Increased tyrosine phosphorylation mediates the cooling-induced contraction and increased vascular reactivity of Raynaud's disease.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 5, May 2004, pp 1578–1585 DOI 10.1002/art.20214 © 2004, American College of Rheumatology Increased Tyrosine Phosphorylation Mediates the Cooling-Induced Contraction and Increased Vascular Reactivity of Raynaud’s Disease Philip B. Furspan, Soumya Chatterjee, and Robert R. Freedman Objective. Increased levels of protein tyrosine kinase (PTK) are mechanistically associated with increased contractile responsiveness to cooling. This study tests the hypothesis that increased PTK activity mediates the increased vascular reactivity to agonists and cooling associated with primary Raynaud’s disease (RD). Methods. The response of dermal arterioles isolated from control (n ⴝ 29) and RD (n ⴝ 29) subjects to contractile and dilatory agents at 37°C and 31°C was characterized using the microvessel perfusion technique. Fluorescence immunohistochemistry was used to measure tyrosine phosphorylation. Results. At 37°C, arteries from RD patients exhibited similar sensitivity to the specific ␣2-adrenergic agonist UK 14,304, to serotonin, and to angiotensin II. At 31°C, however, the response to all 3 agonists was greater in the arterioles from the RD patients than in those from the control subjects. Agonist-induced contraction at both temperatures was reversed by cumulative addition of the PTK inhibitors genistein (1–30 M) and tyrphostin 47 (0.1–10 M). All arterioles from control subjects relaxed slightly in response to cooling, whereas more than half of those from RD patients contracted. This cooling-induced contraction was reversed by the cumulative addition of genistein. The 3 agonists elicited large increases in tyrosine phosphorylation only in arterial segments from RD patients at 31°C. Cooling from 37°C to 31°C elicited a large increase in tyrosine phosphorylation in arterioles from RD pa- tients, but not those from control subjects. All increases in tyrosine phosphorylation could be prevented by genistein. Conclusion. Increased tyrosine phosphorylation mediates cooling-induced contraction and the increased vascular reactivity of skin arterioles from individuals with RD. Raynaud’s disease (RD) is characterized by episodic digital vasospasms that are provoked by cold and/or emotional stress. Lewis (1) hypothesized that a “local fault” caused cold hypersensitivity of the digital blood vessels. A possible mechanistic link between cooling and vasoconstriction/vasospasm is suggested by recent studies investigating the biochemical basis of cold-induced modification of vascular response. These studies have strongly implicated the protein tyrosine kinase (PTK) signal transduction pathway as the mediator of coldinduced vasoconstriction in a variety of blood vessels (2–4). During the last decade, PTK signal transduction pathways have been revealed to play an important role in the mediation of agonist- and growth factor–initiated contractile activity in vascular smooth muscle (5). Wagerle et al (2) demonstrated that the coldinduced contraction of the middle cerebral artery of the lamb could be reversed completely by 2 structurally diverse inhibitors of PTK, genistein and tyrphostin 47, but not by staurosporine, a protein kinase C inhibitor. Conversely, sodium orthovanadate (SOV), an inhibitor of protein tyrosine phosphatase, potentiated the coldinduced contraction. In a subsequent study from the same group, it was observed that whereas rat tail artery did not normally contract in response to cooling from 37°C to 24°C, rat tail artery pretreated with SOV did contract in response to cooling (6). We have corroborated these results (4). Dahdah et al (3) observed that contraction in lamb coronary artery was associated with Supported by a grant from the American Heart Association, Midwest Affiliate (51302Z). Philip B. Furspan, PhD, Soumya Chatterjee, MD, Robert R. Freedman, PhD: Wayne State University, Detroit, Michigan. Address correspondence and reprint requests to Philip B. Furspan, PhD, C. S. Mott Center, Wayne State University, 275 East Hancock Avenue, Detroit, MI 48201. E-mail: email@example.com. Submitted for publication September 5, 2003; accepted in revised form January 23, 2004. 1578 TYROSINE PHOSPHORYLATION AND RAYNAUD’S DISEASE increased tyrosine phosphorylation when the temperature was reduced from 37°C to 7°C. These results suggest that increased levels of PTK activity are mechanistically associated with increased contractile responsiveness to cold. This conclusion is supported by evidence indicating that cooling enhances the contractile effect of G-protein–coupled agonists that activate PTK signal transduction pathways (5,7). PTK pathways have also been implicated in the mediation of vasospasm in other vascular diseases, for example, stroke, heart disease, and atherosclerosis (8–10). The present study was designed to examine the role played by PTK activity in the cooling- and agonistinduced contraction of arterioles from patients with Raynaud’s disease (RD) and from healthy control subjects. Because the reactivity to a number of different agonists has been reported to be enhanced in blood vessels from RD patients compared with those from controls, we have examined the actions of 3 of these agonists. We found that the enhanced reactivity does not result from a defect in the interaction between a specific agonist and its receptor, but rather from an alteration in the PTK signal transduction pathway that mediates their actions. PATIENTS AND METHODS Characteristics of the study subjects. Raynaud’s disease was classified using the Allen and Brown criteria (11): bilateral digital (must include fingers) color changes (2 of the following 3: blanching, cyanosis, rubor), provoked by cold and/or emotional stress, for at least 2 years, in the absence of any identifiable disease process (e.g., scleroderma, rheumatoid arthritis, etc.). Additionally, the 29 premenopausal women with RD (ages 21–54 years) were required to be negative for antinuclear and anticentromere antibodies (on HEp-2 cell substrate). Twenty-nine healthy, age-matched (ages 21–50 years) women with no evidence of any physical disorder were also evaluated in this study. Each study subject was medication-free (at least 2 weeks), had related their medical history, and had completed an extensive symptom questionnaire used in our previous studies (12,13) to ensure the absence of any disorder. All subjects were menstruant. Subjects did not undergo a biopsy at a specific point in their menstrual cycle, since hormone status does not appear to affect the incidence or severity of attacks of vasospasm (14,15). Smokers were not included because smoking impairs vascular function (16). All patients and normal volunteers were required to be free of any cardiovascular disease (e.g., hypertension, heart disease) or other disease that might affect vascular function (e.g., diabetes). All procedures followed in this study were in accordance with Wayne State University guidelines for studies 1579 involving humans. An institutional review committee approved this study, and all subjects gave written informed consent. Vessel preparation. Using lidocaine (without norepinephrine) as a local anesthetic, skin biopsies (6 mm in diameter) were taken from the medial forearm of all study subjects. Arterioles of usable size (mean ⫾ SEM pressurized diameter 136 ⫾ 11 m and 137 ⫾ 10 m in control subjects and RD patients, respectively) were dissected out from the dermal– subcutaneous boundary. Arterioles were used immediately or were stored overnight at 4°C in Eagle’s minimum essential medium (Sigma, St. Louis, MO). Preliminary experiments found no difference in reactivity between vessels that were used the day of the biopsy and vessels from the same biopsy tissue that were used the next day. Similar results have been reported in the literature (17,18). Arterioles were attached to the proximal micropipette using individual strands from 3-0 nylon braided suture and were superfused with physiologic salt solution (118 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4䡠7H2O, 1.6 mM CaCl2䡠2H2O, 25.0 mM NaHCO3, 5.5 mM dextrose, and 1.2 mM CaNa2–EDTA) and aerated with 95% O2 and 5% CO2. A perfusion pressure of ⬃20 mm Hg was used to clear the lumen of blood, followed by attachment of the distal end of the artery segment to the closed distal pipette. The superfusate was warmed to, and maintained at, 37°C. Transmural pressure was controlled with a pressure-servo system. After an equilibration period of 45 minutes at 20 mm Hg, the pressure was increased to 40 mm Hg (19). Precise and rapid (⬍2 minutes) temperature reduction was accomplished by cooling the superfusate as it flowed through a jacketed temperature-exchange coil connected to a second circulating heating/cooling waterbath that was maintained at 31°C. Agents to be evaluated were added directly to the superfusate. Superfusate replacement occurred in less than 2 minutes. Contractile activity of the blood vessels was monitored with a video camera and quantified with a video dimension analyzer (Living Systems Instrumentation, Burlington, VT) that was connected to a video monitor. Changes in diameter were recorded on videotape and a computer-based dataacquisition system (DATAQ Instruments, Akron, OH) and were expressed as the percentage of change from baseline. Experimental protocols. Perfused microvessels. After equilibration, arterioles were exposed to 95 mM K⫹– physiologic salt solution (equimolar substitution of KCl for NaCl). The presence of an intact endothelium was determined by exposing arterioles that had been precontracted with norepinephrine (10⫺6M) to increasing concentrations of acetylcholine (10⫺9–10⫺6M). Concentration-response curves were constructed for the agonists tested: UK 14,304, a specific ␣2-adrenergic agonist (10⫺10–10⫺8M), serotonin (10⫺9– 10⫺6M), and angiotensin II (10⫺10–10⫺8M). All of these agonists have been reported to exert their contractile effects, at least in part, via the PTK pathway (4,20). Responses were measured at 37°C and at 31°C. A low temperature of 31°C was chosen because it represents the temperature reached by the fingers shortly after exposure to moderately cold temperature (10°C) (21) and because preliminary results indicated that lower temperatures significantly inhibited contraction in response to these agonists (i.e., 31°C was the lowest temperature at which responses of arterioles from normal individuals did not exhibit a decrease in responsiveness to agonists). Results 1580 FURSPAN ET AL Figure 1. Contraction of skin arteriole segments from control subjects (solid line) and patients with Raynaud’s disease (broken line) induced by the agonists UK 14,304, serotonin, and angiotensin II at 37°C (open symbols) and 31°C (solid symbols). Values are the mean ⫾ SEM of 6 samples per group. ⴱⴱ ⫽ P ⬍ 0.01; ⴱ ⫽ P ⬍ 0.05. were expressed as the percentage of change in the lumen diameter from baseline. The effect of PTK inhibition on agonist-induced contraction was determined by cumulatively adding genistein (1–100 M) or tyrphostin 47 (0.1–10 M), both of which are inhibitors of PTK activity, to the bath after contraction (⬃60% decrease in diameter) in response to an agonist had reached a plateau. The order of exposure to agonist, antagonist, and temperature was randomized. The response of vessels to cooling from 37°C to 31°C was determined in the absence and presence of SOV (100 M), an inhibitor of tyrosine phosphatase. If contraction occurred, genistein (1–100 M) was added. SOV is able to inhibit tyrosine phosphatase in part because of a similarity in size and charge that it shares with inorganic phosphatase (22). In addition, SOV is able to assume a trigonal bipyramidal coordination that is thought to resemble a phosphate group (22). Fluorescence immunohistochemistry (23). Freshly obtained dermal arteries measuring 1–3 mm in length and 100–200 m in diameter were cleaned of adventitial tissue and cut into pieces ⬃200 m wide. Segments were placed in wells containing 50 l of phosphate buffered saline (PBS) composed of 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8 mM Na2HPO4. After incubation in PBS at 37°C on a thermostatically controlled block for 30 minutes, the pieces were transferred to wells containing PBS and the agents of interest. If indicated, the temperature was reduced to 31°C by adding an equivalent volume of 25°C PBS and the agent of interest. After 5 minutes at the appropriate temperature, the vessel segments were transferred to PBS containing 2% paraformaldehyde for 10 minutes to fix the tissues (this and subsequent steps were performed at room temperature). Next, the tissue was incubated with 0.1% Triton X-100 in PBS for 10 minutes. After a wash in PBS, the pieces were incubated in a solution containing 0.9% sodium citrate, 2% goat serum, 1% bovine serum albumin, 0.05% Triton X-100, 0.025% NaNO3, and tetramethyl rhodamine isothiocyanate– labeled phosphotyrosine monoclonal antibody (1:50 dilution) for 45 minutes. After incubation, the vessel pieces were washed with 0.02% Triton X-100 and 0.9% sodium citrate in PBS and then in PBS alone. The tissue segments were then mounted on glass slides and examined under a microscope equipped with an epifluorescence optical system. Measurement of fluorescence intensity as an indicator of tyrosine phosphorylation was performed using SimplePCI, a Compix (Cranberry Township, PA) image analysis program. Reagents. All drugs and chemicals were obtained from Sigma (St. Louis, MO). Genistein and tyrphostin 47 were dissolved in DMSO. Statistical analysis. Concentration-response curves were calculated as geometric means. Paired and unpaired t-tests and analysis of variance were performed. When necessary, modified analyses were used to allow for multiple testing procedures (e.g., Bonferroni correction for multiple compari- TYROSINE PHOSPHORYLATION AND RAYNAUD’S DISEASE 1581 Figure 2. Effect of genistein and tyrphostin 47 on skin arteriole segments maintained at 31°C from control subjects (solid line) and Raynaud’s disease patients (broken line). Values are the mean ⫾ SEM of 6 samples per group. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.01. sons). In all cases, P values less than 0.05 were considered significant. All results are expressed as the mean ⫾ SEM. RESULTS The contraction of arterioles from RD patients and control subjects in response to UK 14,304, serotonin, and angiotensin II was not significantly different when measured at 37°C (Figure 1). At 31°C, however, the curves for all 3 agonists applied to arterioles from RD patients were shifted to the left compared with those from control subjects (Figure 1). The maximum contraction of arterioles from RD patients at 31°C was not only greater than that of control subjects at both 37°C and 31°C, but it was also greater than the maximum contraction of arterioles from RD patients at 37°C (Figure 1). Contraction in response to 95 mM KCl was equivalent in both groups and at both temperatures (data not shown). The involvement of PTK in the mediation of contraction in response to the 3 agonists is suggested by the relaxation induced by the cumulative addition of the PTK inhibitors genistein (1–30 M) and tyrphostin 47 (0.1–10 M). At 37°C, the response to genistein was similar irrespective of the subject group or the agonist evaluated (data not shown). When applied to arterioles maintained at 31°C, those from RD patients exhibited greater sensitivity to the relaxant effect of both genistein and tyrphostin 47 (Figure 2). Vehicle (DMSO; 1:10,000 to 1:1,000 dilution) was without effect. Genistein and tyrphostin 47 were not effective in reversing the contraction caused by 95 mM KCl (data not shown). In response to cooling the superfusate from 37°C to 31°C, none (0 of 15) of the arterioles from control subjects exhibited contraction (Figure 3). Of the arterioles from RD patients, however, more than half (8 of 15) contracted in response to cooling (Figure 3). The contraction in response to cooling was reversed by the cumulative addition of genistein (1–30 M) (Figure 3). After washout of genistein, the vessel diameter remained at the baseline size. Pretreating the arterioles with SOV (100 M) produced a small contraction (9.2 ⫾ 5.1%, n ⫽ 6) in arterioles from control subjects but did not alter the response of those from RD patients. Concentrations of UK 14,304, serotonin, and angiotensin II that caused maximal contraction of arte- 1582 Figure 3. Change in lumen diameter of skin arteriole segments (only those that responded; see text for details) from control subjects and Raynaud’s disease (RD) patients in response to a reduction in the bath temperature from 37°C to 31°C, and effect of various concentrations of genistein on the contraction induced in the arterioles from the RD patients. Values are the mean and SEM of 15 control samples and 8 RD samples per group. ⴱⴱ ⫽ P ⬍ 0.01 versus control group; # ⫽ P ⬍ 0.05 versus untreated RD group. rioles also caused increases in tyrosine phosphorylation, as indicated by increases in fluorescence intensity (Figure 4). The pattern of increases in tyrosine phosphorylation was similar for the 3 agonists: RD patient arterioles at 31°C ⬎⬎ control subject arterioles at 31°C ⬎ RD patient arterioles at 37°C ⬎ control subject arterioles at 37°C. Only the increases in tyrosine phosphorylation elicited at 31°C in arteriole segments from RD Figure 4. Change in tyrosine phosphorylation (as indicated by fluorescence intensity) of skin arteriole segments from control subjects and Raynaud’s disease (RD) patients in response to 3 different agonists, UK 14,304, serotonin, and angiotensin II, at 37°C and at 31°C, and in segments from RD patients exposed to each of the agonists at 31°C in the presence of 30 M genistein. Values are the mean and SEM of 5 samples per group. ⴱⴱⴱ ⫽ P ⬍ 0.01 versus control group at 31°C. FURSPAN ET AL Figure 5. Change in tyrosine phosphorylation (as indicated by fluorescence intensity) of skin arteriole segments from control subjects and Raynaud’s disease (RD) patients in response to a decrease in the bath temperature from 37°C to 31°C in the absence and presence of 100 M sodium orthovanadate (SOV). Values are the mean and SEM of 5 samples per group. ⴱ ⫽ P ⬍ 0.05 versus untreated control group. Values for the RD groups were not significantly different from each other. patients were significantly greater than the other values. Genistein (30 M) was able to prevent the increases in tyrosine phosphorylation in all cases. (Only the results of its effect on arteriole segments from RD patients at 31°C are shown in Figure 4.) Tyrphostin 47 (3 M) had a similar effect on tyrosine phosphorylation (data not shown). Cooling in the absence of agonists caused a small increase in tyrosine phosphorylation in arteriole segments from control subjects and a large increase in segments from RD patients (Figure 5). The presence of SOV (100 M) increased the level of tyrosine phosphorylation in arteriole segments from control subjects, but did not increase it significantly in segments from RD patients (Figure 5). Because some studies have reported impaired endothelium-mediated vasodilation in individuals with RD (24,25), we examined the response of precontracted (with 10⫺6M norepinephrine) arterioles from RD patients and control subjects to acetylcholine (10⫺8– 10⫺6M). At 37°C, there was no difference in the response to acetylcholine between arterioles from RD patients and those from control subjects (Figure 6). Surprisingly, at 31°C, arterioles from control subjects exhibited a reduced sensitivity and maximal relaxation to acetylcholine compared with those from the other groups (Figure 6). A decrease in sensitivity, but not maximal relaxation, to bradykinin (10⫺11–10⫺8M) was TYROSINE PHOSPHORYLATION AND RAYNAUD’S DISEASE Figure 6. Response of precontracted (with 10⫺6M norepinephrine) skin arterioles from control subjects (solid line) and Raynaud’s disease (RD) patients (broken line) to the cumulative addition of acetylcholine (ACh) at 37°C (open symbols) and 31°C (solid symbols). Values are the mean ⫾ SEM of 7 samples per group. ⴱ ⫽ P ⬍ 0.05 and ⴱⴱ ⫽ P ⬍ 0.01 for the control value at 31°C compared with the RD value at 31°C; ## ⫽ P ⬍ 0.01 for the control value at 31°C compared with the control value at 37°C. also observed in arterioles from control subjects at 31°C (data not shown). DISCUSSION These experiments strongly suggest a causative relationship between increased tyrosine phosphorylation and the increased vascular reactivity at reduced temperature and cooling-induced contraction associated with RD. Contraction in response to the 3 agonists tested and in response to cooling was reversed by the cumulative addition of the PTK inhibitors genistein and tyrphostin: contraction in response to 95 mM KCl was not. The same agonists also elicited increases in tyrosine phosphorylation that could be prevented by genistein and tyrphostin 47. PTK mediation of contraction induced by G-protein–coupled receptor agonists such as those used in this study is well established (5). Thus, the increased tyrosine phosphorylation revealed in the present study may underlie reports of increased vascular reactivity to a variety of agonists in vivo in RD patients (12,13,26). The increased vascular reactivity associated with increased tyrosine phosphorylation may be related to the reported enhancement of myofilament calcium sensitivity in vascular smooth muscle by PTK (27,28). The PTK signal transduction system also has been 1583 implicated in the pathophysiology of other vascular diseases characterized by increased vascular reactivity and vasospasm, such as hypertension (29) and arteriosclerosis (10). Of course, the hallmark of RD is cooling-induced vasospasm of digital blood vessels. Although we did not observe cooling-induced vasospasm (i.e., complete closure of the lumen) in the arterioles we tested, we did observe cooling-induced contraction in more than onehalf the arterioles from RD patients but not control subjects. The lack of a vasospastic response to cooling in all isolated vessels may relate to the absence of conditions in vitro that are required for vasospasm to occur in response to cooling. The inconsistent occurrence of the cooling-induced contractile response suggests that specific conditions may be required for this response to occur, perhaps relating to the balance between contractile and relaxing influences existing at the time the vessel is exposed to the reduction in temperature. Even in vivo, vasospasm in response to laboratory cooling occurs only 80–90% of the time in individuals with RD (30,31). The involvement of PTK in the cooling-induced contraction of arterioles from RD patients is suggested by its reversal by genistein and tyrphostin 47 and by the large increase in tyrosine phosphorylation measured in arteriole segments. The inhibition of an increase in tyrosine phosphorylation by these PTK inhibitors correlates with their reversal of contraction in isolated arterioles (Figures 4 and 2). Although cooling elicited an increase in tyrosine phosphorylation in untreated and SOV-treated arteriole segments from control subjects, only those treated with SOV exhibited contraction. These results suggest that there may be a threshold level of tyrosine phosphorylation that is required for contraction to occur and/or that other conditions need to exist in addition to a specific level of tyrosine phosphorylation. In a previous study, we found that rat tail artery cooled from 37°C to 25°C did not contract, whereas those previously exposed to SOV, the protein tyrosine phosphatase inhibitor, did contract (4). Tyrosine phosphorylation of human skin arterioles uniformly increases in response to cooling. However, the fact that they do not always contract or exhibit vasospasm (either in vitro or in vivo) suggests that tyrosine phosphorylation by itself is required, but is not sufficient, for contraction to occur. Other investigators have reported a role for PTK in the mediation of other vasospastic conditions. Fujikawa et al (9) reported that stimulation of the tyrosine kinase pathway(s) of canine basilar artery by subarachnoid hemorrhage leads to vasospasm. Ito et al (10) 1584 FURSPAN ET AL observed that the specific PTK inhibitor ST 638 blocked coronary vasospasm induced by chronic interleukin-1 treatment in pigs. Because some studies have suggested that impaired endothelium-dependent vasodilation plays a role in the increased vascular reactivity that is characteristic of RD (24,25), we examined the response of precontracted arterioles to acetylcholine. We found that the response to acetylcholine is impaired at 31°C in arterioles from control subjects, whereas the response of arterioles from RD patients at 31°C did not differ from the response at 37°C. This finding is consistent with a report of an enhanced dilatory response, as measured by fingertip blood flow after methacholine infusion, in RD patients compared with control subjects (32). The absence of impairment in the vasodilatory response may represent a compensatory adjustment to the greater vascular reactivity at this temperature. This adjustment may occur as a consequence of an increase in PTK activity in endothelial cells from RD patients paralleling that in vascular smooth muscle cells. 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