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INTRODUCTION: Pathological cardiac fibrosis, through excessive extracellular matrix protein deposition from fibroblasts and pro-fibrotic immune responses and vascular stiffening is associated with most forms of cardiovascular disease. Pathological cardiac fibrosis and stiffening can lead to heart failure and arrythmias and vascular stiffening may lead to hypertension. ROCK, a serine/threonine kinase downstream of the Rho-family of GTPases, may regulate many pro-fibrotic and pro-stiffening signaling pathways in numerous cell types. AREAS COVERED: This article outlines the molecular mechanisms by which ROCK in fibroblasts, T helper cells, endothelial cells, vascular smooth muscle cells, and macrophages mediate fibrosis and stiffening. We speculate on how ROCK could be targeted to inhibit cardiovascular fibrosis and stiffening. EXPERT OPINION: Critical gaps in knowledge must be addressed if ROCK inhibitors are to be used in the clinic. Numerous studies indicate that each ROCK isoform may play differential roles in regulating fibrosis and may have opposing roles in specific tissues. Future work needs to highlight the isoform- and tissue-specific contributions of ROCK in fibrosis, and how isoform-specific ROCK inhibitors in murine models and in clinical trials affect the pathophysiology of cardiac fibrosis and stiffening. This could progress knowledge regarding new treatments for heart failure, arrythmias and hypertension and the repair processes after myocardial infarction.

Background The pathogenesis of vascular stiffening and hypertension is marked by non‐compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho‐associated coiled‐coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice ( Rock1 +/− and Rock2 +/− ) were used to determine the role of ROCK1 and ROCK2 in age‐related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild‐type mice compared with that of Rock1 +/− and Rock2 +/− mice. As a model for age‐related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L‐N ω ‐nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12‐week‐old male Rock1 +/− and Rock2 +/− mice. Similar to advancing age, angiotensin II/L‐N ω ‐nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2 +/− , and to a lesser extent, Rock1 +/− mice. The reduction of aortic stiffening in Rock2 +/− mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor‐β1 or angiotensin II was greatly attenuated in Rock2 −/− mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age‐related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age‐related vascular stiffening.

Cavernous angioma (CA) is a vascular pathology caused by loss of function in one of the 3 CA genes (CCM1, CCM2, and CCM3) that result in rho kinase (ROCK) activation. We investigated a novel ROCK2 selective inhibitor for the ability to reduce brain lesion formation, growth and maturation. We used genetic methods to explore the use of a ROCK2-selective kinase inhibitor to reduce growth and hemorrhage of CAs. The role of ROCK2 in CA was investigated by crossing Rock1 or Rock2 hemizygous mice with Ccm1 or Ccm3 hemizygous mice, and we found reduced lesions in the Rock2 hemizygous mice. A ROCK2-selective inhibitor, BA-1049 was used to investigate efficacy in reducing CA lesions after oral administration to Ccm1(+/−) and Ccm3(+/−) mice that were bred into a mutator background. After assessing the dose range effective to target brain endothelial cells in an ischemic brain model, Ccm1(+/−) and Ccm3(+/−) transgenic mice were treated for three (Ccm3(+/−)) or four months (Ccm1(+/−)), concurrently, randomized to receive one of three doses of BA-1049 in drinking water, or placebo. Lesion volumes were assessed by micro-computed tomography. BA-1049 reduced activation of ROCK2 in Ccm3(+/−)Trp53(−/−) lesions. Ccm1(+/−)Msh2(−/−) (n=68) and Ccm3(+/−)Trp53(−/−) (n=71) mice treated with BA-1049 or placebo showed a significant dose-dependent reduction in lesion volume after treatment with BA-1049, and a reduction in hemorrhage (iron deposition) near lesions at all doses. These translational studies show that BA-1049 is a promising therapeutic agent for treatment of CA, a disease with no current treatment except surgical removal of the brain lesions.

Background and Purpose— Previously, murine models Krit1 +/− Msh2 −/ − and Ccm2 +/ − Trp53 −/ − showed a reduction or no effect on cerebral cavernous malformation (CCM) burden and favorable effects on lesional hemorrhage by the robust Rock (Rho-associated protein kinase) inhibitor fasudil and by simvastatin (a weak pleiotropic inhibitor of Rock). Herein, we concurrently investigated treatment of the more aggressive Pdcd10/Ccm3 model with fasudil, simvastatin, and higher dose atorvastatin to determined effectiveness of Rock inhibition. Methods— The murine models, Pdcd10 +/ − Trp53 −/ − and Pdcd10 +/ − Msh2 −/ − , were contemporaneously treated from weaning to 5 months of age with fasudil (100 mg/kg per day in drinking water, n=9), simvastatin (40 mg/kg per day in chow, n=11), atorvastatin (80 mg/kg per day in chow, n=10), or with placebo (n=16). We assessed CCM volume in mouse brains by microcomputed tomography. Lesion burden was calculated as lesion volume normalized to total brain volume. We analyzed chronic hemorrhage in CCM lesions by quantitative intensity of Perls staining in brain sections. Results— The Pdcd10 +/ − Trp53 −/ − /Msh2 −/ − models showed a mean CCM lesion burden per mouse reduction from 0.0091 in placebos to 0.0042 ( P =0.027) by fasudil, and to 0.0047 ( P =0.025) by atorvastatin treatment, but was not changed significantly by simvastatin. Hemorrhage intensity per brain was commensurately decreased by Rock inhibition. Conclusions— These results support the exploration of proof of concept effect of high-dose atorvastatin on human CCM disease for potential therapeutic testing.

Statins, 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors, have been used for decades for the prevention of coronary artery disease and stroke. They act primarily by lowering serum cholesterol through the inhibition of cholesterol synthesis in the liver, which results in the upregulation of low-density lipoprotein receptors in the liver. This results in the removal of low-density lipoproteincholesterol. Studies have suggested that statins may demonstrate additional effects that are independent of their effects on low-density lipoprotein-cholesterol. These have been termed “pleiotropic” effects. Pleiotropic effects may be due to the inhibition of isoprenoid intermediates by statins. Isoprenoid inhibition has effects on the small guanosine triphosphate binding proteins Rac and Rho which in turn effects nicotinamide adenine dinucleotide phosphate oxidases. Therefore, there are changes in endothelial nitric oxide synthase expression, atherosclerotic plaque stability, pro-inflammatory cytokines and reactive oxygen species production, platelet reactivity, and cardiac fibrosis and hypetrophy development. Recently, statins have been compared to the ezetimibe and the recently published outcomes data on the proprotein convertase subtilisin kexin type 9 inhibitors has allowed for a reexamination of statin pleiotropy. As a result of these diverse effects, it has been suggested that statins also have anti-arrhythmic effects. This review focuses on the mechanisms of statin pleiotropy and discusses evidence from the statin clinical trials as well as examining the possible anti-arrhythmic effects atrial fibrillation and ventricular tachyarrhythmias.

Over the past four decades, no class of drugs has had more impact on cardiovascular health than the 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors or statins. Developed as potent lipid-lowering agents, statins were later shown to reduce morbidity and mortality of patients who are at risk for cardiovascular disease. However, retrospective analyses of some of these clinical trials have uncovered some aspects of their clinical benefits that may be additional to their lipid-lowering effects. Such ‘pleiotropic’ effects of statins garnered intense interest and debate over its contribution to cardiovascular risk reduction. This review will provide a brief background of statin pleiotropy, assess the available clinical evidence for and against their non-lipid-lowering benefits, and propose future research directions in this field.

Background. The blood-brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of microvascular endothelial cells (BMVECs), which form the lining of cerebral blood vessels and are linked via tight junctions (TJs). The BBB is regulated by components of the extracellular matrix (ECM), and matrix metalloproteinase 3 (MMP3) remodels the ECM’s basal lamina, which forms part of the BBB. Oxidative stress is implicated in activation of MMPs and impaired BBB. Thus, we investigated whether MMP3 modulates BBB permeability. Methods. Experiments included in vivo assessments of isoflurane anesthesia and dye extravasation from brain in wild-type (WT) and MMP3-deficient (MMP3-KO) mice, as well as in vitro assessments of the integrity of monolayers of WT and MMP3-KO BMVECs and the expression of junction proteins. Results. Compared to WT mice, measurements of isoflurane usage and anesthesia induction time were higher in MMP3-KO mice and lower in WT that had been treated with MMP3 (WT+MMP3), while anesthesia emergence times were shorter in MMP3-KO mice and longer in WT+MMP3 mice than in WT. Extravasation of systemically administered dyes was also lower in MMP3-KO mouse brains and higher in WT+MMP3 mouse brains, than in the brains of WT mice. The results from both TEER and Transwell assays indicated that MMP3 deficiency (or inhibition) increased, while MMP3 upregulation reduced barrier integrity in either BMVEC or the coculture. MMP3 deficiency also increased the abundance of TJs and VE-cadherin proteins in BMVECs, and the protein abundance declined when MMP3 activity was upregulated in BMVECs, but not when the cells were treated with an inhibitor of extracellular signal related-kinase (ERK). Conclusion. MMP3 increases BBB permeability following the administration of isoflurane by upregulating the ERK signaling pathway, which subsequently reduces TJ and VE-cadherin proteins in BMVECs.

Objectives Cognitive dysfunction and dementia are common following ischemic stroke. Endothelial nitric oxide synthase (eNOS) has been found to play an important role in neurologic function and cognition. The purpose of the present study was to assess the specific role of eNOS in cognitive performance after stroke. Design Male wild-type and mice lacking eNOS (eNOS) underwent middle cerebral artery occlusion or sham-surgery. Primary outcomes were repeated measures of neurologic score, limb asymmetry, sensory/motor function, and spatial memory/learning assessed at intervals up to 28 days postsurgery. Group differences in brain microglia activation and infiltration and levels of interferon-gamma were examined. Results There was no genotype × surgery interaction effect on the pattern of change in neurologic score, limb asymmetry, or sensory motor function across the 28 days postsurgery. In the Morris water maze, eNOS-/- middle cerebral artery occlusion mice displayed learning and memory deficits not evident in wild-type middle cerebral artery occlusion mice. Poorer spatial memory and learning in eNOS-/- middle cerebral artery occlusion mice was associated with a reduction in the number of activated microglia in the striatum on the lesion side and decreased brain tissue levels of interferon-gamma. Conclusions This study's data support a role for eNOS in cognitive performance after stroke. This finding may lead to the development of novel interventions to treat poststroke cognitive deficits.

Traditional informatics in comparative genomics work only with static representations of biomolecules (i.e., sequence and structure), thereby ignoring the molecular dynamics (MD) of proteins that define function in the cell. A comparative approach applied to MD would connect this very short timescale process, defined in femtoseconds, to one of the longest in the universe: molecular evolution measured in millions of years. Here, we leverage advances in graphics-processing-unit-accelerated MD simulation software to develop a comparative method of MD analysis and visualization that can be applied to any two homologous Protein Data Bank structures. Our open-source pipeline, DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations), works in conjunction with existing molecular modeling software to convert any Linux gaming personal computer into a “comparative computational microscope” for observing the biophysical effects of mutations and other chemical changes in proteins. DROIDS implements structural alignment and Benjamini-Hochberg-corrected Kolmogorov-Smirnov statistics to compare nanosecond-scale atom bond fluctuations on the protein backbone, color mapping the significant differences identified in protein MD with single-amino-acid resolution. DROIDS is simple to use, incorporating graphical user interface control for Amber16 MD simulations, cpptraj analysis, and the final statistical and visual representations in R graphics and UCSF Chimera. We demonstrate that DROIDS can be utilized to visually investigate molecular evolution and disease-related functional changes in MD due to genetic mutation and epigenetic modification. DROIDS can also be used to potentially investigate binding interactions of pharmaceuticals, toxins, or other biomolecules in a functional evolutionary context as well.

Background and Purpose— We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in 2 models that recapitulate the human disease. Methods— Two heterozygous murine models, Ccm1 +/− Msh2 − /− and Ccm2 +/− Trp53 −/− , were treated from weaning to 4 to 5 months of age with Fasudil (100 mg/kg per day), simvastatin (40 mg/kg per day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, nonheme iron deposition (as a quantitative measure of chronic lesional hemorrhage), and ROCK activity. Results— Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1 +/− Msh2 −/− mice, and in meta-analysis of both models combined, when compared with mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1 +/− Msh2 −/− mice, and in both models combined, compared with mice given placebo. ROCK activity in mature lesions of Ccm1 +/− Msh2 −/− mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1 +/− Msh2 −/− mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2 +/− Trp53 −/− mice alone, and Fasudil benefit seemed limited to males. Conclusions— ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors and the clinical testing of commonly used statin agents in CCM.

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil-containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element-binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

Aims The endothelium plays an important role during vascular inflammation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD and prevented the development of diabetes and obesity. However, its effect on vascular inflammation and the underlying mechanism is still unknown. Methods and results Leukocyte adhesion was evaluated in-vivo and in-vitro using dynamic flow chamber and intravital microscopy in mice. Expression of adhesion molecules induced by TNFα and adhesion of leukocytes to the vessel wall were inhibited by MnTBAP. The anti-inflammatory effect of MnTBAP was partly mediated by up-regulation of the BMPR-II and Smad dependent pathway. Additionally, MnTBAP decelerated the turn-over of endogenous BMPR-II. Conclusion Our data demonstrate that MnTBAP activates Smad signaling, preserves the turn-over of BMPR-II and elicits anti-inflammatory effects in endothelial cells, partly mediated by BMPR-II. This finding suggests a potential therapeutic impact of MnTBAP in the treatment of vascular inflammation.

Hypertensive cardiac remodeling is characterized by left ventricular hypertrophy and interstitial fibrosis, which can lead to heart failure with preserved ejection fraction. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the serine/threonine protein kinase family, which mediates the downstream effects of the small GTP-binding protein RhoA. There are 2 isoforms: ROCK1 and ROCK2. They have different functions in different types of cells and tissues. There is growing evidence that ROCKs contribute to the development of cardiovascular diseases, including cardiac fibrosis, hypertrophy, and subsequent heart failure. Recent experimental studies using ROCK inhibitors, such as fasudil, have shown the benefits of ROCK inhibition in cardiac remodeling. Mice lacking each ROCK isoform also exhibit reduced myocardial fibrosis in a variety of pathological models of cardiac remodeling. Indeed, clinical studies with fasudil have suggested that ROCKs could be potential novel therapeutic targets for cardiovascular diseases. In this review, we summarize the current understanding of the roles of ROCKs in the development of cardiac fibrosis and hypertrophy and discuss their therapeutic potential for deleterious cardiac remodeling. (Circ J 2016; 80: 1491-1498).

BACKGROUND Rho-associated kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton and endothelial nitric oxide synthase (eNOS). Because the phosphorylation of eukaryotic elongation factor-1A1 (eEF1A1) by ROCK2 is critical for eNOS expression, we hypothesized that this molecular pathway may play a critical role in neuroprotection following focal cerebral ischemia.Methods and Results:Adult male wild-type (WT) and mutant ROCK2 and eNOS-/-mice were subjected to middle cerebral artery occlusion (MCAO), and cerebral infarct size, neurological deficit and absolute cerebral blood flow were measured. In addition, aortic endothelium-dependent response to acetylcholine, NG-nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside were assessed ex vivo. Endothelial cells from mouse brain or heart were used to measure eNOS and eEF1A activity, as well as NO production and eNOS mRNA half-life. In global hemizygous ROCK2+/-and endothelial-specific EC-ROCK2-/-mice, eNOS mRNA stability and eNOS expression were increased, which correlated with enhanced endothelium-dependent relaxation and neuroprotection following focal cerebral ischemia. Indeed, when ROCK2+/-mice were place on an eNOS-/-background, the neuroprotective effects observed in ROCK2+/-mice were abolished. CONCLUSIONS These findings indicate that the phosphorylation of eEF1A1 by ROCK2 is physiologically important for eNOS expression and NO-mediated neuroprotection, and suggest that targeting endothelial ROCK2 and eEF1A may have therapeutic benefits in ischemic stroke and cardiovascular disease.

Ozone causes airway hyperresponsiveness (AHR) and pulmonary inflammation. Rho kinase (ROCK) is a key regulator of smooth muscle cell contraction and inflammatory cell migration. To determine the contribution of the two ROCK isoforms ROCK1 and ROCK2 to ozone-induced AHR, we exposed wild-type, ROCK1+/−, and ROCK2+/− mice to air or ozone (2 ppm for 3 h) and evaluated mice 24 h later. ROCK1 or ROCK2 haploinsufficiency did not affect airway responsiveness in air-exposed mice but significantly reduced ozone-induced AHR, with a greater reduction in ROCK2+/− mice despite increased bronchoalveolar lavage (BAL) inflammatory cells in ROCK2+/− mice. Compared with wild-type mice, ozone-induced increases in BAL hyaluronan, a matrix protein implicated in ozone-induced AHR, were lower in ROCK1+/− but not ROCK2+/− mice. Ozone-induced increases in other inflammatory moieties reported to contribute to ozone-induced AHR (IL-17A, osteopontin, TNFα) were not different in wild-type vs. ROCK1+/− or ROCK2+/− mice. We also observed a dose-dependent reduction in ozone-induced AHR after treatment with the ROCK1/ROCK2 inhibitor fasudil, even though fasudil was administered after induction of inflammation. Ozone increased pulmonary expression of ROCK2 but not ROCK1 or RhoA. A ROCK2 inhibitor, SR3677, reduced contractile forces in primary human airway smooth muscle cells, confirming a role for ROCK2 in airway smooth muscle contraction. Our results demonstrate that ozone-induced AHR requires ROCK. Whereas ROCK1-dependent changes in hyaluronan may contribute to ROCK1's role in O3-induced AHR, the role of ROCK2 is downstream of inflammation, likely at the level of airway smooth muscle contraction.

Aims Angiogenesis is defined as the sprouting of capillaries from pre-existing vasculature. It is a complex process that includes endothelial proliferation, migration, and tube formation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. In addition, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD in various tissues. However, its effect on the vasculature remains unknown. Methods and results HUVECs were treated with MnTBAP. Migration, tube formation, and capillary sprouting assays were performed to evaluate the pro-angiogenic effect in vitro . Matrigel plug assay was performed to assess capillary ingrowth in vivo . Compared to control, treatment with MnTBAP revealed increased cell migration, tube formation and capillary sprouting along with more capillary ingrowth in the Matrigel plug assay. This effect was mediated through a mitofusin (Mfn)-1-dependent pathway. Expression of Tie-2, Ang-2 and VEGF mRNA was increased in muscle tissues after ligation in MnTBAP treated mice. However, revascularization in the hindlimb ischemia model was not statistically significant at day 10 in MnTBAP treated mice. Conclusion In summary, our data demonstrate a strong pro-angiogenic, but less pro-arteriogenic effect of MnTBAP in HUVECs mediated by Mfn-1.

Sox2 has a critical role in embryonic stem (ES) cell maintenance and differentiation. Interestingly, its activity is highly dosage-dependent. Although transcriptional regulation of Sox2 has been extensively studied, the mechanisms orchestrating its degradation remain unclear. In this study, we identified ubiquitin-conjugating enzyme E2S (Ube2s) as a novel effector for Sox2 protein degradation. Ube2s mediates K11-linked polyubiquitin chain formation at the Sox2-K123 residue, thus marking it for proteasome-mediated degradation. Besides its role in fine-tuning the precise level of Sox2, Ube2s reinforces the self-renewing and pluripotent state of ES cells. Importantly, it also represses Sox2-mediated ES cell differentiation toward the neural ectodermal lineage.

Background Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration. Objective To determine the role of ROCK2 acting within CD4+ cells in allergic airways responses. Methods ROCK2 haploinsufficient (ROCK2+/-) and wildtype mice were sensitized with ovalbumin (OVA). ROCK2+/- mice then received either CD4+ cells from ROCK2 sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 hours before challenge with aerosolized OVA. Wildtype mice received saline before challenge. Allergic airway responses were measured 48 hours after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4+ cells (ROCK2CD4Cre mice) versus control (CD4-Cre and ROCK2flox/flox) mice. Results OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2+/- versus wildtype mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2+/- mice, adoptive transfer with CD4+ cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wildtype levels, whereas eotaxin and airway hyperrresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2 insufficient mice, allergic inflammation was not different in ROCK2CD4Cre versus control mice. Conclusion ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non lymphocyte cells involved in lymphocyte activation and migration into the airways. This article is protected by copyright. All rights reserved.

Objective— The pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) independent of cholesterol-lowering effects are thought to be mediated through inhibition of the Rho/Rho-kinase pathway. However, we have previously demonstrated that the pleiotropic effects of regular-dose statins are mediated mainly through inhibition of the Rac1 signaling pathway rather than the Rho/Rho-kinase pathway, although the molecular mechanisms of the selective inhibition of the Rac1 signaling pathway by regular-dose statins remain to be elucidated. In this study, we tested our hypothesis that small GTP-binding protein GDP dissociation stimulator (SmgGDS) plays a crucial role in the molecular mechanisms of the Rac1 signaling pathway inhibition by statins in endothelial cells. Approach and Results— In cultured human umbilical venous endothelial cells, statins concentration-dependently increased SmgGDS expression and decreased nuclear Rac1. Statins also enhanced SmgGDS expression in mouse aorta. In control mice, the protective effects of statins against angiotensin II–induced medial thickening of coronary arteries and fibrosis were noted, whereas in SmgGDS-deficient mice, the protective effects of statins were absent. When SmgGDS was knocked down by its small interfering RNA in human umbilical venous endothelial cells, statins were no longer able to induce Rac1 degradation or inhibit angiotensin II–induced production of reactive oxygen species. Finally, in normal healthy volunteers, statins significantly increased SmgGDS expression with a significant negative correlation between SmgGDS expression and oxidative stress markers, whereas no correlation was noted with total or low-density lipoprotein-cholesterol. Conclusions— These results indicate that statins exert their pleiotropic effects through SmgGDS upregulation with a resultant Rac1 degradation and reduced oxidative stress in animals and humans.

Introduction Activated RhoA/Rho kinase (ROCK) has been implicated in diabetes‐induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear. Aim We tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes. Methods Eight weeks after streptozotocin‐induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho‐p38 MAPK, p38 MAPK, phospho‐MYPT‐1 Thr850 , MYPT‐1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), partial ROCK 2 +/− knockout (KO), and ROCK 2 +/− KO + D mice. Main Outcome Measures The expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT‐1 Thr850 and p38 MAPK, arginase activity/expression, endothelial‐ and nitrergic‐dependent relaxation of CC was assayed. Results Diabetes significantly reduced maximum relaxation (E max ) to both endothelium‐dependent acetylcholine (WT + D: E max ; 61 ± 4% vs. WT: E max ; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho‐MYPT‐1 Thr850 , phospho‐p38 MAPK, arginase II, and activity of corporal arginase (1.6‐fold) in WT diabetic CC. However, this impairment in CC of WT + D mice was absent in heterozygous ROCK 2 +/− KO + D mice for acetylcholine (E max : 80 ± 5%) and attenuated for nitrergic nerve‐induced relaxation. CC of ROCK 2 +/− KO + D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes‐induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh‐ and nitrergic nerve‐induced relaxation and elevation of arginase activity. Conclusion ROCK 2, p38 MAPK and arginase play key roles in diabetes‐induced impairment of CC relaxation.

The Rho-associated coiled-coil containing kinases, ROCK1 and ROCK2, are important regulators of cell shape, migration, and proliferation through effects on the actin cytoskeleton. However, it is not known whether ROCK2 plays an important role in the development of cardiac hypertrophy. To determine whether the loss of ROCK2 could prevent cardiac hypertrophy, cardiomyocyte-specific ROCK2-null (c-ROCK2−/−) were generated using conditional ROCK2flox/flox mice and α-myosin heavy-chain promoter-driven Cre recombinase transgenic mice. Cardiac hypertrophy was induced by Ang II infusion (400 ng/kg/min, 28 d) or transverse aortic constriction (TAC). Under basal conditions, hemodynamic parameters, cardiac anatomy, and function of c-ROCK2−/− mice were comparable to wild-type (WT) mice. However, following Ang II infusion or TAC, c-ROCK2−/− mice exhibited a substantially smaller increase in heart-to-body weight ratio, left ventricular mass, myocyte cross-sectional area, hypertrophy-related fetal gene expression, intraventricular fibrosis, cardiac apoptosis, and oxidative stress compared to control mice. Deletion of ROCK2 in cardiomyocytes leads to increased expression of four-and-a-half LIM-only protein-2 (FHL2) and FHL2-mediated inhibition of serum response factor (SRF) and extracellular signal-regulated mitogen-activated protein kinase (ERK). Knockdown of FHL2 expression in ROCK2-deficient cardiomyocytes or placing ROCK2-haploinsufficient (ROCK2+/−) mice on FHL2+/−-haploinsufficient background restored the hypertrophic response to Ang II. These results indicate that cardiomyocyte ROCK2 is essential for the development of cardiac hypertrophy and that up-regulation of FHL2 may contribute to the antihypertrophic phenotype that is observed in cardiac-specific ROCK2-deficient mice.—Okamoto, R., Li, Y., Noma, K., Hiroi, Y., Liu, P.-Y., Taniguchi, M., Ito, M., Liao, J. K. FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2.

The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.

Aims Rho-associated coiled-coil containing kinase (ROCK)-2 is an important mediator of the actin cytoskeleton. Because changes in the actin cytoskeleton are critical for platelet function, we hypothesized that ROCK2 in platelets will play important role in thrombosis and can be potentially a target for therapeutic intervention in thromboembolic stroke. Methods and results We generated platelet-specific ROCK2-deficient mice (ROCK2Plt-/-) from conditional ROCK2fl°x/fl°x and platelet factor (PF)-4-Cre transgenic mice. Platelets from ROCK2Plt-/- mice were less responsive to thrombin stimulation in terms of pseudopodia formation, collagen adhesion, and in the formation of homotypic and heterotypic aggregates. This corresponded to prolonged bleeding time and delayed vascular occlusion following vessel injury. To determine whether these changes in platelet function could affect thrombotic disease, we utilized a clot-embolic model of ischaemic stroke. When pre-formed clots from ROCK2Plt-/- mice were injected into the middle cerebral artery of control mice, cerebral blood flow recovery occurred more rapidly, leading to decreased cerebral injury and neurological deficits, compared to pre-formed clots from control mice. Interestingly, pre-formed clots from control mice produced similar degree of cerebral injury when injected into control or ROCK2Plt-/- mice, suggesting that platelet ROCK2 deficiency affects clot formation but not propagation. Indeed, in a non-thrombotic intra-filament MCA occlusion model of stroke, platelet ROCK2 deletion was not protective. Furthermore, ROCK2Plt-/- mice exhibit similar atherosclerosis severity and vascular remodeling as control mice. Conclusion These findings indicate that platelet ROCK2 plays important role in platelet function and thrombosis, but does not contribute to the pathogenesis of atherosclerosis and vascular remodeling.

The statins have been used for 30 years to prevent coronary artery disease and stroke. Their primary mechanism of action is the lowering of serum cholesterol through inhibiting hepatic cholesterol biosynthesis thereby upregulating the hepatic low-density lipoprotein (LDL) receptors and increasing the clearance of LDL-cholesterol. Statins may exert cardiovascular protective effects that are independent of LDL-cholesterol lowering called pleiotropic effects. Because statins inhibit the production of isoprenoid intermediates in the cholesterol biosynthetic pathway, the post-translational prenylation of small GTP-binding proteins such as Rho and Rac, and their downstream effectors such as Rho kinase and nicotinamide adenine dinucleotide phosphate oxidases are also inhibited. In cell culture and animal studies, these effects alter the expression of endothelial nitric oxide synthase, the stability of atherosclerotic plaques, the production of proinflammatory cytokines and reactive oxygen species, the reactivity of platelets, and the development of cardiac hypertrophy and fibrosis. The relative contributions of statin pleiotropy to clinical outcomes, however, remain a matter of debate and are hard to quantify because the degree of isoprenoid inhibition by statins correlates to some extent with the amount of LDL-cholesterol reduction. This review examines some of the currently proposed molecular mechanisms for statin pleiotropy and discusses whether they could have any clinical relevance in cardiovascular disease.

To investigate the effect of phospholipid transfer protein(PLTP) on cigarette smoke extract(CSE) induced epithelial-mesenchymal transition(EMT) in rat alveolar Type Ⅱ cells (RLE-6TN).CSE of different concentrations (0%, 0.25%, 0.5% and 1%) was co-cultured for 2 or 3 days with RLE-6TN, either pre-treated or not pre-treated with siRNA-PLTP for 6 h. Expression levels of E-cadherin mRNA and Vimentin mRNA were examined by RT-PCR, while expression levels of PLTP, E-cadherin, N-cadherin and Vimentin were examined by Western blot.Our results showed that the expression of E-cadherin mRNA decreased in CSE-treated groups: 1.01±0.05, 0.74±0.05, 0.65±0.03, 0.30±0.08 respectively at different concentrations of CSE (0 %, 0.25%, 0.5 %, and 1.0%); while the level of Vimentin mRNA increased significantly in 1% CSE treated cells(1.88±0.49), compared with control cells (1.01±0.20). Treatment with CSE at different concentrations (0%, 0.25%, 0.5% and 1%) showed that the protein levels of PLTP were 0.42±0.02, 0.89±0.25, 1.08±0.18, 1.61±0.06 respectively; those of E-cadherin were 1.61±0.04, 1.08±0.10, 0.62±0.08, 0.68±0.17, respectively; those of N-cadherin were 0.60±0.14, 0.57±0.26, 0.88±0.30, 1.94±0.54, respectively; and those of Vimentin were 0.61±0.05, 0.98±0.16, 1.07±0.14, 1.34±0.19, respectively; all P0.05 when the 1% CSE group was compared with the control group. EMT induced by CSE was significantly inhibited by siRNA-PLTP.PLTP may be involved in CSE induced EMT of rat alveolar cells.

Background— Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow–derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined. Methods and Results— The bone marrows from wild-type, ROCK2 +/− , and ROCK2 −/− mice were transplanted into irradiated recipient low-density lipoprotein receptor −/− mice, and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared with wild-type bone marrow–transplanted mice, ROCK2 +/− bone marrow–transplanted and ROCK2 −/− bone marrow–transplanted mice showed substantially less lipid accumulation in the aorta (8.46±1.42% and 9.80±2.34% versus 15.64±1.89%; P 3 μm 2 versus 520.2±125.7×10 3 μm 2 ; P P P Conclusions— ROCK2 contributes to atherosclerosis, in part, by inhibiting peroxisome proliferator-activated receptor-γ–mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.

Stroke is the third leading cause of death and a major cause of long-term disability in the adult population. Growing evidence suggests that inflammation may play an important role in the evolution of stroke. Because Rho-associated coiled-coil containing kinases (ROCKs) are important mediators of inflammation, they may contribute to stroke and stroke recovery.The pathophysiological role of ROCKs in mediating inflammation at different phases of stroke, and the therapeutic opportunities for stroke prevention and stroke treatment with ROCK inhibitors will be discussed.Inflammation is a double-edged sword during the evolution of stroke. Immunomodulation might provide a novel therapeutic approach for stroke prevention and stroke treatment. ROCK plays an important role in mediating the inflammatory response following vascular injury as well as platelet activation and thrombus formation. ROCK inhibitors have been shown to be beneficial in stroke prevention, acute neuroprotection and chronic stroke recovery by affecting inflammatory-mediated platelet and endothelial function, smooth muscle contraction and neuronal regeneration. Thus, ROCK-mediated inflammation could be a potential therapeutic target for stroke prevention and stroke treatment. However, the mechanism by which ROCKs regulate the inflammatory response is unclear, and the role of the two ROCK isoforms in stroke and stroke recovery remains to be determined.

Ultrasound (US) is an effective tool for local delivery of genes into target tumors or organs. In combination with microbubbles, US can temporarily change the permeability of cell membranes by cavitation and facilitate entry of plasmid DNA into cells. Here, we demonstrate that repeated US-mediated delivery of anti-angiogenic genes, endostatin or calreticulin, into muscle significantly inhibits the growth of orthotopic tumors in the liver, brain or lung. US-mediated anti-angiogenic gene therapy also seems to function as an adjuvant therapy that significantly enhances the antitumor effects of the chemotherapeutic drug doxorubicin and adenovirus-mediated cytokine gene therapy. Significantly higher levels of tumor apoptosis or tumor-infiltrating lymphocytes were observed after combined therapy consisting of either anti-angiogenic therapy and chemotherapy, or anti-angiogenic therapy and immunotherapy. Taken together, our experiments demonstrate that intramuscular delivery of anti-angiogenic genes by US exposure can effectively treat distant orthotopic tumors, and thus has great therapeutic potential in terms of clinical treatment.

We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.

Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 +/− and Rock2 +/− mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.

Notch signaling is reported to regulate angiogenesis, interacting with vascular endothelial growth factor (VEGF) signaling. HMG CoA reductase inhibitors (statins) also alter Notch signaling in vascular cells, but the mechanism and involvement of Notch and VEGF signaling in statin-mediated angiogenesis remain unclear. Here, we examined how statins activate the endothelial Notch1, and promote angiogenesis and arteriogenesis. We examined blood flow recovery after hindlimb ischemia in wild-type (WT) and Notch1 mutant mice treated with or without pitavastatin (3 mg/kg/day, p.o.). Although VEGF induction was not altered in ischemic limbs, pitavastatin promoted blood flow recovery in ischemic limbs in control mice but not in Notch1 mutant mice. Furthermore, pitavastatin induced endothelial ephrinB2 downstream of Notch1 and increased the density of both capillaries and arterioles in the ischemic limbs of WT but not of Notch1 mutant mice. Pitavastatin (100 nmol/l) rapidly activated γ-secretase and Notch1 in human umbilical vein endothelial cells without VEGF induction, which was suppressed by pharmacological inhibition and knockdown of Akt. Pitavastatin also augmented endothelial proliferation and tube formation on Matrigel, which were suppressed by either γ-secretase inhibition or knockdown of Notch1. Pitavastatin-induced microvascular sprouting was also impaired in Notch1 mutant aortic explants. Taken together, pitavastatin activates Notch1 through Akt-dependent stimulation of γ-secretase in endothelial cells, and thereby increases vasculogenesis without VEGF induction.

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Background— The signaling mechanisms that regulate the recruitment of bone marrow (BM)–derived cells to the injured heart are not well known. Notch receptors mediate binary cell fate determination and may regulate the function of BM-derived cells. However, it is not known whether Notch1 signaling in BM-derived cells mediates cardiac repair after myocardial injury. Methods and Results— Mice with postnatal cardiac-specific deletion of Notch1 exhibit infarct size and heart function after ischemic injury that is similar to that of control mice. However, mice with global hemizygous deletion of Notch1 (N1 ± ) developed larger infarct size and worsening heart function. When the BM of N1 ± mice were transplanted into wild-type (WT) mice, infarct size and heart function were worsened and neovascularization in the infarct border area was reduced compared with WT mice transplanted with WT BM. In contrast, transplantation of WT BM into N1 ± mice lessened the myocardial injury observed in N1 ± mice. Indeed, hemizygous deletion of Notch1 in BM-derived cells leads to decreased recruitment, proliferation, and survival of mesenchymal stem cells (MSC). Compared with WT MSC, injection of N1 ± MSC into the infarcted heart leads to increased myocardial injury whereas injection of MSC overexpressing Notch intracellular domain leads to decreased infarct size and improved cardiac function. Conclusions— These findings indicate that Notch1 signaling in BM-derived cells is critical for cardiac repair and suggest that strategies that increase Notch1 signaling in BM-derived MSC could have therapeutic benefits in patients with ischemic heart disease.

The Rho/Rho-associated coiled-coil forming kinases (ROCKs) are important regulators of the actin cytoskeleton. Because changes in the actin cytoskeleton underlie vascular contractility and remodeling, inflammatory cell recruitment, and cellular proliferation, it is likely that the Rho/ROCK pathway will play a central role in mediating vascular function. Indeed, increased ROCK activity is observed in cerebral and coronary vasospasm, hypertension, vascular inflammation, arteriosclerosis, and atherosclerosis. Recent experimental and clinical studies suggest that inhibition of ROCK could be a promising target for the treatment of cardiovascular disease. For example, inhibition of ROCK might be the underlying mechanism by which statins or HMG-CoA reductase inhibitors exert their therapeutic benefits beyond cholesterol reduction. In this review, we provide a current understanding of the critical role of RhoA/ROCK pathway in the regulation of vascular function and discuss its therapeutic potential in the treatment of atherosclerosis and vascular disease.

Through local cell-cell interactions, the Notch signaling pathway controls tissue formation and homeostasis during embryonic and adult life. In the heart, Notch1 is expressed in a variety of cell types, such as cardiomyocytes, smooth muscle cells, and endothelial cells. In cardiomyocytes, Notch1 is activated in proliferating embryonic and immature cardiomyocytes, and it is downregulated in the myocardium during postnatal development. However, Notch signaling in the adult myocardium could be activated transiently in response to myocardial injury, suggesting that Notch signaling may contribute to cardiac repair. Indeed, activation of Notch1 intracellular domain blunts the severity of myocardial injury and improves myocardial hemodynamic function. Conversely, genetic ablation of the Notch1 gene, either systemically or in bone marrow-derived cells, leads to impaired cardiac repair following myocardial infarction. In this review, we discuss the complex mechanisms of Notch signaling and its role in cardiac repair and regeneration after myocardial infarction.

Deregulated production of IL-17 and IL-21 plays a key pathogenic role in many autoimmune disorders. A delineation of the mechanisms that underlie the inappropriate synthesis of IL-17 and IL-21 in autoimmune diseases can thus provide important insights into potential therapies for these disorders. Here we have shown that the serine-threonine kinase Rho-associated, coiled-coil–containing protein kinase 2 (ROCK2) becomes activated in mouse T cells under Th17 skewing conditions and phosphorylates interferon regulatory factor 4 (IRF4), a transcription factor that is absolutely required for the production of IL-17 and IL-21. We furthermore demonstrated that ROCK2-mediated phosphorylation of IRF4 regulated the synthesis of IL-17 and IL-21 and the differentiation of Th17 cells. Whereas CD4+ T cells from WT mice activated ROCK2 physiologically under Th17 conditions, CD4+ T cells from 2 different mouse models of spontaneous autoimmunity aberrantly activated ROCK2 under neutral conditions. Moreover, administration of ROCK inhibitors ameliorated the deregulated production of IL-17 and IL-21 and the inflammatory and autoantibody responses observed in these autoimmune mice. Our findings thus uncover a crucial link among ROCK2, IRF4, and the production of IL-17 and IL-21 and support the idea that selective inhibition of ROCK2 could represent an important therapeutic regimen for the treatment of autoimmune disorders.

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting.

Currently available modalities for the treatment of acute ischemic stroke are aimed at preserving or augmenting cerebral blood flow. Experimental evidence suggests that statins, which show 25-30% reduction of stroke incidence in clinical trials, confer stroke protection by upregulation of eNOS and increasing cerebral blood flow. The upregulation of eNOS by statins is mediated by inhibition of small GTP-binding protein RhoA. Our recent study uncovered a unique role for a Rho-family member Rac1 in stroke protection. Rac1 in endothelium does not affect cerebral blood flow. Instead, inhibition of endothelial Rac1 leads to broad upregulation of the genes relevant to neurovascular protection. Intriguingly, inhibition of endothelial Rac1 enhances neuronal cell survival through endothelium-derived neurotrophic factors, including artemin. This review discusses the emerging therapeutic opportunities to target neurovascular signaling beyond the BBB, with special emphasis on the novel role of endothelial Rac1 in stroke protection.

Background— Alteration of the circadian rhythm and increased vascular senescence are linked to cardiovascular disease. Per2 , a circadian gene, is known to regulate endothelium-dependent vasomotion. However, the mechanism by which Per2 affects endothelial function is unknown. We hypothesize that endothelial dysfunction in Per2 mutant (Per2 m/m ) mice is mediated in part by increased vascular senescence and impaired endothelial progenitor cell (EPC) function. Methods and Results— Endothelial cells from Per2 m/m mice exhibit increased protein kinase Akt signaling, greater senescence, and impaired vascular network formation and proliferation. Indeed, Per2 m/m mice have impaired blood flow recovery and developed autoamputation of the distal limb when subjected to hind-limb ischemia. Furthermore, matrigel implantation into Per2 m/m mice resulted in less neovascularization. Because EPCs contribute to angiogenesis, we studied the role of Per2 in these cells using bone marrow transplantation. Basal EPC levels were similar between wild-type and Per2 m/m mice. However, compared with wild-type bone marrow transplantation mice, EPC mobilization was impaired in Per2 m/m bone marrow transplantation mice in response to ischemia or VEGF stimulation. Bone marrow transplantation or infusion of wild-type EPC restored blood flow recovery and prevented autoamputation in Per2 m/m mice. Conclusion— These findings indicate that mutation of Per2 causes Akt-dependent senescence and impairs ischemia-induced revascularization through the alteration of EPC function.

The anaplastic lymphoma kinase (ALK) is an oncogene product involved in hematopoietic and non-hematopoietic malignancies. Recent studies have demonstrated that nucleophosmin (NPM)-ALK, originated from the fusion of NPM and ALK genes, causes cell transformation through diverse mechanisms. Here, we show a novel mechanism by which NPM-ALK transforms lymphoid tumor cells to become resistant to glucocorticoid (GC) or dexamethasone (Dex) treatment. Transformed BaF3 cells by NPM-ALK were much more resistant to Dex compared with their parental cells, and concurrently had a constitutive activation of mammalian target of rapamycin (mTOR) signaling, as evidenced by hyperphosphorylation of its downstream effectors, p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The mTOR inhibitor rapamycin suppressed activation of p70S6K in BaF3/NPM-ALK cells and reversed GC resistance by synergistically inhibiting mTOR signaling pathway, enhancing cell cycle arrest at G(1) phase and promoting apoptotic cell death. In conclusion, our data indicate that the ALK fusion kinase, NPM-ALK, induces GC resistance by activating mTOR signaling, and addition of mTOR inhibitors to the chemotherapeutic regimen of ALK+ lymphomas may improve the prognosis.

Although Rho-associated kinase (ROCK) activity has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of ROCKs in the vascular response to injury are not known. To address the role of ROCKs in this process, we generated haploinsufficient Rock1 (Rock1(+/-)) and Rock2 (Rock2(+/-)) mice and performed carotid artery ligations. Following this intervention, we found reduced neointima formation in Rock1(+/-) mice compared with that of WT or Rock2(+/-) mice. This correlated with decreased vascular smooth muscle cell proliferation and survival, decreased levels proinflammatory adhesion molecule expression, and reduced leukocyte infiltration. In addition, thioglycollate-induced peritoneal leukocyte recruitment and accumulation were substantially reduced in Rock1(+/-) mice compared with those of WT and Rock2(+/-) mice. To determine the role of leukocyte-derived ROCK1 in neointima formation, we performed reciprocal bone marrow transplantation (BMT) in WT and Rock1(+/-) mice. Rock1(+/-) to WT BMT led to reduced neointima formation and leukocyte infiltration following carotid ligation compared with those of WT to WT BMT. In contrast, WT to Rock1(+/-) BMT resulted in increased neointima formation. These findings indicate that ROCK1 in BM-derived cells mediates neointima formation following vascular injury and suggest that ROCK1 may represent a promising therapeutic target in vascular inflammatory diseases.

Alpha-actinins are critical components of the actin cytoskeleton. Here we show that alpha-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). Alpha-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express alpha-actinin-1 and -4, alpha-actinin-4 interacted and colocalized with eNOS. Addition of alpha-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of alpha-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of alpha-actinin-4 by siRNA increased eNOS activity in BAECs. The alpha-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on alpha-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased alpha-actinin-4-eNOS interaction, leading to translocation of alpha-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced alpha-actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with alpha-actinin-4 and calmodulin.

Photoacoustic (PA) imaging has been used mainly for anatomical and functional imaging. Although functionalized nanoparticles also have been developed for PA molecular imaging, only single targeting has been demonstrated. In this study, PA imaging of multiple targets using gold nanorods is demonstrated experimentally using HER2 and CXCR4 as target molecules. The two corresponding monoclonal antibodies were conjugated to two types of gold nanorod with different aspect ratios. Gold nanorods with mean aspect ratios of 5.9 and 3.7 exhibited peak optical absorptions at 1000 and 785 nm, respectively. Appropriate selection of laser irradiation wavelength enhances PA signals by 7-12 dB and allows signals from gold nanorods corresponding to specific bindings to be distinguished. This approach potentially allows the expression levels of different oncogenes of cancer cells to be revealed simultaneously.

The Rho kinase (ROCK) isoforms, ROCK1 and ROCK2, were initially discovered as downstream targets of the small GTP-binding protein Rho. Because ROCKs mediate various important cellular functions such as cell shape, motility, secretion, proliferation, and gene expression, it is likely that this pathway will intersect with other signaling pathways known to contribute to cardiovascular disease. Indeed, ROCKs have already been implicated in the regulation of vascular tone, proliferation, inflammation, and oxidative stress. However, it is not entirely clear how ROCKs are regulated, what some of their downstream targets are, and whether ROCK1 and ROCK2 mediate different cellular functions. Clinically, inhibition of ROCK pathway is believed to contribute to some of the cardiovascular benefits of statin therapy that are independent of lipid lowering (ie, pleiotropic effects). To what extent ROCK activity is inhibited in patients on statin therapy is not known, but it may have important clinical implications. Indeed, several pharmaceutical companies are already actively engaged in the development of ROCK inhibitors as the next generation of therapeutic agents for cardiovascular disease because evidence from animal studies suggests the potential involvement of ROCK in hypertension and atherosclerosis.

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS−/–) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS−/– mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS−/– mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Mammalian cells respond to bacterial lipopolysaccharide (LPS) through a cognate receptor: Toll-like receptor 4 (TLR4). The signaling pathways, which link TLR4 to the proinflammatory transcription factor nuclear factor κB (NF-κB), occur through the intracellular docking proteins MyD88 and Trif. We hypothesize that unlike antigen-presenting cells, vascular endothelial cells (ECs) lack the Trif protein TRAM and are therefore incapable of eliciting Trif-dependent immune responses to LPS. Stimulation of wild-type mice with LPS leads to the activation of NF-κB in ECs and macrophages in vitro and in vivo. In contrast to macrophages, LPS did not activate endothelial NF-κB or NF-κB–dependent genes in MyD88 −/− mice, suggesting the absence of a functional Trif pathway in vascular ECs. Indeed, the Trif-dependent gene cxcl10 was not expressed in ECs after LPS stimulation. This correlated with diminished expression of the Trif accessory TIR protein TRAM in ECs. Overexpression of TRAM cDNA in ECs reconstituted LPS-induced Trif-dependent NF-κB activation and cxcl10 promoter activity. The functional absence of TRAM in vascular ECs restricts TLR4 signaling to MyD88-dependent pathway. This is in contrast to macrophages, which respond to LPS via both Trif- and MyD88-dependent pathways. These findings indicate that vascular ECs do not express the Trif-dependent gene subset. This implies that these genes may be dispensable for the endothelial response to bacterial infection and play no role in the endothelial contribution to the development of atherosclerosis.

Rho-associated kinases (ROCKs), the immediate downstream targets of RhoA, are ubiquitously expressed serine-threonine protein kinases that are involved in diverse cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, and gene expression. Recent studies have shown that ROCKs may play a pivotal role in cardiovascular diseases such as vasospastic angina, ischemic stroke, and heart failure. Indeed, inhibition of ROCKs by statins or other selective inhibitors leads to the upregulation and activation of endothelial nitric oxide synthase (eNOS) and reduction of vascular inflammation and atherosclerosis. Thus inhibition of ROCKs may contribute to some of the cholesterol-independent beneficial effects of statin therapy. Currently, two ROCK isoforms have been identified, ROCK1 and ROCK2. Because ROCK inhibitors are nonselective with respect to ROCK1 and ROCK2 and also, in some cases, may be nonspecific with respect to other ROCK-related kinases such as myristolated alanine-rich C kinase substrate (MARCKS), protein kinase A, and protein kinase C, the precise role of ROCKs in cardiovascular disease remains unknown. However, with the recent development of ROCK1- and ROCK2-knockout mice, further dissection of ROCK signaling pathways is now possible. Herein we review what is known about the physiological role of ROCKs in the cardiovascular system and speculate about how inhibition of ROCKs could provide cardiovascular benefits.

We aimed to clarify responsiveness to angiotensin (Ang) II in the porcine basilar artery and the role of Ang II receptor subtypes by functional, radioligand binding, and cell culture studies. Ang II induced more potent contractions in the proximal part than in the distal part of isolated porcine basilar arteries. The contraction induced by Ang II was inhibited by the Ang II type 1 (AT1) receptor antagonist losartan, but the Ang II type 2 (AT2) receptor antagonist PD123319 enhanced it. After removal of the endothelium, the effect of losartan remained but the effect of PD123319 was abolished. The specific binding site of [3H]Ang II on the smooth muscle membrane was inhibited by losartan, but not by PD123319. Stimulation of angiotensin II increased nitric oxide (NO) production in cultured basilar arterial endothelial cells. This production was inhibited by PD123319 and the NO synthase inhibitor L–NG-nitroarginine. These results suggest that the contraction induced by Ang II might be mediated via the activation of AT1 receptors on the basilar arterial smooth muscle cells and be modulated via the activation of AT2 receptors on the endothelial cells, followed by NO production.

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 +/− haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice. Methods and Results— Litters of ROCK1 −/− mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1 +/− mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1 +/− mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg −1 · min −1 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 +/− mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1 +/− mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1 +/− 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.