Rho激酶在小型猪白介素-1β介导的冠状动脉痉挛中的作用机制

目的探讨Rho激酶途径在冠状动脉痉挛发病中的作用机制。方法小型雄性家猪随机分为对照组(n=8)和模型组(n=8),冠状动脉外膜分别包裹吸附含生理盐水和白介素(IL)-1β琼脂糖微粒悬液的纸巾。2周后,采用冠状动脉造影观察管腔狭窄程度及5-羟色胺诱发冠状动脉痉挛情况。测定冠状动脉包裹血管段Rho激酶mRNA表达、肌球蛋白结合亚基磷酸化表达及光镜病理学改变。结果冠状动脉外膜包裹IL-1β血管段发生不同程度的管腔狭窄,5-羟色胺可诱发病变血管段痉挛。模型组与对照组相比,Rho激酶的mRNA表达病变血管段明显上调[(98·20±7·66)%对(63·70±4·26)%,P<0·05];肌球蛋白轻链磷酸酶的肌球蛋白结合亚基磷酸化表达升高(25485±4745对6510±779,P<0·05)。光镜可见病变血管段内膜增殖及炎症细胞聚集现象。结论Rho激酶参与IL-1β介导的冠状动脉痉挛的发生,其可能是通过增加肌球蛋白结合亚基磷酸化水平,抑制肌球蛋白轻链磷酸酶活性及加强钙增敏途径所致。     

Cellular and molecular mechanisms of coronary artery spasm: lessons from animal models

Coronary artery spasm plays an important role in the pathogenesis of a wide variety of ischemic heart diseases, especially in the Japanese population. Because coronary artery spasm can be induced by a variety of stimuli with different mechanisms of action, the occurrence of the spasm appears to be due to the local hyperreactivity of the coronary artery rather than to an enhanced stimulation with a single mechanism of action. Several lines of evidence indicate that coronary artery spasm is caused primarily by smooth muscle hypercontraction whereas the contribution of endothelial dysfunction may be minimal. In order to elucidate the cellular and molecular mechanisms of the spasm, porcine models of the spasm were developed. In the first model with balloon injury and high-cholesterol feeding, a close topological correlation between the early atherosclerotic lesions and the spastic sites was noted, whereas in the second model with an inflammatory cytokine the potential importance of coronary inflammatory changes, especially at the adventitia, was noted. Subsequent studies in vivo and in vitro demonstrated that protein kinase C (PKC) and Rho-kinase are substantially involved in the intracellular mechanism of the spasm, resulting in increases in the mono- and diphosphorylations of myosin light chain (MLC). Furthermore, molecular biological analyses demonstrated that Rho-kinase is upregulated at the spastic site (at all levels, including mRNA, protein, and activity), resulting in the inhibition of MLC phosphatase through the phosphorylation of its myosin binding subunit and thereby causing the increase in MLC phosphorylations. Preliminary results also suggest that the long-term inhibition of Rho-kinase is effective in inhibiting the development of arteriosclerotic vascular lesions in several porcine models. Thus, Rho-kinase could be regarded as a novel therapeutic target for coronary arteriosclerosis in general and coronary artery spasm in particular.     

Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm

Subarachnoid hemorrhage (SAH) often induces a long-term narrowing of the cerebral artery called cerebral vasospasm. Myosin light chain (MLC) in the spastic basilar artery was reported previously to be phosphorylated by Ca(2+)/calmodulin-dependent MLC kinase. Because Rho-kinase, which is activated by the small GTPase Rho, phosphorylates not only MLC but also myosin phosphatase at its myosin-binding subunit (MBS), thus inactivating myosin phosphatase, we examined whether Rho-kinase is involved in the development of vasospasm. Cerebral vasospasm was produced in the canine basilar artery by a 2-hemorrhage method, and vasocontractions were induced by topical application of 80 mmol/L KCl or 0.5 micromol/L serotonin to the canine basilar artery exposed transclivally. The phosphorylation of MLC in the basilar artery was increased concurrently with an enhancement in the intensity of vasospasm with the passage of time after SAH. In addition, Rho-kinase in the basilar artery was activated concurrently with an increase in the phosphorylation of MBS at Ser854 in vasospasm. The Rho-kinase activation levels in vasospasm on days 0 and 2 were comparable to those in KCl- and serotonin-induced sustained vasocontraction, respectively, and those in vasospasm on day 7 were markedly high. The topical application of Y-27632, a specific inhibitor of Rho-kinase, to the exposed spastic basilar artery on day 7 induced a dose-dependent dilation, and the intensities of vasospasm and the phosphorylation of MBS and MLC were simultaneously decreased by 10 micromol/L Y-27632, although the decrease in MBS phosphorylation was more marked than the decrease in MLC phosphorylation. These results indicate that the activation of Rho-kinase and the phosphorylation of MLC and MBS occur concomitantly during vasospasm induced by SAH and suggest that Rho-kinase is involved in the enhancement of cerebral vasospasm in addition to Ca(2+)/calmodulin-dependent MLC kinase by increasing the phosphorylation of MLC directly or indirectly as a result of the inhibition of myosin phosphatase by its phosphorylation.     

Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo

OBJECTIVE: We recently demonstrated that Rho-kinase/ROK/ROCK is functionally upregulated at the arteriosclerotic coronary lesions and plays a key role for coronary vasospastic responses in our porcine model with interleukin (IL)-1beta. In the present study, we tested our hypothesis that Rho-kinase is involved in the pathogenesis of coronary arteriosclerosis per se in our porcine model. METHODS: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1beta-treated site, as previously reported. Then, animals were randomly divided into two groups; one group was treated with fasudil for 8 weeks followed by 1 or 4 weeks of washout period and another group served as a control. After oral absorption, fasudil is metabolized to hydroxyfasudil that is a specific inhibitor of Rho-kinase. RESULTS: In the fasudil group, coronary stenosis and vasospastic response were progressively reduced in vivo, while the coronary hyperreactivity was abolished both in vivo and in vitro. Furthermore, Western blot analysis showed that in the fasudil group, the Rho-kinase activity (as evaluated by the extent of phosphorylation of myosin binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase) was significantly reduced, while histological examination demonstrated a marked regression of the coronary constrictive remodeling. CONCLUSIONS: These results indicate that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by long-term inhibition of the molecule in vivo. Thus, Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular disease.     

Low Dose Alcohol Contracts the Human Umbilical Artery in Vitro

Beginning at very low concentrations (0.001 g/100 ml), alcohol elicited dose-dependent contractions of the human umbilical artery in vitro. Additionally, 16 of the 108 arteries tested had a 5-10-min spasm in response to alcohol. Alcohol (0.2 g/100 ml) also increased tension developed in response to all angiotensin II doses, but had no effect on serotonin-induced contractions. These results suggest that alcohol may increase umbilicoplacental resistance in vivo, thus decreasing fetal-placental blood flow.     

Agonist-induced regulation of myosin phosphatase activity in human platelets through activation of Rho-kinase

Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti-myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPgammaS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPgammaS-RhoA-dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase-mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.     

Recent insights into the mechanisms, predisposing factors, and racial differences of coronary vasospasm

Coronary vasospasm is currently considered to be an exaggerated contractile nonspecific response of the vascular smooth muscle in the large coronary artery to various agonists or stimulation, that is established after the process of inflammation and fibrocellular proliferation. Endothelial dysfunction with reduced nitric oxide bioavailability has been reported in angiographically normal coronary arteries in Japanese patients with coronary spastic angina. Recently, several interesting findings concerning the exact mechanism of calcium hypersensitivity of spastic vascular smooth muscle have been reported. In animal models with coro-nary spasm Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin light chain phosphatase, resulting in enhancement of its phosphorylation. Also, oxidative stress has been given attention as an important mediator of the spastic conversion of vascular smooth muscle cell phenotype. The incidence of coronary spastic angina in the Japanese population is reported to be remarkably high compared with that in Caucasians. Clinical and pathophysiological differences between Japanese and Caucasian patients with respect to coronary vasospasm are characterized by a lower prevalence of fixed coronary artery stenoses and diffuse coronary hyperreactivity in the Japanese patients. Recently, several distinct characteristics have been recognized to be associated with coronary vasospasm in studies analyzing data obtained from Japanese patients. In the present review, we will discuss our point of view on the mechanisms and predisposing factors in coronary vasospasm. Predisposing factors include smoking, lipid metabolic disorders, and gene expression, all of which may be interrelated issues.     

Coronary artery spasm and the polymorphisms of the endothelial nitric oxide synthase gene.

BACKGROUND: Coronary artery spasm plays an important role in the pathogenesis of vasospastic angina, and contributes to the development of several acute coronary syndromes. Endothelial nitric oxide synthase (ecNOS) catalyzes the synthesis of nitric oxide, which regulates vascular tone, and may be related to coronary vasospasm. The present study investigated whether coronary spasm is related to particular polymorphisms of the ecNOS gene. METHODS AND RESULTS: Spasm provocation by serial infusions of acetylcholine was performed on 165 patients who were clinically suspected of having angina. In both study patients and healthy controls (n=400), genomic polymorphisms of the ecNOS gene were determined by using polymerase chain reaction. Quantitative luminal diameter measurements of the 3 major coronary arteries were initially obtained before and after acetylcholine injection, and then after isosorbide dinitrate injection, by using a computer-assisted analysis system. Logistic multiple regression analysis identified the a/a or a/b genotype in intron 4 of ecNOS (NOS4a: p=0.0431, odds ratio (OR) 2.43) and diabetes mellitus (p=0.0060, OR 4.88) as significant predictors of coronary spasm. In the patients with NOS4a, both the induced and spontaneous contractions were augmented. CONCLUSION: The present study results indicated that NOS4a could be a good marker for coronary artery spasm.     

Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells

Recent studies have demonstrated that upregulated Rho-kinase plays an important role in the pathogenesis of arteriosclerosis and vasospasm in both animals and humans. However, little is known about the molecular mechanism(s) involved in the Rho-kinase upregulation. Since inflammatory mechanisms have been implicated in the pathogenesis of arteriosclerosis and vasospasm, we examined whether inflammatory stimuli upregulate Rho-kinase in vitro and in vivo. In cultured human coronary vascular smooth muscle cells (hcVSMC), inflammatory stimuli, such as angiotensin II and interleukin-1beta, increased Rho-kinase expression (at both mRNA and protein levels) and function (as evaluated by the extent of the phosphorylation of the ERM (the ezrin/radixin/moesin) family, substrates of Rho-kinase) in a time- and concentration-dependent manner. The expression of Rho-kinase was inhibited by blockades of protein kinase C (PKC) (by either GF109253 or prolonged treatment with phorbol myristate acetate for 24 h) and an adenovirus-mediated gene transfer of dominant-active Ikappa-B, suggesting an involvement of PKC and NF-kappaB in the intracellular signal transduction pathway for the Rho-kinase expression. Furthermore, coronary vascular lesion formation (characterized by medial thickening and perivascular fibrosis) induced by a long-term administration of angiotensin II was markedly suppressed in NF-kappaB(-/-) mice with reduced expression and activity of Rho-kinase in vivo. These results indicate that the expression and function of Rho-kinase are upregulated by inflammatory stimuli (e.g. angiotensin II and IL-1beta) in hcVSMC with an involvement of PKC and NF-kappaB both in vitro and in vivo.     

Rho-kinase inhibition in the therapy of cardiovascular disease

Rho is a GTPase known to be a major mediator in the formation of stress fibers and focal adhesions, cell morphology, and smooth muscle contraction. Its role in smooth muscle contraction has led to exploration into the connection between Rho-mediated kinase activity and cardiovascular disease. The role of Rho-kinase in calcium sensitization for vascular smooth muscle contraction has recently been characterized. Inappropriate coronary artery vasoconstriction resulting from increased Rho-kinase in the vascular system is likely involved in the pathogenesis of exercise-induced myocardial ischemia, spontaneous coronary artery spasm, and hypertension. In clinical trials, Rho-kinase inhibitors such as fasudil and Y-27632 have demonstrated antiischemic, antivasospastic, and antihypertensive effects. These compounds have also exhibited the ability to blunt progression of cardiomyocyte hypertrophy and cardiac remodeling in heart failure. As such, Rho-kinase inhibition represents a potential novel therapeutic approach in cardiovascular disease.     

Rho激酶与动脉血管再狭窄的研究进展

Rho激酶是具有信息传导和分子开关功能的信号多肽,与动脉粥样硬化、高血压、冠脉痉挛、心肌缺血等主要心血管病的发生和发展关系密切,并通过多种途径参与血管损伤后内膜增殖和再狭窄的调控过程。现简要综述再狭窄发病过程中Rho激酶的作用机制及Rho激酶抑制剂预防再狭窄的实验结果。     

Rupture of coronary vasa vasorum as a trigger of acute myocardial infarction

Some controversy has always existed regarding the presence and extent of the vasa vasorum--the nutrient vessels in the wall of the human aorta--in the coronary arteries. Now, cinemicrographic studies using silicone polymer injections in cleared human hearts have identified the vasa vasorum of coronary arteries, revealing evidence of neovascularization in the region of atherosclerotic plaques. These studies suggest an important role for the vasa vasorum in the pathogenesis of coronary atherosclerosis and its sequelae, especially intramural hemorrhage and vascular spasm. The wall of the human coronary artery in regions of atherosclerotic injury may be particularly rich in capillary vessels of the vasa vasorum. From this, the evidence suggests that with the morning increase in blood pressure, fragile neovascular structures of the vasa vasorum may be more prone to rupture and may be responsible, in part, for the circadian variation in myocardial infarction.     

Lokalisation und Ausdehnung der Arteriosklerose

BACKGROUND: Arteriosclerosis is generally considered to be a generalized disease, involving different vascular regions to approximately the same extent. PATIENTS AND METHODS: In order to assess the extent of ateriosclerosis quantitatively, all arteriosclerotic lesions and additionally the complicated (calcified, ulcerated, thrombosed) arteriosclerotic lesions of the abdominal aorta, the superficial femoral arteries, the left descending coronary artery and the internal carotid arteries were measured by planimetry in 102 autopsies (64 male, 38 female; mean age: 62 +/- 16 years). RESULTS: Arteriosclerosis increased with age. The overall extent of arteriosclerotic lesions of each artery was classified to one of the following 4 degrees of intimal involvement (0-25, 26-50, 51-75%, 76-100%). When comparing the 4 different vascular regions of each patient we found maximal deviations by 1 degree in 34%, by 2 degrees in 41% and by 3 degrees in 20%. In only 5% of the patients studied were all arteriosclerotic lesions of the same degree. In order to analyze the complicated plaques 7 degrees were used (intimal involvement: 0, 1-10, 11-20, 21-30, 31-40, 41-50% and > 50%). In 14 patients we found no complicated plaques. The remaining 88 showed complicated lesions in at least 1 vascular region. Deviations by 1 degree were found in 22%, by 2 degrees in 32%, and by 3 or more degrees in 46%. In the paired arteries (carotid, femoral) there were no significant differences comparing the mean values of the extent of arteriosclerotic lesions on the right and left side. In many cases, however, remarkable intraindividual differences occurred. Patients with main risk factors usually had more pronounced arteriosclerotic lesions than those without. In patients who died from coronary artery disease the extent of arteriosclerotic lesions was also usually larger in the other vascular regions as compared to those patients who died from other reasons. Comparing postmortem and intravital extent of arteriosclerotic lesions some peculiarities of postmortem specimens should be considered. CONCLUSIONS: A uniform involvement of all vascular regions by arteriosclerosis occurs only rarely. Considerable intraindividual differences are the rule and a severe, generalized uniform affection is an exception. This, however, does not exclude in patients with severe arteriosclerosis of one region that a larger involvement of other vascular regions may also occur, as this was found for patients with severe arteriosclerosis in coronary arteries. Regarding the extent of arteriosclerosis there are artery- and additional localization-specific features.     

The role of vascular failure in coronary artery spasm

Coronary artery spasm plays an important role in the pathogenesis of angina pectoris as well as acute coronary syndrome and sudden death. The prevalence of coronary spasm is greater in East Asian populations than in other parts of the world. Although the mechanism of coronary spasm is still unclear, both endothelial and smooth muscle dysfunction have been reported to play a role. We recently proposed a new concept termed 'vascular failure' that represents an integration of endothelial and smooth muscle abnormalities. Thus, vascular failure is the primary cause of coronary artery spasm.     

Cholesterol primes vascular smooth muscle to induce Ca2 sensitization mediated by a sphingosylphosphorylcholine-Rho-kinase pathway: possible role for membrane raft

Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca(2+) sensitization. Here we report the strong linkage between cholesterol and the Ca(2+) sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum. However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by beta-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca(2+) sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca(2+) sensitization of VSM mediated by a SPC/Src-TK/Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia.     

Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm

OBJECTIVE: We recently demonstrated in our swine model of coronary artery spasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono- and diphosphorylations) play a central role in the pathogenesis of the spasm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we addressed these points using hydroxyfasudil, a novel inhibitor of protein kinases, which we found preferentially inhibits Rho-kinase. METHODS: The specificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MRCK beta and PKC were examined by kinase assay in vitro. The left porcine coronary artery was chronically treated with interleukin-1 beta (IL-1 beta, 2.5 micrograms). Two weeks after the operation, coronary artery vasomotion was examined both in vivo and in vitro. MLC phosphorylations were examined by Western blot analysis and the sites for the phosphorylations by anti-phosphorylated MLC antibodies that identified the monophosphorylation site as Ser19 and diphophorylation sites as Ser19/Thr18 of MLC. RESULTS: Inhibitory effects of hydroxyfasudil was at least 100 times more potent for Rho-kinase as compared with other protein kinases tested. Intracoronary serotonin (10 micrograms/kg) caused coronary hyperconstriction at the IL-1 beta-treated site in vivo, which was dose-dependently inhibited by hydroxyfasudil (p < 0.01). The coronary segment taken from the spastic site also showed hypercontractions to serotonin in vitro, which were again dose-dependently inhibited by hydroxyfasudil (p < 0.01). Western blot analysis showed that MLC monophosphorylation was significantly greater in the spastic segment than in the control segment, while MLC diphosphorylation was noted only at the spastic segment (p < 0.01). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC, respectively. Both types of MLC phosphorylations at the spastic segment were markedly inhibited by hydroxyfasudil (p < 0.01). CONCLUSION: These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm.     

Role of calcium antagonists in cerebral arterial spasm

Spasm of the large cerebral arteries at the base of the brain causes delayed ischemic neurologic deficits in approximately 30% of patients after a subarachnoid hemorrhage from an intracranial aneurysm. In vitro chamber studies have shown that both dog and human large cerebral artery segments contract to a variety of vasoactive agents, and the dog and human segments are remarkedly similar in their responses. The source of calcium necessary to initiate contraction was found to be extracellular for large cerebral arteries. In contrast, systemic arteries such as the femoral artery use a bound intracellular pool of calcium for contraction. The calcium antagonists nifedipine and nimodipine were found to selectively inhibit the contractions of large cerebral arteries but not the femoral artery. In vivo experiments demonstrated that both nifedipine and nimodipine, given sublingually, would prevent and reverse cerebral arterial spasm in the dog after a subarachnoid hemorrhage. Nimodipine was found to be more potent, both in the chamber and in the live dog experiments. Nimodipine significantly decreased the occurrence of severe neurologic deficits from spasm alone in a multi-institutional, prospective, double-blind, randomized, placebo-controlled trial.     

Sphingosylphosphorylcholine is a novel messenger for Rho-kinase-mediated Ca2+ sensitization in the bovine cerebral artery: unimportant role for protein kinase C

Although recent investigations have suggested that a Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase-mediated Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC50=3.0 micromol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester-induced Ca2+ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase-mediated Ca2+ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.     

Adiponectin is a novel humoral vasodilator

OBJECTIVES: Perivascular adipose tissue secretes an adipocyte-derived relaxing factor(s) (ADRF) that opens K(v) channels in rat arteries. Visceral fat accumulation causes adipocyte dysfunction, including hyposecretion of adiponectin. We tested the hypothesis that ADRF might be adiponectin and that adiponectin plays a role in the paracrine control of vascular tone by perivascular adipose tissue. METHODS AND RESULTS: We studied Sprague-Dawley rats, wild-type and adiponectin gene-deficient (Apn 1-/-) mice, and New Zealand obese (NZO) mice. In rat aortas, recombinant adiponectin at serum levels (2-5 microg/ml) inhibited serotonin-induced contractions. The effects were abolished by K(v) channel inhibition with 4-aminopyridine (4-AP, 2 mM). Similar effects were observed in NZO mouse mesenteric arteries. To study vascular function in Apn 1-/- mice, the mesenteric vascular bed was isolated, cannulated, and perfused at a constant 4-5-ml/min flow in the absence and presence of serotonin. 4-AP (2 mM) induced a similar increase in perfusion pressure in the Apn 1-/- perfused isolated mesenteric vascular bed, compared to wild-type mice. Removal of perivascular fat increased the vasoconstrictor responses, but abolished the 4-AP effects. The anti-contractile effects of perivascular fat were similar in mesenteric artery and aortic rings from Apn 1-/- and wild-type mice. Despite high adiponectin levels, the anti-contractile effects of perivascular fat were diminished in mesenteric arteries of NZO mice with age. CONCLUSION: Adiponectin is a novel humoral vasodilator that relaxes aortic and mesenteric rings by opening K(v) channels. Similar to the rat, perivascular adipose tissue of the mouse harbors an ADRF, which is malfunctional in NZO mice and is not adiponectin.     

Coronary Artery Spasm Induced by Stent Implantation: Studies in a Swine Model

Mechanical stimulation is a known cause of arterial spasm. Since stent implantation in the coronary arteries imparts mechanical trauma, we investigated, in an animal model that simulates human coronary anatomy and physiology, the frequency of occurrence and factors that influence the development of spasm. In 28 Hanford miniature swine, 9 of which were atherosclerotic and 19 of which were nonatherosclerotic, spasm occurred in 15 (54%). Stent-induced spasm directly caused the death of one animal and was implicated in the death of two others. Factors that significantly contributed to coronary spasm were the stent: artery ratio (more spasm with oversized stents) and the size of the artery (more spasm with smaller arteries). There was a trend toward more spasm in atherosclerotic arteries. Spasm occurred despite the use of antispasmodic agents before, during, and after the scenting procedure. Our data suggest that coronary artery spasm may be an important clinical accompaniment to stent implantation in man and may be minimized by the judicious selection of stent size to avoid overdistention of the artery and the avoidance of stent placement in smaller arteries . (J Interven Cardiol 1993; 6:149–155)