Satoshi Shirao

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 &mgr;mol/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.

Involvement of Src Family Protein Tyrosine Kinases in Ca2+ Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway

Abstract— We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca2+ sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca2+]i levels and the Ca2+-dependent contraction induced by high K+ depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase–dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca2+ sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.

Evaluation of Cerebral Perfusion parameters measured by Perfusion CT in chronic Cerebral ischemia: Comparison with xenon CT

Purpose The purpose of this work was to evaluate the usefulness of perfusion CT in the evaluation of patients with chronic cerebral ischemia by comparing it with xenon CT (Xe-CT). Method Cerebral blood flow (CBF) of perfusion CT (CBFper) and time to peak (TTP) were compared with the CBF of Xe-CT (CBFxe) in 18 patients. Cerebral blood volume (CBV) was compared with cerebral vascular reserve (CVR) in 10 of 18 patients who underwent pre- and postacetazolamide Xe-CT. Results CBFper and TTP demonstrated a high correlation with CBFxe in relative values by side-to-side comparisons (r = 0.743, p < 0.0001 and r = −0.760, p < 0.0001, respectively). There was a negative correlation between relative CBV and relative CVR (r = −0.637, p = 0.0025). Visually, territories with delayed TTP corresponded well to those of decreased CBFxe, but these territories tended to be larger in TTP maps. Conclusion Perfusion CT is a useful tool to evaluate chronic hemodynamic disturbance and can be an alternative method for those using acetazolamide challenge.

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 Ca2+ 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 Ca2+ sensitization. Here we report the strong linkage between cholesterol and the Ca2+ 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 &bgr;-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca2+ 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 Ca2+ 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.

Preoperative Prediction of Outcome in 283 Poor-Grade Patients with Subarachnoid Hemorrhage: A Project of the Chugoku-Shikoku Division of the Japan Neurosurgical Society

Background: The management of patients with poor-grade subarachnoid hemorrhage (SAH) continues to be controversial. The objective of this study was to examine predictors of outcome of poor-grade SAH after surgical obliteration of the aneurysm. Methods: The study was performed as a retrospective review of 283 patients with poor-grade SAH who underwent surgical obliteration of the aneurysm at multiple centers in Chugoku and Shikoku, Japan. Results: A favorable outcome at discharge was achieved in 97 of the 283 patients (34.3%). Age (p < 0.001), World Federation of Neurosurgical Societies (WFNS) grade V at admission (p = 0.002), improvement in WFNS grade after admission (p = 0.002), Fisher grade (p = 0.039) and a low-density area (LDA) associated with vasospasm on computed tomography (CT; p < 0.001) showed a significant association with outcome. Further analysis of WFNS grades indicated that most patients who only improved to preoperative grade IV from grade V at admission did not have a favorable outcome. Multivariate analysis identified age (especially of ≧65 years; p < 0.001), WFNS grade V (p < 0.001) and LDA associated with vasospasm on CT (p < 0.001) as predictors of a poor outcome, and improvement in WFNS grade (p = 0.001) as a predictor of a favorable outcome after surgical obliteration of the aneurysm. Conclusions: Advanced age, WFNS grade V, improvement in WFNS grade, and LDA associated with vasospasm on CT were found to be independent predictors of clinical outcome, whereas rebleeding, early aneurysm surgery and treatment modality (surgical clipping or Guglielmi detachable coil embolization) were not independently associated with outcome in patients with poor-grade aneurysm.

Cooling of the epileptic focus suppresses seizures with minimal influence on neurologic functions.

Purpose:  Focal brain cooling is effective for suppression of epileptic seizures, but it is unclear if seizures can be suppressed without a substantial influence on normal neurologic function. To address the issue, a thermoelectrically driven cooling system was developed and applied in free‐moving rat models of focal seizure and epilepsy.

Inhibitory Effects of Eicosapentaenoic Acid on Chronic Cerebral Vasospasm after Subarachnoid Hemorrhage: Possible Involvement of a Sphingosylphosphorylcholine-Rho-Kinase Pathway

Background and Purpose: Rho-kinase (ROK)-mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). We previously demonstrated that sphingosylphosphorylcholine (SPC) induces Ca2+ sensitization through sequential activation of the Src family protein tyrosine kinases (Src-PTKs) and ROK in vitro, and that Ca2+ sensitization is inhibited by eicosapentaenoic acid (EPA) through the selective inactivation of Src-PTK. In this study, we examined whether SPC induced CV in vivo, and, if it did, whether EPA would inhibit CV, as induced by SPC or in an in vivo model of subarachnoid hemorrhage (SAH). Methods: Changes in the diameter of the canine basilar artery were investigated by angiography after administering SPC into the cisterna magna. Then, Y27632, a specific Rho-kinase inhibitor, or EPA was injected intracisternally and the effects of both agents were investigated. In another experiment using a single-hemorrhage model, Y27632 or EPA was injected on day 7 after SAH and the changes in the diameter of the canine basilar artery were investigated. Results: At cerebrospinal fluid concentrations of 100 and 300 µmol/l, SPC induced severe vasoconstriction (maximum vasoconstriction by SPC (100 µmol/l): 61.8 ± 8.2%), which was markedly reversed by Y27632 (96.3 ± 4.4%) or EPA (92.6 ± 12.8%). SAH caused severe vasospasm on day 7 (67.6 ± 7.8%), which was significantly blocked by Y27632 (95.5 ± 10.6%) or EPA (90.0 ± 4.4%). Conclusions: SPC is a novel mediator of ROK-induced CV in vivo. The inhibition of CV induced by SPC or after SAH by EPA suggests beneficial roles of EPA in the treatment of CV. Our findings are compatible with the notion that the SPC-ROK pathway may be involved in CV.

Multicenter Prospective Cohort Study on Volume Management After Subarachnoid Hemorrhage Hemodynamic Changes According to Severity of Subarachnoid Hemorrhage and Cerebral Vasospasm

Background and Purpose— Systemic circulation management has not been established for patients with poor grade aneurysmal subarachnoid hemorrhage (SAH) or delayed cerebral ischemia (DCI) after SAH. The aims of the study were to examine hemodynamic variables in these patients and to establish treatment strategies. Methods— A multicenter prospective cohort study of hemodynamic variables from days 1 to 14 was performed using a transpulmonary thermodilution system (PiCCO Plus). Parameters were analyzed by Mann–Whitney test. Multivariate analysis was performed to identify parameters involved in onset of DCI. Results— The subjects were 204 patients, including 138 with poor grade SAH (World Federation of Neurological Surgeons grades IV and V) and 52 who developed DCI. The extravascular lung water index, pulmonary vascular permeability index, and systemic vascular resistance index were significantly greater in patients with poor grade SAH compared with those with good grade SAH (World Federation of Neurological Surgeons I–III) on day 2 (P=0.049, P=0.039, and P=0.038). Cardiac index was significantly lower in patients with poor grade SAH on days 1 and 2 (P=0.027 and P=0.011). In patients with DCI, the global end-diastolic volume index was significantly lower than in those without DCI on days 3 to 5 (P=0.0053; P=0.048; and P=0.048). In multivariate analysis, median global end-diastolic volume index, cardiac index, and systemic vascular resistance index at an early stage of SAH (days 3–6) were independently related to onset of DCI (P=0.023, P=0.013, and P=0.003). Conclusions— Patients with poor grade SAH developed heart failure–like afterload mismatch at an early stage, and those with DCI had decreased global end-diastolic volume index (hypovolemia) in the early stage of SAH. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: UMIN000003794.

Does eicosapentaenoic acid (EPA) inhibit cerebral vasospasm in patients after aneurysmal subarachnoid hemorrhage

Background –  Cerebral vasospasm following subarachnoid hemorrhage (SAH) is a significant cause of morbidity and mortality and recent studies indicate that Rho‐kinase plays an important role in the occurrence of such cerebral vasospasm. Eicosapentaenoic acid (EPA), an n‐3 polyunsaturated fatty acid, inhibits sphingosylphosphorylcholine (SPC)‐induced Rho‐kinase activation in vitro, so this study examined whether EPA prevented cerebral vasospasm occurrence after SAH in patients.

Elevated concentrations of sphingosylphosphorylcholine in cerebrospinal fluid after subarachnoid hemorrhage: A possible role as a spasmogen

This study investigates the role of sphingosylphosphorylcholine (SPC) in the mechanisms underlying cerebral vasospasm after subarachnoid hemorrhage (SAH). The levels of SPC were measured in cerebrospinal fluid (CSF) of patients with SAH and also in an experimental canine model. CSF samples were collected from 11 patients with SAH, and from dogs that had received an injection of SPC into the cisterna magna to examine SPC kinetics in the CSF. SPC was assayed using solid-phase extraction and triple quadrupole mass spectrometry. The SPC concentrations in SAH patients on days 3, 8, and 14 after the onset of SAH were significantly higher than those in normal CSF. In the canine model, rapid dilution of SPC in CSF was observed. In combination with data from previous studies, these results suggest that SPC is involved in the development of cerebral vasospasm. Rapid dilution of SPC in CSF suggests that SPC is released into CSF at higher concentrations than those measured in the present study.