877 Upregulation of Proteinase-Activated Receptor-2and Increased Response to Trypsin in EndothelialCells after Exposure to Oxidative Stress in RatAortas Murasaki Aman Mayumi Hirano Hideo Kanaide Katsuya Hirano Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences,Kyushu University, Fukuoka, Japan   than WKY and responded to trypsin without serum-free in-cubation. Treatment with ascorbic acid attenuated the tryp-sin-induced relaxation and the PAR 2 expression in SHR. Con-clusion: This study provides the first evidence that oxidativestress upregulates PAR 2 in endothelial cells, thereby enhanc-ing the endothelium-dependent relaxant response to PAR 2  agonists in rat aortas. Copyright © 2010 S. Karger AG, Basel Introduction Proteinase-activated receptors (PARs) form a uniquefamily of G protein-coupled receptors which mediate thecellular effects of serine proteinases such as thrombinand trypsin [1–3]. The activation of PARs is initiated by proteolytic cleavage of the extracellular region at the spe-cific site [1, 2]. Therefore, one proteinase could activatemultiple receptors, while one type of receptor could beactivated by multiple proteinases. Among the 4 membersof PARs, PAR  1  , PAR  3 and PAR  4 serve as receptors forthrombin, while PAR  1  , PAR  2 and PAR  4 serve as receptorsfor trypsin [1–3]. In terms of thrombin or trypsin recep-tor, PAR  2 serves as a specific receptor for trypsin. How-ever, the role of PAR  1 as a trypsin receptor is controversial[2, 4, 5]. Trypsin has been shown to cleave and inactivate Key Words Endothelial cell Proteinase-activated receptor Rat aortaVasodilation Oxidative stress Trypsin Abstract Background/Aims: The effects of oxidative stress on thevascular responsiveness to the agonists of proteinase-acti-vated receptors (PARs) were investigated. Methods: Serum-free incubation was utilized to impose oxidative stress toisolated rat aortas. Spontaneously hypertensive rats (SHR)were investigated as a model of in vivo oxidative stress. Re- sults: Thrombin, trypsin, PAR 1  -activating peptide (PAR 1  -AP),PAR 2  -AP and PAR 4  -AP induced little or no effect in the aortasof female Wistar-Kyoto rats (WKY). Serum-free incubation in-duced endothelium-dependent relaxant responses to PAR 2  agonists, but not PAR 1 or PAR 4 agonists, in a manner sensitiveto diphenyleneiodonium or ascorbic acid. In male aortas,trypsin and PAR 2  -AP induced a transient endothelium-de-pendent relaxation without serum-free incubation. The ace-tylcholine-induced endothelium-dependent relaxation andthe sodium nitroprusside-induced endothelium-indepen-dent relaxation remained unchanged. Immunoblot analysesrevealed the upregulation of PAR 2 in endothelial cells, whichwas abolished by either diphenyleneiodonium or ascorbicacid. Aortas of female SHR expressed a higher level of PAR 2   Received: April 26, 2009Accepted after revision: December 10, 2009Published online: April 30, 2010 Dr. Katsuya HiranoDivision of Molecular Cardiology, Research Institute of Angiocardiology Graduate School of Medical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 (Japan) Tel. +81 92 642 5550, Fax +81 92 642 5552, E-Mail khirano @ molcar.med.kyushu-u.ac.jp © 2010 S. Karger AG, Basel1018–1172/10/0476–0494$26.00/0Accessible online at:www.karger.com/jvr   PAR  2 Upregulation by Oxidative Stress J Vasc Res 2010;47:494–506 495 PAR  1 in rat myometrium and vascular endothelial cells[4, 5], while it was also reported to activate PAR  1 at rela-tively higher concentrations than those required to acti- vate PAR  2 [2]. The different sets of PARs are expressed in a tissue-specific manner and mediate the tissue-specific respons-es to agonist proteinases [1, 2]. In vascular tissues, endo-thelium-dependent relaxation is the most frequently re-ported vascular effect of thrombin and trypsin, whileendothelium-dependent contraction or direct smoothmuscle contraction has also been reported depending onthe type of blood vessels [3, 6–8]. In addition to the vaso-motor function, PAR  1 stimulation has been reported toinduce endothelial barrier dysfunction, production of re-active oxygen species, cytokine release and alteration of the expression of various genes, including cell adhesionmolecules and tissue factor [1–3]. PAR  2 stimulation hasalso been reported to induce cytokine release, leukocyteadhesion and exocytosis of a Weibel-Palade body in en-dothelial cells [9–12]. These effects mediated by PAR  1 andPAR  2 are thus suggested to contribute to the pathogenesisand pathophysiology of vascular diseases. Various chemical factors and physical stimulations aswell as pathological conditions have been shown to mod-ulate the expression of PARs [3, 13]. For example, the ex-pression of PAR  1 has been reported to be upregulated by the action of growth factors or in vascular lesions of bal-loon injury, atherosclerosis and subarachnoid hemor-rhage [14–20]. On the other hand, the PAR  2 expressionhas been reported to be upregulated by inflammatory stimuli such as tumor necrosis factor- ␣  , interleukin-1 ␣  and 1 ␤ and lipopolysaccharide [21–23]. The upregulationof PARs is considered to be a critical step for these recep-tors to contribute to the pathogenesis and pathophysiol-ogy of vascular diseases, especially for those expressedat a low level under physiological conditions [3]. On theother hand, oxidative stress is one of the critical factorscontributing to the pathogenesis and pathophysiology of vascular disease, such as hypertension, vasospasm,atherosclerosis and subarachnoid hemorrhage [24–26]. Itis therefore suggested that the modulation of the expres-sion of PARs is one of the mechanisms underlying thepathological effects of oxidative stress in vascular diseas-es. Cyclic strain has been reported to upregulate PAR  1 insmooth muscle in a manner sensitive to a NADPH oxi-dase inhibitor [27]. However, the effects of oxidative stresson the responses to PARs agonists and the expression of PARs in vascular tissues still remain to be investigated. The present study thus investigated the effect of oxida-tive stress on the vascular responsiveness to thrombin,trypsin, PAR  1  -activating peptide (PAR  1  -AP) and PAR  2  -AP in vascular tissues. For this purpose, 24-hour incuba-tion in serum-free media was utilized to impose oxidativestress to the isolated vascular tissues, as previously re-ported [28, 29]. Spontaneously hypertensive rats (SHR)have been reported to be exposed to a higher degree of oxidative stress than Wister-Kyoto rats (WKY) [30]. SHR were thus investigated as a model of in vivo oxidativestress. Since the responsiveness to the stimulation of PAR  2  , but not PAR  1 or PAR  4  , was observed after serum-free incubation, the expression of PAR  2 was then directly investigated by an immunoblot analysis. The presentstudy thereby demonstrates, for the first time, that oxida-tive stress upregulates the PAR  2 expression and enhancesthe response to the PAR  2 activation in vascular endothe-lial cells.  Methods Tissue Preparation WKY (Kyudo, Saga, Japan) and SHR (Charles River, Yokoha-ma, Japan), ranging in age from 9 to 11 weeks, were euthanized by intraperitoneal injection of 100 mg sodium pentobarbital/kgweight, according to the protocol approved by the Animal Careand the Ethical Committee of Kyushu University. The study wasperformed mainly with virgin female rats, unless otherwise spec-ified. The descending thoracic aorta was excised and the adventi-tial tissues were mechanically removed under a binocular micro-scope. The aortas were opened longitudinally and then cut intocircular strips measuring ϳ  1 mm in width and ϳ  4 mm in length.The strips with an intact endothelium were mainly used in thestudy, unless otherwise specified. When the study was conductedin the absence of endothelium, the luminal surface was rubbed off with a cotton swab to remove the endothelium. The vascularstrips were equilibrated in a physiological saline solution (PSS)consisting of 123 mmol/l NaCl, 4.7 mmol/l KCl, 1.25 mmol/lCaCl 2  , 1.2 mmol/l MgCl 2  , 1.2 mmol/l KH 2  PO 4  , 15.5 mmol/lNaHCO 3 and 11.5 mmol/l D  -glucose, aerated with 95% O 2 and 5%CO 2  .  Protocol for Imposing Oxidative Stress to the Isolated Strips The strips were incubated for 24 h at 37° C in serum-free Dul-becco’s modified Eagle’s medium (DMEM) supplemented withantibiotics in a CO 2 incubator, as previously reported [28, 29]. Thestrips were then thoroughly washed and equilibrated in PSS be-fore starting the experimental protocols. For the control, thestrips were used in the experiments on the day of preparation andwithout incubation in serum-free DMEM at 37 ° C for 24 h. Thesecontrol strips were kept in PSS at room temperature until use.  Tension Measurement of the Isolated Strips The strips were mounted vertically in an organ bath and con-nected to a force transducer (TB612-T, Nihon Koden, Japan), aspreviously described [31]. The change in the tension was thenmonitored at 37 ° C under a 300-mg resting load. The endotheli-   Aman/Hirano/Kanaide/Hirano J Vasc Res 2010;47:494–506 496 um-dependent relaxant responses were examined during the sus-tained phase of the pre-contraction induced by 100 nmol/lU46619, a thromboxane A 2 analogue. The value of tension wasexpressed as a percentage, assigning the levels of tension at restand during the contraction induced by 100 nmol/l U46619 to be0 and 100%, respectively. Acetylcholine was used as a control stimulation to induce anendothelium-dependent relaxation in rat aortas. In the strips of female WKY aortas before serum-free incubation, 10 ␮  mol/l ace-tylcholine induced an ϳ  20% reduction in the U46619-inducedpre-contraction. This relaxant effect was weaker than the previ-ously reported relaxant effect of acetylcholine in female rat aortas( ϳ  70–90% reduction) [32–34]. However, preliminary experi-ments revealed that this weak response to acetylcholine was notdue to usage of vascular strips, because 10 ␮  mol/l acetylcholineinduced a similar relaxation during the U46619-induced pre-con-traction in ring preparations (28.1 8 7.7% reduction of the pre-contraction in ring preparations vs. 26.9 8 4.9% reduction in vascular strips, n = 4). On the other hand, during pre-contractioninduced by 10 ␮  mol/l phenylephrine, 10 ␮  mol/l acetylcholine in-duced a 58.9 8 9.6% reduction in tension in ring preparations and46.1 8 1.6% reduction in vascular strips (n = 4). Acetylcholinewas reported to induce 50–90% relaxation during the phenyleph-rine-induced pre-contraction, depending on the report [32–38].Therefore, the relaxant effect seen during the phenylephrine-in-duced contraction was consistent with the lower side of the re-ported values [35–38].  Immunoblot Analysis of the Expression of PAR 2 and eNOS The tissue lysates were prepared in the lysis buffer consistingof 50 mmol/l Tris-HCl, pH 7.2, 1% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% sodium dodecyl sulfate, 500 mmol/l NaCl,10 mmol/l MgCl 2  , 10 ␮  g/ml leupeptin, 10 ␮  g/ml aprotinin, 10 ␮  mol/l 4-aminidophenylmethane sulfonyl fluoride, as previously reported [15]. Total protein (20 ␮  g) was subjected to an immuno-blot analysis with a mouse monoclonal anti-PAR  2 antibody (sc-13504, SAM11; Santa Cruz Biotechnology, Santa Cruz, Calif.,USA) and a polyclonal anti-PAR  2 antibody (sc-5597, H99; SantaCruz Biotechnology) at 0.2 and 0.4 ␮  g/ml, respectively, and ahorseradish peroxidase-conjugated secondary antibody (Sigma,St. Louis, Mo., USA) in an immunoreaction enhancer solution(Can-Get-Signal; Toyobo, Osaka, Japan). The expression of endo-thelial NO synthase (eNOS) was detected with anti-eNOS anti-body (No. 610296; BD Biosciences, San Jose, Calif., USA) at 250-fold dilution. The immune complex was detected with an en-hanced chemiluminescence technique (GE Healthcare, Tokyo,Japan). The chemiluminescence signal was detected and analyzedwith the ChemiDoc XRS-J image analysis system (Bio-Rad, To-kyo, Japan). After performing immunoblot detection, the mem-branes were stained with naphthol blue black to visualize the actinbands. As shown in figure 8c, the level of actin did not signifi-cantly differ between the presence and absence of endothelium,and also before and after serum-free incubation, thus suggestinga negligible contribution of the endothelial actin to the level of total actin. The levels of eNOS and PAR  2 were therefore evaluatedby normalizing the sample loading by the level of actin.   N -Glycosidase F Treatment  The tissue lysates were treated with and without (for control)7.5 units N   -glycosidase F/10 ␮  g protein at 37 ° C for 3 h, accordingto the manufacturer’s instruction. Thereafter, the lysates weresubjected to an immunoblot analysis for PAR  2 as described above.  Drugs and Solutions Thrombin (bovine plasma), trypsin (bovine pancreas),DMEM, acetylcholine, sodium nitroprusside (SNP), dipheny-leneiodonium chloride (DPI), N  ␻   -nitro- L  -arginine methyl ester( L  -NAME), U46619 and N   -glycosidase F were purchased fromSigma. Superoxide dismutase was from Calbiochem (La Jolla,Calif., USA). TFLLR-NH 2 (PAR  1  -AP), SLIGRL-NH 2 (PAR  2  -AP),and AYPGKF-NH 2 (PAR  4  -AP) were from Bachem (Bubendorf,Switzerland). L  (+)-ascorbic acid was from Wako (Osaka, Japan).  Statistical Analysis All data are expressed as the mean 8 SEM of the indicatednumber of experiments. One strip obtained from one animal wasused for each experiment, and therefore the number of experi-ments indicates the number of animals. Unpaired Student’s t-testand a one-way ANOVA followed by Scheffé’s post hoc test wereused to determine any significant differences. A value of p ! 0.05was considered to be statistically significant.  Results Enhancement of the PAR 2  -Mediated Relaxation after 24-Hour Incubation in Serum-Free Media in WKY  Aortas In the strips with endothelium obtained from the fe-male WKY aortas and before incubating in serum-freemedia, 100 nmol/l U46619 induced a sustained contrac-tion (fig. 1a, d). Trypsin and PAR  2  -AP had no effect onthis contraction (fig. 1a, d). After 24-hour incubation inserum-free media at 37 ° C, trypsin and PAR  2  -AP induceda significant relaxation in rat aortas (fig. 1b, e, 2e). The sustained phase of relaxation was frequently accompa-nied by oscillatory responses (fig. 1b, e). When incubatedat 4 ° C in either serum-free DMEM or PSS for 24 h, tryp-sin and PAR  2  -AP induced no responses (fig. 1c, f). On the other hand, after 24-hour incubation with 5% serum,trypsin and PAR  2  -AP induced 28.5 and 25.6% reductions(n = 2) in the U46619-induced pre-contraction, respec-tively. It is therefore conceivable that ex vivo incubationat 37 ° C but not serum deprivation was responsible for theobserved changes in the PAR  2  -mediated responses. Thechanges in the relaxant responses were therefore com-pared between before and after serum-free incubation. The relaxations seen with trypsin and PAR  2  -AP afterserum-free incubation were abolished either by the treat-ment with L  -NAME or the removal of endothelium(fig. 2). L  -NAME was applied 30 min before initiating thepre-contraction by U46619. The endothelium was re-moved after 24-hour serum-free incubation of the strips   PAR  2 Upregulation by Oxidative Stress J Vasc Res 2010;47:494–506 497 with endothelium. The successful removal of functionalendothelium was confirmed by the loss of the relaxantresponse to acetylcholine (fig. 2b, d). In female WKY aor-tas, acetylcholine induced a relaxant response in the pres-ence of endothelium (fig. 5). The evaluation of the con-centration-dependent responses revealed that the relax-ant response to trypsin was significantly enhanced afterthe incubation in serum-free media (fig. 3). In contrast,the relaxant response to SNP, a NO donor, remained un-altered (fig. 3). In male WKY aortas, trypsin and PAR  2  -AP induced atransient but significant relaxation without serum-freeincubation (fig. 4). However, this relaxation was similarly enhanced by 24-hour serum-free incubation as observedwith female aortas and became a sustained relaxation(data not shown). Since female rat aortas did not respondto PAR  2 agonists without serum-free incubation, whilethe enhancement of the relaxant response to PAR  2 ago-nists was similarly observed in males and females, femalerats were primarily used in the present investigation.  Acetylcholine induced an endothelium-dependentrelaxation in female WKY aortas, which remained un-changed after serum-free incubation (fig. 5a, b). Throm-bin, even at 3 units/ml, had no effect on the U46619-induced contraction both before and after 24-h incuba-tion in serum-free media (fig. 5c, d). This concentrationof thrombin has been shown to be sufficient for induc-ing endothelium-dependent responses in other arteries 5 min0.1 g100100 nmol/l U46619Before1 μmol/l trypsin a 0     %     T   e   n   s    i   o   n 5 min0.1 g100100 nmol/l U46619After, 37°C1 μmol/l trypsin b 0     %     T   e   n   s    i   o   n 5 min0.1 g100100 nmol/l U46619After, 4°C1 μmol/l trypsin10 μmol/l ACh c 0     %     T   e   n   s    i   o   n 5 min0.1 g100100 nmol/l U46619Before100 μmol/l PAR 2 -AP d 0     %     T   e   n   s    i   o   n 5 min0.1 g100100 nmol/l U46619After, 37°C100 μmol/l PAR 2 -AP e 0     %     T   e   n   s    i   o   n 5 min0.1 g100100 nmol/l U46619After, 4°C200 μmol/l PAR 2 -AP10 μmol/l ACh f  0     %     T   e   n   s    i   o   n   Fig. 1. Induction of the relaxant responseto PAR  2 agonists after 24-hour incubationin serum-free media in female WKY aor-tas. Representative recordings of the re-sponse to trypsin ( a–c  ) or PAR  2  -AP ( d–f   )during the U46619-induced contraction inthe strips with an endothelium, before ( a  , d  ) and after 24-hour incubation in serum-free media at 37 ( b  , e  ) and 4 ° C ( c  , f   ). Thelevels of tension obtained at rest and justprior to application of trypsin or PAR  2  -APduring the U46619-induced contractionwere assigned values of 0 and 100%, re-spectively. Similar results were observed in5 ( a  , b  , d  , e  ) and 3 ( c  , f   ) independent ex-periments.
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