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.

Cerebral Artery Remodeling in Rodent Models of Subarachnoid Hemorrhage.

Vasospasm is known to contribute to delayed cerebral ischemia following subarachnoid hemorrhage (SAH). We hypothesized that vasospasm initiates structural changes within the vessel wall, possibly aggravating ischemia and leading to resistance to vasodilator treatment. We therefore investigated the effect of blood on cerebral arteries with respect to contractile activation and vascular remodeling. In vitro experiments on rodent basilar and middle cerebral arteries showed a gradual contraction in response to overnight exposure to blood. After incubation with blood, a clear inward remodeling was found, reducing the caliber of the passive vessel. The transglutaminase inhibitor L682.777 fully prevented this remodeling. Translation of the in vitro findings to an in vivo SAH model was attempted in rats, using both a single prechiasmatic blood injection model and a double cisterna magna injection model, and in mice, using a single prechiasmatic blood injection. However, we found no substantial changes in active or passive biomechanical properties in vivo. We conclude that extravascular blood can induce matrix remodeling in cerebral arteries, which reduces vascular caliber. This remodeling depends on transglutaminase activity. However, the current rodent SAH models do not permit in vivo confirmation of this mechanism.

Memantine Attenuates Delayed Vasospasm after Experimental Subarachnoid Hemorrhage via Modulating Endothelial Nitric Oxide Synthase.

Delayed cerebral vasospasm is an important pathological feature of subarachnoid hemorrhage (SAH). The cause of vasospasm is multifactorial. Impairs nitric oxide availability and endothelial nitric oxide synthase (eNOS) dysfunction has been reported to underlie vasospasm. Memantine, a low-affinity uncompetitive N-methyl-d-aspartate (NMDA) blocker has been proven to reduce early brain injury after SAH. This study investigated the effect of memantine on attenuation of vasospasm and restoring eNOS functionality. Male Sprague-Dawley rats weighing 350-450 g were randomly divided into three weight-matched groups, sham surgery, SAH + vehicle, and SAH + memantine groups. The effects of memantine on SAH were evaluated by assessing the severity of vasospasm and the expression of eNOS. Memantine effectively ameliorated cerebral vasospasm by restoring eNOS functionality. Memantine can prevent vasospasm in experimental SAH. Treatment strategies may help combat SAH-induced vasospasm in the future.

Possible Role of Raf-1 Kinase in the Development of Cerebral Vasospasm and Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

This study aims to clarify the potential role of Raf-1 kinase in cerebral vasospasm (CVS) and early brain injury (EBI) after subarachnoid hemorrhage (SAH). Two experimental SAH models in rats, including cisterna magna double injection model for CVS study and prechiasmatic cistern single injection model for EBI study, were performed in this research. As a specific inhibitor of Raf-1, BAY 43-9006 was used in this study. In CVS study, time course study showed that the basilar artery exhibited vasospasm after SAH and became most severe at day 5, and the phosphorylation of Raf-1 had the same trends, while both vasospasm and the phosphorylation of Raf-1 induced by SAH were inhibited by BAY 43-9006 treatment. In addition, BAY 43-9006 treatment significantly reversed the phosphorylation of ERK1/2 and the activation of NF-κB induced by SAH and decreased the messenger RNA (mRNA) levels of IL-6 and IL-1ß. In EBI study, BAY 43-9006 treatment significantly suppressed the brain injury induced by SAH. Besides, BAY 43-9006 inhibited the phosphorylation of Raf-1 and ERK1/2; decreased the protein levels of COX-2, VEGF, and MMP-9; and reversed the activation of NF-κB induced by SAH. These results demonstrate that Raf-1 kinase contributes to CVS and EBI after SAH by enhancing the activation of the Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathways, and that the inhibition of these pathways might offer new treatment strategies for CVS and EBI.

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.

Genetic elimination of eNOS reduces secondary complications of experimental subarachnoid hemorrhage.

Delayed complications of subarachnoid hemorrhage (SAH) such as angiographic vasospasm, cortical spreading ischemia, microcirculatory dysfunction, and microthrombosis are reported in both patients and animal models of SAH. We demonstrated previously that SAH is associated with increased oxidative stress in the brain parenchyma, and that this correlates with dysfunction of endothelial nitric oxide synthase (eNOS) (homodimeric uncoupling). Uncoupling of eNOS exacerbated oxidative stress and enhanced nitric oxide (NO) depletion, and was associated with multiple secondary complications such as microthrombosis, neuronal apoptosis, and release of reactive oxygen species. Thus, we hypothesized that genetic abbrogation of eNOS would confer a beneficial effect on the brain after SAH. Using a prechiasmatic injection model of SAH, we show here that eNOS knockout (KO) significantly alleviates vasospasm of the middle cerebral artery and reduces superoxide production. Endothelial nitric oxide synthase KO also affected other nitric oxide synthase isoforms. It significantly increases neuron nitric oxide synthase expression but has no effect on inducible nitric oxide synthase. Endothelial nitric oxide synthase KO decreases Zn(2+) release after SAH, reduces microthrombi formation, and prevent neuronal degeneration. This work is consistent with our findings where, after SAH, increased oxidative stress can uncouple eNOS via Zn(2+) thiolate oxidation, or theoretically by depletion or oxidation of tetrahydrobiopterin, resulting in a paradoxical release of superoxide anion radical, further exacerbating oxidative stress and microvascular damage.

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.

Inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway attenuates cerebral vasospasm following experimental subarachnoid hemorrhage in rabbits.

The p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptosis and is also involved in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Here, we sought to examine whether inhibition of p38 MAPK could attenuate cerebral vasospasm and investigate the underlying mechanisms in a rabbit SAH model. SAH was established in rabbits (n=12/group) using the double-hemorrhage method. We observed apparent vasospasm in the basilar arteries of rabbits with SAH, which was significantly attenuated by SB203580, a selective p38MAPK inhibitor. Immunoblotting assays showed enhanced phosphorylation of p38 MAPK and ATF2 and increased caspase-3 cleavage following SAH, which were, however, markedly suppressed by SB203580. TUNEL staining further revealed significant apoptosis in the basilar arteries of rabbits with SAH, which was scantly present in rabbits treated with SB203580. Our results demonstrated that p38 MAPK was activated in cerebral vasospasm and associated with increased apoptosis in the basilar arteries and p38 MAPK inhibition suppressed apoptosis, suggesting that p38 MAPK could be a novel therapeutic target for cerebral vasospasm.

Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage.

BACKGROUND: Late cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH. RESULTS: Here we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ETB, 5-HT1B and AT1 receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score. CONCLUSION: These results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy.

Endothelial nitric oxide synthase mediates endogenous protection against subarachnoid hemorrhage-induced cerebral vasospasm.

BACKGROUND AND PURPOSE: Vasospasm-induced delayed cerebral ischemia remains a major source of morbidity in patients with aneurysmal subarachnoid hemorrhage (SAH). We hypothesized that activating innate neurovascular protective mechanisms by preconditioning (PC) may represent a novel therapeutic approach against SAH-induced vasospasm and neurological deficits and, secondarily, that the neurovascular protection it provides is mediated by endothelial nitric oxide synthase (eNOS). METHODS: Wild-type mice were subjected to hypoxic PC or normoxia followed 24 hours later by SAH. Neurological function was analyzed daily; vasospasm was assessed on post-surgery Day 2. Nitric oxide availability, eNOS expression, and eNOS activity were also assessed. In a separate experiment, wild-type and eNOS-null mice were subjected to hypoxic PC or normoxia followed by SAH and assessed for vasospasm and neurological deficits. RESULTS: PC nearly completely prevented SAH-induced vasospasm and neurological deficits. It also prevented SAH-induced reduction in nitric oxide availability and increased eNOS activity in mice with and without SAH. PC-induced protection against vasospasm and neurological deficits was lost in wild-type mice treated with the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester and in eNOS-null mice. CONCLUSIONS: Endogenous protective mechanisms against vasospasm exist, are powerful, and can be induced by PC. eNOS-derived nitric oxide is a critical mediator of PC-induced neurovascular protection. These data provide strong "proof-of-principle" evidence that PC represents a promising new strategy to reduce vasospasm and delayed cerebral ischemia after SAH.

New regulatory, signaling pathways, and sources of nitric oxide.

Discovered in 1980 by the late Robert F. Furchgott, endothelium-derived relaxing factor, nitric oxide (NO), has been in the forefront of vascular research for several decades. What was originally a narrow approach, has been significantly widened due to major advances in understanding the chemical and biological properties of NO as well as its signaling pathways and discovering new sources of this notorious free radical gas. In this review, recent discoveries regarding NO and their implications on therapy for delayed cerebral vasospasm are presented.

Nitric oxide synthase inhibitors and cerebral vasospasm.

L-arginine is a source of nitric oxide (NO) that is cleaved from the terminal guanidino nitrogen atom by nitric oxide synthase (NOS). NO evokes, because of its free radical properties and affinity to heme, ferrous iron and cysteine, a wide spectrum of physiological and pathophysiological effects. For many years, different exogenous NOS inhibitors were used to elucidate the role of NOS and NO in health and disease. Later, endogenous NOS inhibitors, as asymmetric dimethylarginine (ADMA) were discovered. Endogenous inhibitors as ADMA are produced by post-translational methylation of L-arginine which is catalyzed by a family of protein N-methyltransferases (PRMT), using S-adenosylmethionine as a methyl group donor. ADMA is eliminated by dimethylarginine dimethylaminohydrolases (DDAH I or II). ADMA hydrolysis increases NOS activity and NO production. Furthermore, L-citrulline, a by-product of ADMA hydrolysis as well as of NO production by NOS, can in turn inhibit DDAH. Therefore, endogenous inhibition of NOS can be modified via different ways (1) changing the availability of L-arginine and/or of L-citrulline; (2) stimulating or inhibiting DDAH activity; (3) modifying methylation via regulating availability of adenosylmethionine; or (4) modifying PRMT activity. Research elucidating the role of NOS inhibitors in respect of delayed cerebral vasospasm after subarachnoid hemorrhage is summarized.

Mitogen-activated protein kinases in cerebral vasospasm after subarachnoid hemorrhage: a review.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) have been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. The goal of this review is to bring together recent diverse data concerning the roles of MAPKs in cerebral vasospasm and to consider the future research. METHOD: A review of publications in the National Library of Medicine and National Institutes of Health database was conducted in August 2009 using specific keyword search terms pertaining to subarachnoid hemorrhage and MAPKs. FINDINGS: There are nine in vitro studies and 17 in vivo studies published. Most of previous studies used MAPK inhibitors or their upstream molecule inhibitors, and showed that MAPK inhibitions prevented vasospasm. The MAPK cascade appears to interact with other signaling molecules, and MAPK may be an important final common pathway for the signaling transduction during cerebral vasospasm. However, the mechanism by which MAPK causes sustained vascular smooth muscle contraction remains unclear. In addition, the role of endogenous MAPK inhibitors, MAPK phosphatases, has not been investigated in cerebral vasospasm. CONCLUSIONS: The experimental data support the causative role of MAPK in cerebral vasospasm and warrant further research.

Mechanisms of statin treatment in cerebral vasospasm.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, commonly known as statins, are widely used clinically for their lipid lowering properties. Recent experimental evidence shows that statins are also effective in ameliorating cerebral vasospasm, which occurs as sequelae of subarachnoid hemorrhage. This literature review focuses on the literature-based putative mechanisms involved in statin mediated attenuation of cerebral vasospasm, such as eNOS, vascular inflammation, apoptosis, especially the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway from our experimental study.

Effect of a free radical scavenger, edaravone, on free radical reactions: related signal transduction and cerebral vasospasm in the rabbit subarachnoid hemorrhage model.

OBJECTIVE: it is hypothesized that free radical reactions evoked by oxyhemoglobin (oxyHb) cause cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH), even though the detailed mechanisms have not yet been fully established. The aims of this study were thus to investigate, through the use of the double-hemorrhage rabbit model, the possibility that free radical reactions play a role in cerebral vasospasm and to delineate the mechanism of signal transduction that causes cerebral vasospasm. METHODS: in the SAH group, SAH was simulated using the double-hemorrhage rabbit model. In the treatment group, edaravone (0.6 mg/kg), a potent free radical scavenger, was injected into the central ear vein twice a day. Four days after SAH, the basilar artery was excised. The degree of cerebral vasospasm was evaluated by measuring the diameter of each basilar artery, and the expression of Rho-kinase in the vascular wall was examined by western blotting. RESULTS: the diameter of the basilar artery in the edaravone-treated group was 0.64 ± 0.06 mm, which was statistically significantly larger than that in the nontreated SAH group (0.50 ± 0.03 mm; p < 0.01). The expression of Rho-kinase in the edaravone-treated group was statistically significantly reduced in comparison to that of the nontreated SAH group. CONCLUSION: results from this study have indicated for the first time that free radical reactions mediated by oxyHb may play an important role in the pathogenesis of cerebral vasospasm through the expression of Rho-kinase.

Uncoupling of endothelial nitric oxide synthase after experimental subarachnoid hemorrhage.

We studied whether endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled in large cerebral arteries after subarachnoid hemorrhage (SAH) and also whether this causes cerebral vasospasm in a mouse model of anterior circulation SAH. Control animals underwent injection of saline instead of blood (n=16 SAH and n=16 controls). There was significant vasospasm of the middle cerebral artery 2 days after SAH (lumen radius/wall thickness ratio 4.3 ± 1.3 for SAH, 23.2 ± 2.1 for saline, P<0.001). Subarachnoid hemorrhage was associated with terminal deoxynucleotidyl transferase dUTP nick-end labeling, cleaved caspase-3, and Fluoro-Jade-positive neurons in the cortex and with CA1 and dentate regions in the hippocampus. There were multiple fibrinogen-positive microthromboemboli in the cortex and hippocampus after SAH. Transgenic mice expressing lacZ under control of the eNOS promoter had increased X-gal staining in large arteries after SAH, and this was confirmed by the increased eNOS protein on western blotting. Evidence that eNOS was uncoupled was found in that nitric oxide availability was decreased, and superoxide and peroxynitrite concentrations were increased in the brains of mice with SAH. This study suggests that artery constriction by SAH upregulates eNOS but that it is uncoupled and produces peroxynitrite that may generate microemboli that travel distally and contribute to brain injury.

Potential role of Ras in cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits.

Previous studies have demonstrated that mitogen-activated protein kinase (MAPK) is involved in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). Ras, an upstream regulator of MAPK, may be activated following SAH. The aim of this study was to investigate the role of Ras in cerebral vasospasm in a rabbit model of SAH. We first investigated the time course of Ras and ERK1/2 activation in the basilar artery after SAH. Next, for the time point at which Ras was maximally activated, we assessed the effect of FTI-277 (a Ras farnesyltransferase inhibitor) on cerebral vasospasm. SAH was induced by injecting autologous blood into the cisterna magna on both day 0 and day 2. FTI-277 was injected into the cisterna magna every 24 hours, beginning 30 minutes after blood injection to the last day of the experiment. Elevated expression of Ras-GTP and phosphorylated ERK1/2 was detected in the basilar artery after SAH and expression peaked on day 3. FTI-277 administration resulted in lower Ras-GTP and phosphorylated ERK1/2 levels and markedly attenuated vasospasm in the basilar arteries relative to animals that did not receive FTI-277. Our results suggest that Ras protein is activated in the arterial wall after SAH and contributes to vasospasm development.

Effect of statin treatment on vasospasm, delayed cerebral ischemia, and functional outcome in patients with aneurysmal subarachnoid hemorrhage: a systematic review and meta-analysis update.

BACKGROUND AND PURPOSE: A recent meta-analysis investigating the efficacy of statin treatment in patients with aneurysmal subarachnoid hemorrhage reported a reduced incidence of vasospasm, delayed cerebral ischemia, and mortality in statin-treated patients. However, the meta-analysis was criticized for its methodology, and several retrospective studies found no beneficial effect. We present the results of a new systematic review, which differs from the previous systematic review in its methodology, and by inclusion of the results of a fourth randomized, placebo-controlled trial. Summary of Review- All randomized, placebo-controlled trials investigating the effect of statins on vasospasm, delayed cerebral ischemia, and functional outcome in patients with aneurysmal subarachnoid hemorrhage were included. Outcomes were the number of patients with transcranial Doppler vasospasm, delayed cerebral ischemia, poor outcome, and mortality during follow-up. Effect sizes were expressed in (pooled) risk ratio estimates. Data were pooled using random-effects models. RESULTS: In 4 studies, a total of 190 patients were included. No statistically significant effect was observed on transcranial Doppler vasospasm (pooled risk ratio, 0.99 [95% CI, 0.66 to 1.48]), delayed cerebral ischemia (pooled risk ratio, 0.57 [95% CI, 0.29 to 1.13]), poor outcome (pooled risk ratio, 0.92 [95% CI, 0.68 to 1.24]), or mortality (pooled risk ratio, 0.37 [95% CI, 0.13 to 1.10]). CONCLUSIONS: The results of the present systematic review do not lend statistically significant support to the finding of a beneficial effect of statins in patients with aneurysmal subarachnoid hemorrhage as reported in a previous meta-analysis.

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.

Nitric oxide synthase gene therapy: progress and prospects.

NOS gene therapy has been the focus of extensive research as dysfunction of this enzyme has been implicated in several cardiovascular diseases. Research has concentrated on comparing the effect of gene delivery of NOS isoforms (eNOS, iNOS and nNOS) in healthy and diseased animal models on intimal hyperplasia, restenosis, vascular tone and ischemia-reperfusion injury. Most results demonstrate therapeutic benefits following vascular gene delivery of all NOS in pre-clinical models of cardiovascular disease. eNOS has been shown to have particular promise as it promotes re-endothelialisation and inhibits intimal hyperplasia in injured blood vessels. The ultimate goal is to translate the benefit of NOS gene therapy in animal models into clinical practise. To develop NOS gene therapy for clinical use further work needs to be undertaken to improve delivery systems and vectors to minimise detrimental side-effects and enhance positive treatment outcomes. This review focuses on current research on NOS gene therapy in cardiovascular disease and identifies the next steps that would be necessary to lead to clinical trials.