SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway.

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

Changes of the Expression and Activity of Phosphodiesterase V in the Basilar Artery Before and After Cerebral Vasospasm in a Rabbit Model.

OBJECTIVE: To examine changes of expression and activity of phosphodiesterase V (PDE V) in the basilar artery following cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) in a rabbit model. METHODS: A rabbit model of CVS after SAH was constructed by double blood injection into the cisterna magna. Subjects were divided into 3 groups: blank control group, normal saline group, and SAH group. Transcranial Doppler and selective vertebrobasilar digital subtraction angiography were performed to identify changes of CVS. Changes of PDE V expression and activity were examined. RESULTS: Mean basilar arterial blood flow rate measured by transcranial Doppler was significantly increased in the SAH group compared with the blank control group and normal saline group. Mean basilar artery diameter measured by digital subtraction angiography in the SAH group was narrower than in the other 2 groups. Compared with the other 2 groups, the expression of PDE V in the SAH group was significantly upregulated, and the activity was significantly enhanced. CONCLUSIONS: The rabbit model of SAH-induced CVS was successfully constructed through double blood injection method. Increased basilar artery blood flow, narrowing of the basilar artery, increased PDE V expression, and enhanced PDE V activity in the basilar artery were detected in the CVS rabbits, suggesting that PDE V has the potential to be used as a target for CVS therapy.

Involvement of Hydrogen Sulfide in Endothelium-Derived Relaxing Factor-Mediated Responses in Rat Cerebral Arteries.

BACKGROUND/AIM: H2S is a novel vasoactivator. To verify the hypothesis that H2S may act as an endothelium-derived hyperpolarizing factor (EDHF) in the rat cerebrovasculature, the role of H2S in endothelium-derived relaxing factor (EDRF)-mediated responses was investigated. METHODS: Cystathionine-γ-lyase (CSE) was knocked down with an siRNA technique. Artery diameter, hyperpolarization and Ca2+-activated K+ (KCa) current were measured. RESULTS: CSE knockdown was indicated by a decrease in protein and mRNA expression in the rat middle cerebral artery (MCA) and cerebral basilar artery (CBA). Acetylcholine (ACh) induced significant hyperpolarization and vasodilation in endothelium-intact MCA and CBA. Removal of the endothelium abolished these responses. The nitric oxide (NO) synthase inhibitor L-NAME, but not the PGI2 production inhibitor indomethacin, significantly inhibited ACh-induced hyperpolarization and vasodilation in the CBA. In the presence of L-NAME and indomethacin, ACh-induced hyperpolarization and vasodilation in the MCA and CBA were attenuated. The non-NO/PGI2-mediated responses were abolished by the KCa channel blockers charybdotoxin and apamin. In the cerebral arteries from the CSE knockdown rat, non-NO/PGI2-mediated responses were significantly attenuated, and the remaining responses were abolished by charybdotoxin and apamin or the CSE inhibitor propargylglycine. CSE knockdown did not affect L-NAME-sensitive responses in the CBA. Sodium hydrosulfide (NaHS) augmented the KCa current in CBA vascular smooth muscle cells. CONCLUSION: EDHF-mediated responses in rat cerebral arteries were due to H2S activating the KCa channel.

The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery.

Alteration of basilar artery rho-kinase and soluble guanylyl cyclase protein expression in a rat model of cerebral vasospasm following subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). Previous results showed that CGS 26303, an endothelin converting enzyme (ECE) inhibitor, effectively prevented and reversed arterial narrowing in animal models of SAH. In the present study, we assessed the effect of CGS 26303 on neurological deficits in SAH rats. The involvement of vasoactive pathways downstream of ET-1 signaling in SAH was also investigated. METHODS: Sprague-Dawley rats were divided into five groups (n = 6/group): (1) normal control, (2) SAH, (3) SAH+vehicle, (4) SAH+CGS 26303 (prevention), and (5) SAH+CGS 26303 (reversal). SAH was induced by injecting autologous blood into cisterna magna. CGS 26303 (10 mg/kg) was injected intravenously at 1 and 24 hr after the initiation of SAH in the prevention and reversal protocols, respectively. Behavioral changes were assessed at 48 hr after SAH. Protein expression was analyzed by Western blots. RESULTS: Deficits in motor function were obvious in the SAH rats, and CGS 26303 significantly improved the rate of paraplegia. Expressions of rho-kinase-II and membrane-bound protein kinase C- δ and rhoA were significantly increased, while those of soluble guanylyl cyclase α 1 and ß 1 as well as protein kinase G were significantly decreased in the basilar artery of SAH rats. Treatment with CGS 26303 nearly normalized these effects. CONCLUSIONS: These results demonstrate that the rhoA/rho-kinase and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. It also shows that ECE inhibition is an effective strategy for the treatment of this disease.

Morphological changes of cerebral vessels and expression patterns of MMP-2 and MMP-9 on cerebrovascular wall of alcoholic rats.

Alcohol abuse increases the incidence of cerebral accidents, which correlates with cerebrovascular structural changes. The present study was designed to observe the cerebrovascular remodeling of drinking rats with light microscopy and transmission electron microscopy (TEM). Short-term alcohol administration induced apparent amplification of perivascular spaces around small vessels in brain tissue, while long-term administration caused pathological changes of basilar arteries (BAs), including endothelial exfoliation, inner elastic lamina (IEL) fragmentation and thickening of tunica media and adventitia. In addition, the relationship between cerebrovascular remodeling and MMP-2 and MMP-9 synthesized by endothelial cells and vascular smooth muscle cells was explored by immunohistochemistry. The two protein expression in cerebral vessels changed dynamically, peaking at 1-2 weeks after treatment, and decreasing as treatment continued. These results suggest that MMP-2 and MMP-9 may play a significant role in blood-brain barrier disruption after alcohol abuse. But the chronic changes of cerebral arteries resulted from drinking are not coincident with time course of MMP-2 and MMP-9 expression in situ.

CaMKII and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture.

BACKGROUND: Cerebral ischemia induces transcriptional upregulation of inflammatory genes in the brain parenchyma and in cerebral arteries, thereby contributing to the infarct development. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CaMKII) II and extracellular signal-regulated kinase1/2 (ERK1/2) on inflammatory mediators in rat cerebral arteries using organ culture as a method for inducing ischemic-like vascular wall changes. METHODS: Rat basilar arteries were cultured in serum-free medium for 0, 3, 6 or 24 hours in the presence or absence of the CaMKII inhibitor KN93 or the MEK1/2 inhibitor U0126. Protein expression of activated CaMKII, ERK1/2, and inflammatory-associated protein kinases and mediators were examined with western blot and immunohistochemistry. Caspase-3 mRNA levels in basilar arteries were studied with real-time PCR. RESULTS: Western blot evaluation showed that organ culture induced a significant increase in phosphorylated ERK1/2 at 3, 6 and 24 hours, while CaMKII was found to be already activated in fresh non-incubated arteries and to decrease with incubation time. The addition of U0126 or KN93 decreased levels of phosphorylated c-Jun N-terminal kinase and p-p38, as evaluated by immunohistochemistry. KN93 affected the increase in caspase-3 mRNA expression only when given at the start of incubation, while U0126 had an inhibitory effect when given up to six hours later. Tumor necrosis factor receptor 1 was elevated after organ culture. This inflammatory marker was reduced by both of the two different protein kinase inhibitors. CONCLUSIONS: The novel findings of the present study are that the cross-talk between the two protein kinases and the inhibition of CaMKII or MEK1/2 in a time-dependent manner attenuates inflammatory-associated protein kinases and mediators, suggesting that they play a role in cerebrovascular inflammation.

Tonic arterial contraction mediated by L-type Ca2+ channels requires sustained Ca2+ influx, G protein-associated Ca2+ release, and RhoA/ROCK activation.

KCl-evoked sustained contraction requires L-type Ca(2+) channel activation, metabotropic Ca(2+) release from the sarcoplasmic reticulum (mechanism denoted calcium channel-induced Ca(2+) release) and RhoA/Rho associated kinase activation. Although high K(+) solutions are used to depolarize myocytes, these solutions can stimulate other signaling pathways such as those triggered by the activation of muscarinic and purinergic receptors. The present study examines the functional role of calcium channel-induced Ca(2+) release under pharmacological activation of L-type Ca(2+) channel without significant membrane depolarization. It also analyzes the role of the "steady-state" Ca(2+) influx through L-type Ca(2+) channels on myocyte sustained contraction. Measurement of contractility in arterial rings was done on a vessel myograph. Membrane potential was measured by fluorescence techniques loading intact myocytes with a membrane potential sensitive dye, and a reversible permeabilization method was used to load myocytes in intact arteries with GDPßS and Ca(v)1.2 siRNA. Application of an L-type Ca(2+) channel agonist, without effect on membrane potential, evoked sustained contraction via G-protein induced Ca(2+) release from the sarcoplasmic reticulum and RhoA/Rho associated kinase activation. Tonic myocyte contractions mediated by L-type Ca(2+) channel activation required sustained Ca(2+) influx through the channels and Ca(2+) uptake by the sarcoplasmic reticulum. Because L-type Ca(2+) channels participate in numerous pathophysiological processes mediated by maintained arterial contraction, our data could help to optimize therapeutic treatment of arterial vasospasm.

Potential contribution of matrix metalloproteinase-9 (mmp-9) to cerebral vasospasm after experimental subarachnoid hemorrhage in rats.

This study investigated the possible involvement of matrix metalloproteinase 9 (MMP-9) in cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) in rats. The CVS model was established by injection of fresh autologous nonheparinized arterial blood into the cisterna magna. Experiment 1 aimed to investigate the timecourse of the MMP-9 expression in the basilar artery after SAH. In Experiment 2, we chose the maximum time point of vasospasm (Day 3) and assessed the effect of SB-3CT (a selective MMP- 9 inhibitor) on the regulation of cerebral vasospasm. The cross-sectional area of basilar artery was measured by H&E staining and the MMP-9 expression was assessed by immunohistochemistry analysis. The elevated expression of MMP-9 was detected in the basilar artery after SAH and peaked on day 3. After intracisternal administration of SB-3CT, the vasospasm was markedly attenuated after blood injection on day 3. Our results suggest that MMP-9 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in this rat experimental model of SAH and that the administration of the specific MMP-9 inhibitor could prevent or reduce cerebral vasospasm caused by SAH.

Interactive role of protein phosphatase 2A and protein kinase Calpha in the stretch-induced triphosphorylation of myosin light chain in canine cerebral artery.

The interactive role of protein kinase C (PKC) isoforms and protein phosphatase 2A (PP2A) in the mechanisms underlying the gradual reduction in stretch-induced contraction through triphosphorylation of 20-kDa myosin light chain (MLC(20)) was investigated in the canine basilar artery. In the presence of 5 mM tetraethylammonium, stretching at a rate of 1 mm/s from the initial length (L(i)) to 1.5 L(i) produced a contraction. Maintaining the stretched state for 15 min (15-min stretch) produced triphosphorylation of MLC(20 )at Ser-19, Thr-18 and Thr-9, and a gradual reduction in the contraction, both of which were reversed by Gö6976 (1 microM), an inhibitor of conventional PKC. The 15-min stretch increased PKCalpha activity whereas it decreased PP2A activity, both of which were blocked by Y-27632, an inhibitor of rho kinase. Okadaic acid (OA; 1 microM), a PP2A inhibitor, also produced triphosphorylation of MLC(20) at the same amino acid residues and activated PKCalpha, which was inhibited by Gö6976. Stretching and OA increased phosphorylation of 17-kDa PKC-potentiated inhibitory phosphoprotein (CPI-17), and this phosphorylation was inhibited by Gö6976. The present results suggest that activation of PKCalpha mediated by an inhibitor of PP2A is involved in the stretch-induced triphosphorylation of MLC(20), and that this triphosphorylation counteracts the stretch-induced contraction.

The endothelium of basilar artery of diabetic rat treated with epoetin delta.

Erythropoiesis-stimulating agents (ESAs) are used to treat anemia associated with renal failure. It is now known that these agents also show a broad range of cell- and tissue-protective effects. In the current study, we explored whether an ESA, epoetin delta, affects vascular pathology linked to diabetes mellitus (DM). In a rat model of streptozotocin-induced DM, we investigated, by pre-embedding electron-immunocytochemistry, whether epoetin delta affects DM-induced structural changes in cerebrovascular endothelium of the rat basilar artery and influences the subcellular distribution of endothelial nitric oxide synthase (eNOS). Epoetin delta treatment influenced DM-induced changes to the distribution of eNOS in, and the structure of, the endothelial cell. This may indicate potential beneficial effects of epoetin delta on cerebrovascular endothelium and suggests eNOS as a possible target molecule of epoetin delta in DM.

Effect of smoking on rat basilar artery: correlation with inducible nitric oxide synthase and endothelin converting enzyme-1.

AIM: Smoking is an extremely important risk factor for subarachnoid hemorrhage and seems to increase rupture risk of unruptured aneurysms by accelerating their growth rate. The aim of the study was to investigate the effect of smoking on the luminal diameter with wall thicknesses of rat basilar arteries and to detect alterations of inducible nitric oxide synthase and endothelin-converting enzyme -1 in the endothelial cells. MATERIAL AND METHODS: Rats were divided into two groups. The level of middle pons slices were embedded in paraffin before they were stained with hematoxylin and eosin. Rabbit anti-human inducible nitric oxide synthase and endothelin converting enzyme-1 antibodies were used. RESULTS: Significant decrease of the vessel luminal diameter and increase of the vessel wall thickness were found in chronic smokers in our study. There was a linear and significant (p= 0,023, r =0,704) correlation between thickness of the wall and endothelin converting enzyme-1 immune reaction. Correlation was not found with inducible nitric oxide synthase (p > 0.05). CONCLUSION: This study on the comparison of vessel luminal diameter and vessel wall thickness with inducible nitric oxide synthase and endothelin converting enzyme-1 immune reactions revealed that the main effect of smoking on the vessel wall is associated with endothelin converting enzyme-1.

Multifaceted effects of selective inhibitor of phosphodiesterase III, cilostazol, for cerebral vasospasm after subarachnoid hemorrhage in a dog model.

BACKGROUND: Selective inhibition of phosphodiesterase type III (PDE III) may be involved in the pathophysiology of vasospasm and a PDE III inhibitor, cilostazol, is thus expected to attenuate vasospasm after subarachnoid hemorrhage (SAH). We tested the therapeutic effects of cilostazol on angiographic and morphological vasospasm. METHOD: Twenty-one mongrel dogs were divided into 4 groups: (1) control (n = 3); (2) SAH (n = 6); (3) SAH with low-dose treatment (n = 6), and (4) SAH with high-dose treatment (n = 6). We used the established double-hemorrhage model of SAH achieved by injecting autologous blood. Angiography was performed on day 0 and day 7. The animals were euthanized after a second angiogram, and Western blotting was performed to analyze phenotypic changes in smooth muscle cells of the basilar artery. The basilar artery was sectioned for immunohistochemistry of SM1, SM2 and SMemb to analyze phenotypic changes (SM1, SM2 for the contractile type of smooth muscle myosin heavy chain and SMemb for the synthetic type). Intact endothelial cells were counted under a microscope. RESULTS: Severe vasospasm was obtained in the SAH group (42 +/- 1%). Cilostazol attenuated angiographic vasospasm in both treatment groups (63 +/- 2 and 74 +/- 4%, respectively). Prevention of endothelial damage and phenotypic changes in smooth muscle cells were observed in both treatment groups (p < 0.05 vs. control, ANOVA). CONCLUSION: Cilostazol attenuates vasospasm following SAH in dogs by suppressing phenotypic changes in the basilar artery and preventing endothelial damage. Therefore, we anticipate that cilostazol may be useful in the management of vasospasm.

Role of ERK1/2 and vascular cell proliferation in cerebral vasospasm after experimental subarachnoid hemorrhage.

BACKGROUND: Although there are still some unresolved aspects, current research has revealed that vascular cell proliferation probably plays an important part in the pathological formation process of cerebral vasospasm. Using a "two-hemorrhage" model of subarachnoid hemorrhage (SAH), this study investigated the function of ERK1/2 and vascular wall cell proliferation in pathological development of cerebral vasospasm. METHODS: Fifty rabbits were randomly divided into five groups: (1) SAH day 1, (2) SAH day 3, (3) SAH day 7, (4) SAH + DMSO (dimethyl sufoxide) solution, (5) SAH + PD98059 (a mitogen-activated protein kinase inhibitor) dissolved in DMSO solution. In the SAH + PD98059/DMSO group and SAH + DMSO control group, PD98059 in DMSO (2 mmol/l) or an equal quantity of DMSO, respectively, was injected into the cisterna magna, once a day from SAH day 1 to day 3. Western protein blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in each group's basilar arteries. Light microscopy and electron microscopy were used for dynamic histological detection at each observation point of the SAH vascular wall under the effects of SAH and the mitogen-activated protein kinase inhibitor. Another 18 rabbits were randomly divided into three groups: SAH, SAH + DMSO and SAH + PD98059/DMSO; cerebral angiograpathy was conducted on SAH days 1 and 7, and the progression of angiographic vasospasm evaluated. RESULTS: Compared with the control group, the extent of vasospasm after SAH increased with time. PD98059 significantly reduced angiographic and morphological vasospasm. In cerebral vasospasm, the expression of T-ERK1/2 showed no significant change. However, expression of p-ERK1/2 and PCNA began to increase significantly on day 3, and achieved a peak on day 7. PD98059 significantly inhibited the expression of p-ERK1/2 and PCNA (p < 0.05). CONCLUSIONS: Cell proliferation on the vascular wall plays an important part in the pathological formation process of cerebral vasospasm. ERK1/2 phosphorylation, as an important signaling pathway, taking part in the process of vascular-wall pathological proliferation of cerebral vasospasm.

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells.

OBJECTIVES: Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. MATERIALS AND METHODS: Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. RESULTS: ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) induced by ET-1. CONCLUSION: Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.

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 micromol/l, SPC induced severe vasoconstriction (maximum vasoconstriction by SPC (100 micromol/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.

Acetylsalicylic acid provides cerebrovascular protection from oxidant damage in salt-loaded stroke-prone rats.

Inflammatory processes may play a pivotal role in the pathogenesis of cerebrovascular injury in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Recent reports revealed that acetylsalicylic acid (aspirin) has anti-oxidative properties and elicits nitric oxide release by a direct activation of the endothelial NO synthase. The present study was designed to determine whether low-dose aspirin might prevent cerebrovascular injury in salt-loaded SHRSP by protecting oxidative damage. Nine-week-old SHRSP were fed a 0.4% NaCl or a 4% NaCl diet with or without treatment by naproxen (20 mg/kg/day), salicylic acid (5 mg/kg/day), or aspirin (5 mg/kg/day) for 5 weeks. Blood pressure, blood brain barrier impairment, mortality, and the parameters of cerebrovascular inflammation and damage were compared among them. High salt intake in SHRSP significantly increased blood brain barrier impairment and early mortality, which were suppressed by treatment with aspirin independent of changes in blood pressure. Salt loading significantly increased superoxide production in basilar arteries of SHRSP, which were significantly suppressed by treatment with aspirin. Salt loading also significantly decreased NOS activity in the basilar arteries of SHRSP, which were significantly improved by treatment with aspirin. At 5 weeks after salt loading, macrophage accumulation and matrix metalloproteinase-9 activity at the stroke-negative area in cerebral cortex of SHRSP were significantly reduced by treatment with aspirin. These results suggest that low-dose aspirin may exert protective effects against cerebrovascular inflammation and damage by salt loading through down-regulation of superoxide production and induction of nitric oxide synthesis.

Activation of c-jun in the rat basilar artery after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) initiates a series of cellular and molecular events, some of which involve a mitogen activated protein kinase, c-jun N-terminal kinase (JNK). However, precise details regarding activation of c-jun in the vessel wall after SAH largely remain to be elucidated. In this study, we therefore investigated the localization and time-dependent expression of c-jun in the rat basilar artery after SAH in a rat single-hemorrhage model featuring infusion of autologous arterial blood. Basilar arteries were obtained at 2, 6 and 12h and 1, 2, 4 and 7 days after SAH, as well as from controls. Western blot analysis with c-jun, phosphorylated c-jun at Ser(63), and actin antibodies revealed that c-jun was immediately phosphorylated at Ser(63) within 2h, thereafter gradually becoming dephosphorylated, while total c-jun and actin levels remained almost unchanged. Immunohistochemistry demonstrated phosphorylation of c-jun at Ser(63) to occur in smooth muscle cells of the basilar artery 2h after SAH. These results indicate that c-jun is activated in the basilar artery immediately after the onset of SAH, presumably resulting in transcription of immediate early genes and smooth muscle cell proliferation.

NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke.

Reactive oxygen species play a role in neuronal damage following cerebral ischemia-reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebral arteries within, adjacent and distant from the ischemic core. The right middle cerebral artery (MCA) of conscious rats was temporarily occluded by perivascular injection of endothelin-1 to induce stroke (ET-1; n=19). Control rats were injected with saline (n=9). At 24 h or 72 h post-administration of ET-1, the MCA and its branches within the ipsilateral penumbra and infarcted core, corresponding arteries in the contralateral hemisphere, and basilar artery were excised. Anatomically similar arteries were excised from saline-injected rats. At 24 h after stroke, NADPH-stimulated superoxide production by arteries from the infarcted core did not differ from levels generated by arteries from control rats, whereas levels were significantly lower 72 h after stroke. However, at both time points after stroke, superoxide production by arteries from the ischemic penumbra was 8-fold greater than levels generated by arteries from control rats. Surprisingly, even in the non-ischemic arteries from the contralateral hemisphere and in the basilar artery, superoxide production was increased approximately 4- to 6-fold at 24 h, but had returned to normal 72 h after stroke. The NADPH-oxidase inhibitor, diphenyleneiodonium, virtually abolished superoxide production by all arteries. Thus, the activity of NADPH-oxidase is enhanced in cerebral arteries from the ischemic penumbra at 24 h and 72 h following cerebral ischemia. Additionally, NADPH-oxidase activity is temporarily enhanced after cerebral ischemia within arteries from non-ischemic parts of the brain.

Separate development of nitric oxide synthase- and vasoactive intestinal polypeptide-immunoreactive nerves arising from the vertebral artery in the rat.

Development of nitric oxide synthase (NOS)-and vasoactive intestinal polypeptide (VIP)-immunoreactive (-IR) nerves supplying the basilar and vertebral arteries (BA and VA) was investigated in White Wistar rats, using double immunohistochemistry. NOS-IR and VIP-IR nerves via the anterior circulation (AC), which mostly expressed NO(+)/VIP(+), extended to the BA during the second postnatal week, and usually reached as far as the rostral two third of the BA on PND 20. NOS-IR nerves were completely lack in the cBA and the VA on PND10, and often absent from these arterial regions even at PND 20. Nevertheless, a small number of VIP(+)/NOS(-) nerves were localized in the walls from the caudal BA (cBA) to the VA on PND 5. On PND 20, they frequently met with the descending NOS-IR and VIP-IR nerves via the AC around the lower portion of the middle BA. Fiber bundles containing NOS(+)/VIP(+) axons were first visualized on the caudal VA at PND 30 and observed frequently at PND 80, with a distinct increase in number of NOS-IR and VIP-IR nerves supplying the cBA and the VA. Thus, NOS-IR nerves coming from the VA develop through its own characteristic sequence that lags markedly behind the time of appearance for VIP-IR nerves from the same vascular route and for NOS-IR and VIP-IR nerves via the AC.