Published online before print December 18, 2008, doi: 10.1161/STROKEAHA.108.530196
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(Stroke. 2009;40:591.)
© 2009 American Heart Association, Inc.
Original Contributions |
Enhanced Contractile Response of the Basilar Artery to Platelet-Derived Growth Factor in Subarachnoid Hemorrhage
From the Division of Molecular Cardiology, Research Institute of Angiocardiology (Y.M., K.H., M.H., Y.K., K.K., H.K.) and Department of Neurosurgery (Y.M., T.S.), Graduate School of Medical Sciences, and the 21st Century COE program (H.K.), Kyushu University, Fukuoka, Japan.
Correspondence to Katsuya Hirano, MD, PhD, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail khirano{at}molcar.med.kyushu-u.ac.jp
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Abstract |
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Background and Purpose— The level of platelet-derived growth factor (PDGF) in cerebrospinal fluid is elevated in subarachnoid hemorrhage (SAH). Therefore, the contractile effect of PDGF on the basilar artery was examined in SAH.
Methods and Results— A rabbit double-hemorrhage SAH model was used. In the medial layers of the control basilar artery, PDGF had no effect on contraction up to 1 nmol/L, whereas 3 nmol/L PDGF induced slight contraction. In SAH, PDGF induced an enhanced contraction with an increase in [Ca2+]i at 1 nmol/L and higher concentrations. The levels of [Ca2+]i and tension induced by 1 nmol/L PDGF in SAH were 17% and 20%, respectively, of those obtained with 118 mmol/L K+ depolarization. The PDGF-induced elevation of [Ca2+]i and contraction seen in SAH were abolished in the absence of extracellular Ca2+. In 
-toxin–permeabilized strips of SAH animals, PDGF induced no further development of tension during contraction induced by 300 nmol/L Ca2+, suggesting no direct effect on myofilament Ca2+ sensitivity. Genistein at 10 µmol/L completely inhibited the tension induced by 1 nmol/L PDGF. The level of myosin light-chain phosphorylation was significantly increased by 1 nmol/L PDGF.
Conclusions— These results show that the contractile response to PDGF of the basilar artery was enhanced in SAH. The PDGF-induced contraction depended mostly on tyrosine phosphorylation and Ca2+-dependent myosin light-chain phosphorylation. The enhancement of the responsiveness to PDGF may therefore contribute to the development of cerebral vasospasm after SAH.
Key Words: subarachnoid hemorrhage • vasospasm • platelet-derived growth factor
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Introduction |
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Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells (VSMCs),1,2 thereby contributing to neointimal formation and the development of vascular lesions in atherosclerosis.3,4 PDGF is known to activate intracellular signal transduction pathways, such as the mitogen-activated protein kinase pathway, the phosphatidylinositol 3-kinase pathway, and the Ca2+ signal pathway.1,5 PDGF has been shown to induce a sustained elevation of cytosolic Ca2+ concentrations ([Ca2+]i) in cultured VSMCs.6 Although the elevation of [Ca2+]i is a primary signal that elicits smooth muscle contraction,7 the contractile effect of PDGF has been reported only in limited types of arteries, such as the rat aorta8 and rabbit ear artery.9 It is conceivable that most "normal" vascular tissues have no responsiveness to PDGF. However, the phenotype of SMCs may be altered in vascular lesions, so that smooth muscle may attain the responsiveness to PDGF and exert a contractile effect in vascular diseases.
A number of subarachnoid hemorrhage (SAH) patients have SAH-associated delayed posthemorrhagic cerebral vasospasm, which plays a critical role in determining the prognosis of both life and neurologic function.10 However, the molecular mechanism for the pathogenesis of cerebral vasospasm still remains to be elucidated. Either the increased production of spasmogens or changes in the vascular responsiveness to the spasmogens may contribute to the development of posthemorrhagic vasospasm. In SAH, expression of the recombinant human BB isoform of PDGF (PDGF-BB) has been found to be elevated in SMCs of the basilar artery.11 The level of PDGF-BB in the cerebrospinal fluid during the acute phase of SAH, which was associated with symptomatic cerebral vasospasm, was significantly higher than that observed in SAH without symptomatic cerebral vasospasm.12 It is conceivable that PDGF could serve as a spasmogen in posthemorrhagic vasospasm in SAH. However, so far there has been no report regarding the contractile effect of PDGF in SAH. Because no contractile effect of PDGF has been reported in normal cerebral arteries, it is possible that the responsiveness of the cerebral artery toward PDGF increases in SAH. However, such a possibility remains to be elucidated. Furthermore, the mechanism of the PDGF-induced contraction remains to be elucidated, although a [Ca2+]i elevation has been suggested to play a primary role in mediating the PDGF-induced contractile effect.9,13
The present study examined the contractile effect of PDGF in SAH in the basilar artery isolated from double-hemorrhage rabbits. In addition, the effects of PDGF on the phosphorylation of myosin light chain (MLC) and the Ca2+ sensitivity of the contractile apparatus were examined. The results demonstrated for the first time that the responsiveness to PDGF was increased in SAH. The PDGF-induced contraction is suggested to be mainly dependent on the Ca2+ signal and MLC phosphorylation, whereas the increase in Ca2+ sensitivity of the contractile apparatus plays only a negligible role in PDGF-induced contraction.
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Materials and Methods |
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Preparation of the SAH Rabbit Model
A double-hemorrhage SAH model was prepared in male Japanese white rabbits (2.5 to 3.0 kg), as previously described.14 During the operative procedures, the animals were anesthetized with ketamine (40 mg/kg IM) and pentobarbital sodium (20 mg/kg IV). Rabbits also received an intracisternal injection of 2.5 mL autologous blood on days 0 and 2. The control animals received injections of the same volume of saline. Seven days after the first injection, the rabbits were heparinized (1000 U) and then euthanized by an overdose of pentobarbital sodium (120 mg/kg IV); the carotid artery was subsequently exsanguinated. The basilar artery was immediately excised, and arterial rings measuring 500 µm wide were prepared. The endothelium was mechanically removed by rubbing the internal surface with a tungsten wire. The absence of a functional endothelium was confirmed by observing that 1 µmol/L carbachol induced no relaxation during contraction induced by 118 mmol/L K+ depolarization.15
Measurement of [Ca2+]i and Tension Development in Intact Smooth Muscle Preparations
The arterial rings were loaded with the Ca2+ indicator dye fura-2 as previously described.15 The rings were mounted horizontally between 2 tungsten wires by passing the wires through the arterial lumen in an organ bath set on the stage of an inverted fluorescence microscope (TMD 56; Nikon, Tokyo, Japan). The preparation was then equilibrated at 37°C for 60 minutes under a resting tension of 50 mg. Tension development was measured with a force transducer U gauge (Minebea, Nagano, Japan). The changes in the fura-2 fluorescence intensities obtained with 340 nm (F340) and 380 nm (F380) excitation and their ratio (F340/F380) were simultaneously monitored with a spectrophotometer (CAM 220, Tokyo, Japan) as previously described.16,17 The tension and fluorescence ratio were expressed as percentages, whereas the values obtained in normal physiologic saline solution (PSS; 5.9 mmol/L K+) and 118 mmol/L K+-PSS were assigned 0% and 100%, respectively, unless otherwise specified.
Measurement of Tension Development in Permeabilized Preparations
The arterial rings were permeabilized by 1-hour treatment with 5000 U/mL staphylococcal -toxin in Ca2+-free cytosolic substitution solution (CSS), as previously described.18 The arterial rings then were mounted horizontally between 2 tungsten wires as described earlier, pulled to 1.2-fold their resting length, and then allowed to relax completely in Ca2+-free CSS for 30 minutes. Force development was recorded with a force transducer U gauge (Minebea). The levels of tension seen in the Ca2+-free CSS (resting state) and 10 µmol/L Ca2+-containing CSS (maximum contraction) were assigned values of 0% and 100%, respectively, unless otherwise specified.
Measurement of MLC Phosphorylation
The extent of MLC phosphorylation was determined with the urea-glycerol gel electrophoresis technique, followed by immunoblot detection of 20-kDa MLC, both unphosphorylated and phosphorylated, with a specific mouse monoclonal anti-MLC antibody, as previously described.19 The immune complex was detected with the enhanced chemiluminescence technique (ECL plus kit; Amersham, Buckinghamshire, UK). X-OMAT AR film (Kodak, Rochester, NY) was used to detect light emission. After obtaining the image of the x-ray film with a gel documentation system equipped with a CCD camera, Printgraph AE-6911CX (Atto, Tokyo, Japan), the density of unphosphorylated and phosphorylated MLCs was determined by gel plotting macros of the NIH Image version 1.61 software (National Institutes of Health, Bethesda, Md). The percentage of the phosphorylated form in total MLC (sum of unphosphorylated and phosphorylated forms) was calculated to indicate the extent of MLC phosphorylation.
Drugs and Solutions
The composition of normal PSS was as follows: 123 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L NaH2PO4, 1.2 mmol/L MgCl2, 1.25 mmol/L CaCl2, and 11.5 mmol/L D-glucose. High-K+ PSS was prepared by replacing NaCl with equimolar KCl. A Ca2+-free PSS was made by omitting CaCl2 from normal PSS. PSS was bubbled with a mixture of 95% O2 and 5% CO2, with the resulting pH being 7.4. The composition of Ca2+-free CSS was as follows: 10 mmol/L EGTA, 100 mmol/L potassium methanesulfonate, 3.38 mmol/L MgCl2, 2.2 mmol/L Na2ATP, 10 mmol/L creatine phosphate, and 20 mmol/L Tris-maleate (pH 6.8). The Ca2+ CSS containing the indicated concentration of free Ca2+ was prepared by adding an appropriate amount of CaCl2 to the Ca2+-free CSS, according to the EGTA-Ca2+–binding constant of 106 (mol/L)–1.20 Fura-2-acetoxymethyl ester and EGTA were purchased from Dojindo Laboratories (Kumamoto, Japan). The PDGF-BB was purchased from Upstate Biotechnology (Lake Placid, NY). PDGF-BB was used in the present study because this isoform activates all 3 types of PDGF receptors.1 Endothelin-1 was purchased from the Peptide Institute (Osaka, Japan). Genistein was purchased from Calbiochem (San Diego, Calif). Staphylococcus aureus -toxin, a monoclonal anti-MLC antibody (clone MY-21), and a secondary antibody were purchased from Sigma (St. Louis, Mo).
Data Analysis
Data are expressed as mean±SEM. Student t test was used to determine statistical significance between the 2 groups, and an ANOVA with the Bonferroni/Dunn post hoc test was used to determine the effects of PDGF, endothelin-1, and Ca2+. Probability values <0.05 were considered statistically significant. All data were collected with a computerized data acquisition system (MacLab; Analog Digital Instruments, Australia; and Macintosh Apple Computer, Cupertino, Calif).
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Results |
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Enhancement of the Contractile Response to PDGF in SAH
We have demonstrated that the endothelium-dependent relaxation induced by acetylcholine and thrombin remained unchanged after SAH.14,21 The present study focused on the contractile responses of the smooth muscle of the basilar artery toward PDGF. The investigation was thus conducted in the absence of an endothelium. First, we evaluated the [Ca2+]i elevation and tension development induced by 118 mmol/L K+ in controls and SAH preparations (data not shown). The [Ca2+]i was calibrated by assigning the maximal and minimal levels of [Ca2+]i obtained with 50 µmol/L ionomycin in 1.25 mmol/L Ca2+-containing PSS and in 2 mmol/L EGTA-containing Ca2+-free PSS to be 100% and 0%, respectively. The level of [Ca2+]i at rest (19.0±2.6%, n=5) and that seen during the 118 mmol/L K+ depolarization (32.8±3.3%, n=5) in controls did not significantly (P>0.05) differ from those seen in SAH (19.2±1.9% and 33.2±3.6%, respectively, n=5). The extent of tension development induced by 118 mmol/L K+ in SAH (308±15 mg, n=6) did not significantly (P>0.05) differ from that seen in controls (353±25 mg, n=6). As a result, [Ca2+]i elevation and tension development induced by 118 mmol/L K+ were similar between controls and SAH preparations. Therefore, the response to 118 mmol/L K+ was used as a reference response (100% level) in the following evaluations, as described in the Methods section.
In the basilar artery obtained from control rabbits, PDGF induced no contraction up to 1 nmol/L (Figure 1a), whereas 3 nmol/L PDGF slightly contracted the control artery (Figure 1d). In SAH, 1 nmol/L PDGF induced a significant increase in [Ca2+]i and development of tension (Figures 1b and 1c). The tension gradually developed after stimulation with PDGF while reaching the maximum at 30 minutes (Figure 1b). The level of [Ca2+]i elevation and tension development induced by 1 nmol/L PDGF in SAH was 16.6±0.5% (n=4) and 20.4±3.3% (n=10), respectively, of those obtained with 118 mmol/L K+. Evaluation of the concentration-dependent response indicated significant enhancement of the contractile response to PDGF at 1 nmol/L and 3 nmol/L in comparison with that seen in controls (Figure 1d).
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Requirement of Extracellular Ca2+ and Tyrosine Kinase Activity for PDGF-Induced Contraction
To elucidate the mechanism of the contraction induced by PDGF, the effects of the removal of extracellular Ca2+ on PDGF-induced increases in [Ca2+]i and tension were examined. When the strips were exposed to Ca2+-free PSS containing 2 mmol/L EGTA, the resting level of [Ca2+]i decreased from 0 to –99.7±8.1% (n=3) within 5 minutes, with no significant change in resting tension. In a Ca2+-free PSS, 1 nmol/L PDGF did not induce any significant increase in [Ca2+]i and tension (Figure 2a). Because tyrosine phosphorylation plays a pivotal role in signal transduction after PDGF stimulation,5,22 the effect of the tyrosine kinase inhibitor genistein on PDGF-induced contraction was examined. When 10 µmol/L genistein was applied during the sustained phase of PDGF-induced contraction, [Ca2+]i and tension decreased from 16.0±0.5% and 15.7±1.3% (n=3), respectively, to resting levels (Figure 2b). However, genistein had no significant effect on the resting level of tension or the sustained level of 118 mmol/L K+–induced [Ca2+]i elevation and tension development (data not shown).
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Effect of PDGF on Ca2+ Sensitivity of the Contractile Apparatus in -Toxin–Permeabilized Preparations
To further elucidate the mechanism of PDGF-induced contraction, the effect of PDGF on Ca2+ sensitivity of the contractile apparatus was examined in -toxin–permeabilized arterial rings of SAH preparations. The arteries were precontracted with 300 nmol/L Ca2+, which produced 35.1±2.5% of the tension seen with 10 µmol/L Ca2+. The addition of 1 nmol/L PDGF during the 300 nmol/L Ca2+–induced contraction induced no contraction (Figures 3a and 3c), whereas 100 nmol/L endothelin-1 induced significant contraction (Figures 3b and 3c). Endothelin-1 also induced significant contraction during the 300 nmol/L Ca2+–induced contraction in the control basilar artery (Figure 3c). The extent of contraction seen with endothelin-1 in SAH did not significantly differ from that seen in controls (Figure 3c).
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Effect of PDGF on MLC Phosphorylation
In the basilar artery of SAH, 14.0±7.7% of MLC was phosphorylated in the resting state. On stimulation with 1 nmol/L PDGF, MLC phosphorylation increased and reached its peak at 15 minutes, and thereafter it decreased to the resting level within 30 minutes (Figure 4a). The level of MLC phosphorylation at 15 minutes was significantly (P<0.05) higher than the resting level (Figure 4b).
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Discussion |
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PDGF has been shown to elicit the intracellular Ca2+ signal,1,5,6 whereas its role in PDGF-induced cell proliferation still remains controversial.6,23 On the other hand, the intracellular Ca2+ signal serves as a primary signal to elicit smooth muscle contraction.7,24 However, the contractile effect of PDGF in the normal artery has only been reported in limited types of arteries, such as the rat aorta and rabbit ear artery.8,9 The present study therefore provides the first evidence that PDGF contracted the basilar artery of control rabbits, although the contractile effect of PDGF in the control artery was small and it was observed only at higher concentrations. These observations in the control artery are, therefore, consistent with those of a previous report that showed that intracisternal injection of PDGF contracted the normal rabbit basilar artery, as evaluated by angiography.11 Therefore, the most important and novel finding of the present study is that the contractile response to PDGF was significantly augmented in SAH.
The contractile response to 118 mmol/L K+ depolarization seen in SAH was similar to that seen in controls. The Ca2+-sensitizing effect of endothelin-1 observed in SAH was also similar to that seen in controls. These observations thus ruled out the possibility that general enhancement of the Ca2+-dependent contractile mechanism or Ca2+ sensitization of the contractile apparatus played a major role in enhancement of the contractile response to PDGF in SAH. Enhancement of the contractile response to PDGF was associated with enhancement of the [Ca2+]i elevation in SAH, thus suggesting that the mechanism is located upstream of the Ca2+ signal. Accordingly, it is most likely that the function of the PDGF receptor increased in the basilar artery of SAH. Such phenotypic conversion in terms of PDGF receptor function may contribute not only to the enhanced contractile response to PDGF, as observed in the present study, but also to the wall thickening seen in SAH.25
Although PDGF elicits the Ca2+ signal, the mechanism of PDGF-induced smooth muscle contraction still remains elusive. Tyrosine phosphorylation plays a pivotal role as an initial step of intracellular signal transduction after PDGF stimulation.5 Indeed, the tyrosine kinase inhibitor tyrphostin has been shown to inhibit PDGF-induced contraction in the rat aorta and MLC phosphorylation in human platelets.26 Consistent with these reports, the tyrosine kinase inhibitor genistein almost completely inhibited PDGF-induced contraction in the present study. Therefore, tyrosine phosphorylation is also suggested to play a critical role in PDGF-induced contraction of the rabbit basilar artery in SAH. PDGF has been reported to activate phospholipase C-
and inositol 1,4,5-triphosphate production, thereby eliciting Ca2+ release.27 In fact, the initial phase of the PDGF-induced [Ca2+]i elevation seen in cultured rat aortic VSMCs was mainly attributed to Ca2+ release.6,28 In the present study, the removal of extracellular Ca2+ almost completely abolished the PDGF-induced [Ca2+]i elevation and contraction, thus indicating that PDGF-induced contraction was mostly dependent on extracellular Ca2+. This result is consistent with the observation of the rabbit ear artery in a previous report, which also showed the dependence of the contractile effect of PDGF on extracellular Ca2+.9 Indeed, PDGF has also been reported to activate several mechanisms of Ca2+ influx, including voltage-operated Ca2+ channels or other Ca2+ channels.29–31
Ca2+-dependent MLC phosphorylation and the alteration in Ca2+ sensitivity of the contractile apparatus play a primary role in the regulation of contraction of VSM.7,32 The simultaneous measurement of [Ca2+]i and tension in the intact preparations did not suggest a Ca2+-sensitizing effect of PDGF in the basilar artery, because the relation between [Ca2+]i elevation and developed tension seen with 1 nmol/L PDGF was similar to that seen with 118 mmol/L K+ depolarization. The effect of contractile stimulation on myofilament Ca2+ sensitivity was also evaluated in the -toxin–permeabilized preparations. Endothelin-1 induced contraction at fixed concentrations of Ca2+ (300 nmol/L), thus indicating that endothelin-1 increased the Ca2+ sensitivity of the contractile apparatus. In contrast, PDGF failed to induce any further development of tension. These observations therefore suggest that an increase in Ca2+ sensitivity played a negligible role, if any, in the PDGF-induced contraction in the rabbit basilar artery of SAH. It should be also noted that the sustained phase of PDGF-induced contraction (30 minutes after stimulation) was associated with an increase in [Ca2+]i (Figure 2a) but not of MLC phosphorylation (Figure 4). These observations suggest the involvement of some contractile mechanism that is dependent on Ca2+ but independent of MLC phosphorylation during the sustained phase of PDGF-induced contraction.
The mean concentration of PDGF in cerebrospinal fluid under physiologic conditions has been reported to be 885.0±104.5 pg/mL (
0.06 nmol/L), whereas the level of PDGF was 1917.5±459.4 pg/mL (0.12 nmol/L), reaching more than twice that of controls.12 These levels of PDGF are close to the lower level of PDGF required to induce significant contraction in SAH in the present study. Therefore, the contractile effect of PDGF may be functionally relevant, and therefore it may contribute, at least in part, to the development of posthemorrhagic cerebral vasospasm.
In conclusion, the contractile response to PDGF was shown to be augmented in the basilar artery in SAH. The PDGF-induced contraction was therefore suggested to be mainly dependent on the [Ca2+]i elevation due to Ca2+ influx and the resultant increase in MLC phosphorylation, whereas the increase in myofilament Ca2+ sensitivity plays a negligible role in PDGF-induced contraction. PDGF in the cerebrospinal fluid has been shown to be elevated in SAH; the enhanced contractile response to PDGF observed in the SAH model may thus contribute to the development of posthemorrhagic vasospasm.
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Acknowledgments |
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We thank Brian Quinn for linguistic comments and help with the manuscript.
Sources of Funding
This study was supported in part by a grant from the 21st Century COE Program and Grants-in-Aids for Scientific Research (Nos. 17590744, 18209045, 18791022) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Yokoyama Rinshoyakuri Foundation.
Disclosures
None.
Received June 30, 2008; accepted July 22, 2008.
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