Background and Purpose-Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo. Methods-Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 mol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured. Results-In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9-and 1.6-fold increase, respectively, at 10 mol/L (PϽ0.05 for both). This correlated with a 1.5-and 2.3-fold increase in eNOS activity and NO production, respectively (PϽ0.05 for both). Fasudil increased cerebral blood flow to both ischemic and nonischemic brain areas, reduced cerebral infarct size by 33%, and improved neurologic deficit score by 37% (PϽ0.05). This correlated with inhibition of brain and vascular ROCK activity and increased eNOS expression and activity. Another ROCK inhibitor, Y-27632, also showed similar effects. The neuroprotective effects of fasudil were absent in eNOS-deficient mice. Conclusions-These findings indicate that the neuroprotective effect of ROCK inhibition is mediated by endotheliumderived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke.

Objective The sphingosine-1-phosphate receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. Methods We used rodent models of middle cerebral artery occlusion and cell culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. Results In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and ICAM-1-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by TNFalpha. Interpretation These findings suggest that anti-inflammatory mechanisms, and possibly vasculo-protection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Background-Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1 ϩ/Ϫ haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice. Methods and Results-Litters of ROCK1Ϫ/Ϫ mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1 ϩ/Ϫ mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1 ϩ/Ϫ mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg Ϫ1 · min Ϫ1 for 28 days) or treatment with N G -nitro-L-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1 ϩ/Ϫ mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1ϩ/Ϫ mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-␤, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1 ϩ/Ϫ mice compared with WT littermates. Conclusions-These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct.

Tumor necrosis factor-alpha (TNFa) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFa, Fas, or both (TNFa/FasÀ/À), we hypothesized that TNFa and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFa/FasÀ/À mice had improved motor performance from 1 to 4 days (P < 0.05), improved spatial memory acquisition at 8 to 14 days (P < 0.05), and decreased brain lesion size at 2 and 6 weeks after CCI (P < 0.05). Protection in TNFa/FasÀ/À mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFa before CCI, and TNFaÀ/À mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice (P < 0.05). Tumor necrosis factor-alpha/FasÀ/À mice had decreased the numbers of cortical cells with plasmalemma damage at 6 h (P < 0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFa and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P < 0.05 versus WT), suggesting that the beneficial effects of TNFa/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFa and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFa/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.

Vascular smooth muscle cells (SMCs) have been implicated in the pathophysiology of stroke, the third most common cause of death and the leading cause of long-term neurological disability in the world. However, there is little insight into the underlying cellular pathways that link SMC function to brain ischemia susceptibility. Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.ischemia ͉ Notch3 ͉ vascular smooth muscle ͉ CADASIL

Abstract-Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1 ϩ/Ϫ ) using Cre-loxP technology. The EC-Rac1 ϩ/Ϫ mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1 ϩ/flox ) mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1 ϩ/Ϫ mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of L-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction. (Circ Res. 2008;103:360-368.) Key Words: angiogenesis Ⅲ endothelium Ⅲ hypertension Ⅲ nitric oxide synthase Ⅲ signal transduction E ndothelium-derived nitric oxide (NO) is an important determinant of endothelium-dependent vasodilation and contributes to the anti-inflammatory and antithrombotic properties of the endothelium. Endothelial dysfunction is characterized by impaired synthesis, release, and activity of endothelialderived NO and is thought to contribute to the development and progression of various vascular disorders such as vasoconstriction, hypertension, atherosclerosis, and peripheral artery occlusive disease. 1 Therefore, understanding the pathophysiological mechanisms of endothelial dysfunction would provide important insights into therapeutic strategies for preventing cardiovascular disease.Endothelial NO synthase (eNOS) is the primary source of endothelium-derived NO. The steady-state mRNA level of eNOS is one of the principal determinants of eNOS expression and activity and NO bioavailability. 2 Numerous exogenous stimuli and conditions have been shown to alter eNOS mRNA level through both transcriptional and posttranscriptional mechanisms. For example, the eNOS mRNA is upregulated by laminar flow, vascular endothelial growth factor, transforming growth factor-beta and lysophosphatidylcholine and downregulated by tumor necrosis factor-␣ and oxidized low-density lipoprotein. 2 The enzymatic activity of eNOS is further regulated by mul...

Abstract-Dipyridamole (DP) is a phosphodiesterase inhibitor that increases the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP) by preventing their conversion to AMP and GMP, respectively. By increasing cAMP and cGMP levels in platelets, DP reversibly inhibits platelet aggregation and platelet-mediated thrombotic disease. In addition, DP may potentiate some of the vascular protective effects of endothelium-derived nitric oxide (NO), which increases cGMP by stimulating soluble guanylyl cyclase. Endotheliumderived NO is an important regulator of vascular tone, blood flow, and tissue perfusion. Indeed, endothelial NO synthase-deficient (eNOS Ϫ/Ϫ ) mice exhibit elevated systemic blood pressure and have larger myocardial and cerebral infarct size after ischemic injury. Other NO/cGMP-dependent effects that may be potentiated by DP include inhibition of vascular smooth muscle proliferation and prevention of endothelial-leukocyte interaction. In addition, DP increases local concentrations of adenosine and prostacyclin, which could affect vascular tone and inflammation. Finally, DP has antioxidant properties, which could stabilize platelet and vascular membranes as well as prevent the oxidation of low-density lipoprotein. These platelet and nonplatelet actions of DP may contribute to some of its therapeutic benefits in vascular disease. (Arterioscler Thromb Vasc Biol. 2008;28:s39-s42)

Complement component C4 mediates C3-dependent tissue damage after systemic ischemiareperfusion injury. Activation of C3 also contributes to the pathogenesis of experimental and human traumatic brain injury (TBI); however, few data exist regarding the specific pathways (classic, alternative, and lectin) involved. Using complement knockout mice and a controlled cortical impact (CCI) model, we tested the hypothesis that the classic pathway mediates secondary damage after TBI. After CCI, C4c and C3d immunostaining were detected in cortical vascular endothelial cells in wild-type (WT) mice; however, C4c and C3d immunostaining were also detected in C1q À/À mice, and C3d immunostaining was detected in C4 À/À mice. After CCI, WT and C1q À/À mice had similar motor deficits, Morris water maze performance, and brain lesion size. Naive C4 À/À and WT mice did not differ in baseline motor performance, but C4 À/À mice had reduced postinjury motor deficits (days 1 to 7, P < 0.05) and decreased brain tissue damage (days 14 and 35, P < 0.05) versus WT. Reconstitution of C4 À/À mice with human C4 (hC4) reversed their protection against postinjury motor deficits (P < 0.05 versus vehicle), but administration of hC4 did not impair postinjury motor performance (versus vehicle) in WT mice. The protective effects of C4 À/À were functionally distinct from the classic pathway and terminal complement, as C1q À/À and C3 À/À mice had postinjury tissue damage and motor dysfunction similar to WT. Thus, C4 contributes to motor deficits and brain tissue damage after CCI by mechanism(s) fundamentally different from those involved in experimental systemic ischemia-reperfusion injury.Journal of Cerebral Blood Flow & Metabolism (2007) 27, 1954-1964 doi:10.1038/sj.jcbfm.9600497; published online 25 April 2007 Keywords: complement; mice; behavior; traumatic brain injury; inflammation; genetics Introduction Traumatic brain injury induces an acute inflammatory response that contributes to posttraumatic cell death and functional neurologic deficits (Schmidt et al, 2005). Activation of complement amplifies local inflammation and exacerbates tissue damage and organ dysfunction after systemic and central nervous system ischemia-reperfusion injury (Chan et al, 2003;del Zoppo, 1999;Zhou et al, 2000), and studies in humans and in experimental animal models provide convincing evidence that Stahel et al, 2000). Complement signaling in injured brain parenchyma may also be contributed by serum components that enter the brain through a compromised blood-brain barrier.To date, only four published studies have examined a role for complement in traumatic cerebral contusion models. The investigators showed beneficial effects on histopathologic or functional outcome of pharmacologic C3 convertase inhibitors (Kaczorowski et al, 1995;Leinhase et al, 2006b), genetic inhibition of factor B (a component of the alternative pathway) (Leinhase et al, 2006a), or of astrocyte-specific overexpression of an endogenous C3 convertase inhibitor (Rancan et al, 2003). These studies...