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.

Adenovirus-mediated transfer of dominant-negative rho-kinase induces a regression of coronary arteriosclerosis in pigs in vivo.

Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1beta, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of beta-galactosidase, into the interleukin-1beta-treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRHOK: These results indicate that Rho-kinase is substantially involved in coronary constrictive remodeling and vasospastic responses, both of which can be reversed by the selective inhibition of the molecule in our porcine model in vivo.

Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in pigs in vivo.

We have previously shown that long-term treatment with an inflammatory cytokine from the adventitia causes the development of coronary vascular lesions, with the accumulation of macrophages. Recent studies in vitro have suggested that small G-protein Rho and its effector, Rho-kinase/ROK/ROCK, may be the key molecules for various cellular functions, including cell adhesion and movement. In this study, we examined whether adventitia-derived macrophages cause the formation of coronary vascular lesions in vivo and, if so, whether Rho-kinase is involved in the process. Porcine coronary segments from the adventitia were chronically treated with monocyte chemoattractant protein-1 alone, oxidized low density lipoprotein alone, or both. Vascular lesion formation (neointimal formation and development of vascular remodeling) was mostly enhanced at the coronary segment cotreated with monocyte chemoattractant protein-1 and oxidized low density lipoprotein, where the phosphorylation of myosin binding subunit of myosin phosphatase was increased, indicating an increased activity of Rho-kinase in vivo. Histological examination demonstrated that macrophages were accumulated at the adventitia and thereafter migrated into the vascular wall. Long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor (hydroxyfasudil) after oral absorption, markedly inhibited the myosin binding subunit phosphorylation, the macrophage accumulation and migration, and the coronary lesion formation in vivo. These results indicate that Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in our porcine model in vivo.

Different vasculoprotective roles of NO synthase isoforms in vascular lesion formation in mice.

NO is known to have several important vasculoprotective actions. Although NO is synthesized by 3 different NO synthase (NOS) isoforms, the vasculoprotective action of individual NOS isoforms remains to be clarified. Permanent ligation of the left common carotid artery was performed in control, endothelial NOS (eNOS) knockout (eNOS-KO), and inducible NOS (iNOS) knockout (iNOS-KO) mice. Four weeks after the procedure, neointimal formation and reduction of cross-sectional vascular area (constrictive remodeling) were noted in the left carotid artery. In the eNOS-KO mice, the extent of neointimal formation was significantly larger than in the control or iNOS-KO mice, whereas the extent of vascular remodeling was the highest in the iNOS-KO mice compared with other 2 strains. Antiplatelet therapy with aspirin or antihypertensive treatment with bunazosin failed to inhibit the accelerated neointimal formation in the eNOS-KO mice. These results indicate that eNOS and iNOS have different vasculoprotective actions against the vascular lesion formation caused by blood flow disruption in vivo: NO derived from eNOS inhibits neointimal formation, whereas NO derived from iNOS suppresses the development of constrictive remodeling.

Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs.

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or beta-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.

Local adenovirus-mediated transfer of C-type natriuretic peptide suppresses vascular remodeling in porcine coronary arteries in vivo.

OBJECTIVE: This study was designed to examine whether or not adenovirus-mediated gene transfer of C-type natriuretic peptide (CNP) can prevent coronary restenotic changes after balloon injury in pigs in vivo. BACKGROUND: Gene therapy to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) might be useful but requires a method applicable for in vivo gene delivery into the coronary artery as well as the efficient vector encoding a potent antiproliferative substance. We tested whether the adenovirus-mediated gene transfer of CNP by use of an infiltrator angioplasty balloon catheter (IABC) might prevent the coronary restenotic changes after balloon injury. METHODS: Balloon angioplasty was performed in the left anterior descending and the left circumflex coronary artery in pigs. Immediately after the balloon injury, adenovirus solution encoding either CNP (AdCACNP) or beta-galactosidase (AdCALacZ) gene was injected with IABC into the balloon-injured coronary segments. Expression of CNP was assessed by immunohistochemical staining and cyclic guanosine 3',5'-monophosphate (cGMP) measurement. Coronary restenotic changes were evaluated by both angiographic and histological examinations. RESULTS: CNP was highly expressed in the media and the adventitia of the coronary artery at the AdCACNP-transfected but not at the AdCALacZ-transfected segment. In the AdCALacZ-transfected segment, vascular cGMP levels tended to be reduced as compared with the untreated segment, whereas in the AdCACNP-transfected segment, vascular cGMP levels were restored. Angiographic coronary stenosis was significantly less at the AdCACNP-transfected than at the AdCALacZ-transfected segment. Histological examination revealed that this was achieved primarily by the marked inhibition of the geometric remodeling of the coronary artery by the CNP gene transfer. CONCLUSIONS: Adenovirus-mediated CNP gene transfer with the IABC system may be a useful gene therapy to prevent restenosis after PTCA in vivo.

Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta.

BACKGROUND: We recently demonstrated that the Rho-kinase-mediated pathway plays an important role for coronary artery spasm in our porcine model with interleukin-1beta (IL-1beta). In this study, we examined whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction. METHODS AND RESULTS: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta-bound microbeads. Two weeks after the operation, as reported previously, intracoronary serotonin repeatedly induced coronary hypercontractions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by Y-27632, one of the specific inhibitors of Rho-kinase. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of Rho-kinase mRNA was significantly increased in the spastic compared with the control segment. Western blot analysis showed that during the serotonin-induced contractions, the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase (MBS), one of the major substrates of Rho-kinase, was significantly greater in the spastic than in the control segment and that the increase in MBS phosphorylations was also markedly inhibited by Y-27632. There was a highly significant correlation between the extent of MBS phosphorylations and that of contractions. CONCLUSIONS: These results indicate that Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin phosphatase through the phosphorylation of MBS in our porcine model.

Sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, inhibits serotonin-induced coronary artery spasm in a porcine model.

Serotonin is one of the most important vasoactive substances and has been implicated in the pathogenesis of coronary artery spasm and of acute coronary syndrome. We have recently demonstrated that local and long-term treatment with interleukin-1beta(IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses to serotonin in pigs in vivo. However, it remains to be examined which serotonergic (5-HT) receptor subtype mediates coronary spasm and whether alterations in serotonergic receptors are involved in the abnormality. In this study, we examined the inhibitory effect of sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, on the serotonin-induced coronary spasm as well as the possible alterations of serotonergic receptors in our porcine model. A segment of the porcine coronary artery was carefully dissected and aseptically wrapped with cotton mesh absorbing IL-1beta-bound microbeads from the adventitia. Two weeks after the procedure, angiographic study was performed, followed by binding assay for 5-HT1B and 5-HT2A serotonergic receptors and reverse transcription-polymerase chain reaction (RT-PCR) analysis for mRNA of those receptors. Angiographic study showed coronary vasospastic responses to serotonin at the IL-1beta-treated site. Sarpogrelate dose-dependently inhibited the serotonin-induced coronary spasm, but it did not affect the prostaglandin F2alpha-induced vasoconstriction. Radiolabeled receptor-binding assay showed that receptor affinity or receptor number of the 5-HT1B, or 5-HT2A receptors did not differ significantly between the spastic and the control sites. Furthermore, RT-PCR analysis showed that the expression of neither 5-HT2A nor 5-HT1B receptor mRNA was significantly altered at the spastic site. These results indicate that serotonin-induced coronary spasm is mediated primarily by 5-HT2A receptor in our porcine model, although the 5-HT2A receptor was not up-regulated, suggesting that alteration in the signal-transduction pathway for vascular smooth muscle contraction beyond the 5-HT2A receptor plays a primary role in the pathogenesis of coronary spasm in our porcine model.

Changes in left ventricular end-diastolic area, end-systolic wall stress, and fractional area change during anesthetic induction with propofol or thiamylal.

PURPOSE: To elucidate the mechanisms of the more profound hypotensive effects of propofol relative to thiamylal, we monitored changes in left ventricular (LV) preload, afterload, and contractility during the course of anesthetic induction with propofol and thiamylal. METHODS: Thirty-two patients (ASA I) were randomly assigned into two groups and injected with propofol (2 mg.kg(-1)) or thiamylal (4 mg.kg(-1)) as anesthetic induction agents. Transthoracic echocardiography (TTE) was used to assess LV performance before and during induction by the two anesthetics. The LV end-diastolic area (EDA) and LV end-systolic wall stress (ESWS) were used as indices of LV preload and LV afterload, respectively, while LV contractility was assessed by the fractional area change (FAC). RESULTS: Both propofol and thiamylal significantly reduced EDA and ESWS without significant change in FAC. Propofol-induced reductions in EDA and ESWS were significantly greater than those of thiamylal. CONCLUSION: The more profound hypotension observed during induction of anesthesia with propofol is due to the greater decrease in preload and afterload than with thiamylal, but not to a decrease in LV contractility.

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.

Massive retroperitoneal hemorrhage associated with femoral vein cannulation.

The right external iliac artery was inadvertently punctured during attempted right femoral vein catheterization. Severe hypotension developed several minutes after the arterial puncture. Concurrently, a large right lower abdominal quadrant swelling (approximately 5 cm in diameter) became apparent. Laparoscopic observation of the retroperitoneal region immediately revealed massive retroperitoneal hemorrhage. Abdominal computerized tomographic scan indicated development of a huge (approximately 10 cm in the maximum diameter) retroperitoneal hematoma along the right psoas muscle from the level of the right external iliac vessels up to the level of the upper pole of the right kidney. Consideration of the anatomy of the "right" femoral and retroperitoneal vessels (ie, mediolateral relationship between the vein and artery) led us to conclude that the site for insertion of the needle was too proximal and the angle for advancement of the needle too low in our patient, allowing the needle to reach and injure the "incompressible" external iliac artery, thereby causing massive retroperitoneal hemorrhage. Although femoral vein catheterization has generally been considered a relatively safe method of intravenous access, a life-threatening serious complication can occur with the inappropriate technique used in our case.

Hemidiaphragmatic paralysis following subclavian vein catheterization.

The right subclavian artery was inadvertently punctured during attempted preoperative insertion of a right subclavian venous catheter in a 59-yr-old woman undergoing radical hysterectomy. Large supraclavicular swelling became apparent soon after the arterial puncture. The postoperative chest X-ray obtained approximately 24 h after the catheterization revealed significant elevation of the right hemidiaphragm, which was further augmented on the 2nd to 4th postoperative days; oxygenation was concurrently impaired during these days. It was clinically judged that the hemidiaphragmatic paralysis was responsible for the elevated diaphragm. Both chest roentogenogram and arterial blood gas analyses started to improve on the 5th day, finally returning to normal on the 6th day. It is unlikely that the surgical procedure caused the paralysis, because it dealt only with the lower abdomen. Rather, the attempts at the subclavian venous catheterization probably caused the phrenic nerve paralysis, because the phrenic nerve travels very close to the subclavian vessels. Both the large haematoma formation following the arterial puncture and the time course of the paralysis suggest that compression of the right phrenic nerve by the haematoma, rather than needle trauma, was responsible for the paralysis.