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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.

Charles Darwin viewed eyes as the epitome of evolution by natural selection, describing them as organs of extreme perfection and complication. The visual system is therefore fertile ground for teaching fundamental concepts in optics and biology, subjects with scant representation during the rise and spread of immersive technologies in K-12 education. The visual system is an ideal topic for three-dimensional (3D) virtual reality learning environments (VRLEs), and here we describe a 3D VRLE that simulates the vision of a tarsier, a nocturnal primate that lives in southeast Asia. Tarsiers are an enduring source of fascination for having enormous eyes, both in absolute size and in proportion to the size of the animal. Our motivation for developing a tarsier-inspired VRLE, or Tarsier Goggles, is to demonstrate the optical and selective advantages of hyperenlarged eyes for nocturnal visual predation. In addition to greater visual sensitivity, users also experience reductions in visual acuity and color vision. On a philosophical level, we can never know the visual world of another organism, but advances in 3D VRLEs allow us to try in the service of experiential learning and educational outreach.

The main objective of this study was to determine the genetic diversity of Xinjiang wild apricot germplasm resources, and lay the foundation for the development and utilization of apricot resources. A total of 61 accessions of Daxigou wild apricot were collected as the research material in Huocheng, Xinjiang. The external morphological characters of the floral organs were observed. The data processing software DPS7.05 was used to calculate the diversity index and for principal component analysis, and MEGA3.0 software was used for cluster analysis. We analyzed the wild apricot flower organs for differences in 17 phenotypic traits. The average diversity index of the Daxigou wild apricot flower organs, which showed abundant morphological diversity, was 5.8872. The diversity of the morphological characters with numeric indices was better than that of the characters with non-numeric indices. The coefficient of variation of the morphological characters of flower organs varied from 6.25 to 97.93%. Through multivariate principal component analysis, we determined that the first seven principal components of the 17 traits represented most of the information on organ morphology of Daxigou wild apricot; the cumulative contribution rate was 72.6815%. Based on the clustering analysis of morphological characters, 61 wild apricot germplasm resources were finally gathered into one class. The diversity of the floral traits of Daxigou wild apricot was greater. The corolla diameter, ovary size, petal size, anther size, petal color, pedicel length, petal shape, and pollen quantity were the main factors contributing to the diversity of Daxigou wild apricot.

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.

Introduction Detection of recurrent genetic abnormalities is of great significance for a refined diagnosis and assessment of prognosis in leukemia. Conventional nested reverse transcription PCR is labor intensive and time-consuming. Methods We have developed a novel dual-color TaqMan probe-based real-time PCR method for the simultaneous screening of 45 fusion transcripts in 12 parallel reactions. The method was tested and validated with cell lines carrying known fusion transcripts and patient samples. Results A multiplex real-time PCR method was successfully developed for rapid detection of 45 fusion genes and validated for 15 of the more commonly detected fusion genes. Intra-assay reproducibility assessed for the most frequent rearrangements ranged from 0.41% to 0.74% for the coefficient of variation (CV) of cycle threshold (Ct) and the interassay reproducibility ranged from 1.62% to 2.83% in five separate experiments. The lowest detection limit for the translocations tested ranged between 1 : 16 000 and 1 : 32 000. Validation of the method with 213 patient samples showed 100% specificity and excellent consistence with conventional nested RT-PCR. Conclusion Overall, we believe that this method is easily applicable, cost-effective, and clinically useful for a rapid screening of fusion genes in the initial diagnostic phase of leukemia. Its use can also be extended to the monitoring of minimal residual disease.

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.

The epidemiology of candidaemia varies between hospitals and geographic regions. Although there are many studies from Asia, a large-scale cross-sectional study across Asia has not been performed. We conducted a 12-month, laboratory-based surveillance of candidaemia at 25 hospitals from China, Hong Kong, India, Singapore, Taiwan and Thailand. The incidence and species distribution of candidaemia were determined. There were 1601 episodes of candidaemia among 1.2 million discharges. The overall incidence was 1.22 episodes per 1000 discharges and varied among the hospitals (range 0.16–4.53 per 1000 discharges) and countries (range 0.25–2.93 per 1000 discharges). The number of Candida blood isolates and the total number of fungal isolates were highly correlated among the six countries ( R ² = 0.87) and 25 hospitals ( R ² = 0.77). There was a moderate correlation between incidence of candidaemia and the intensive care unit (ICU)/total bed ratio ( R ² = 0.47), although ICUs contributed to only 23% of candidaemia cases. Of 1910 blood isolates evaluated, Candida albican s was most frequently isolated (41.3%), followed by Candida tropicalis (25.4%), Candida glabrata (13.9%) and Candida parapsilosis (12.1%). The proportion of C. tropicalis among blood isolates was higher in haemato-oncology wards than others wards (33.7% versus 24.5%, p 0.0058) and was more likely to be isolated from tropical countries than other Asian countries (46.2% versus 18.9%, p 0.04). In conclusion, the ICU settings contribute, at least in part, to the incidence variation among hospitals. The species distribution is different from Western countries. Both geographic and healthcare factors contribute to the variation of species distribution.

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.

Recent animal and human studies have demonstrated the importance of the ROCK (RhoA/Rho-associated kinase) pathway in IsST (ischaemic stroke). Whether the genetic variation within ROCK-associated genes modulates the risk of IsST remains elusive. The association between 66 tSNPs [tagging SNPs (single nucleotide polymorphisms)] of three ROCK-associated genes [ROCK1, ROCK2 and ARHGEF10 (Rho guanine-nucleotide-exchange factor 10)] and the incidence of IsST was investigated in 23294 Caucasian female participants of the prospective WGHS (Women's Genome Health Study). All were free of known cancer and cardiovascular disease at baseline. During a 15-year follow-up period, 323 participants developed their first ever IsST. Multivariable Cox regression analysis was performed to investigate the relationship between genotypes and risk of IsST assuming an additive genetic model. Haplotype-block analysis was also performed. A total of ten tSNPs were associated with the risk of IsST (three in ARHGEF10 and seven in ROCK1; P

SummaryChanges in the severity of pulp browning, endogenous abscisic acid (ABA) and ethylene concentrations, and related gene expression in peach (Prunus persica L. ‘Bayuecui’) fruit stored at the biological freezing-point temperature (BFT; 1oC), at 4oC, or at room temperature (20oC; as a control) were investigated.The results showed that fruit stored at room temperature showed climacteric changes in ethylene and ABA concentrations and in levels of expression of the corresponding ethylene synthetase genes (PpACS1, PpACO1) and the gene for a key enzyme of ABA synthesis (PpNCED), concomitant with a significant decrease in fruit firmness and an increase in juice yield. Meanwhile, two sucrose synthase genes (PpSUS1 and PpSUS2) were expressed at relatively low levels. However, fruit firmness decreased slightly and chilling injury (CI) increased significantly 40 d after storage (DAS) at 4oC, with a significant accumulation of proline [from 8.33 µg g–1 (at t = 0 d) to 9.125 µg g–1 (at t = 40 d)]. In contrast, fr...

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.

Background and Rationale: The actin cytoskeleton directs many pro-fibrotic cellular responses in pulmonary fibrosis, including epithelial cell apoptosis, endothelial cell barrier disruption and fibroblast migration and differentiation. The Rho kinases ROCK1 and ROCK2 regulate the actin cytoskeleton, making them logical targets for anti-fibrotic therapy. We used a genetic approach to identify the …

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.

Isoetes sinensis is a critically endangered quillwort. To facilitate studies on the conservation genetics of this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 50,063 unigenes were predicted by transcriptome sequencing, 5294 (10.6%) of which significantly matched 3011 Gene Ontology annotations and 2363 were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways. Most of these (2297) were involved in metabolism. A total of 1982 SSR motifs were identified, with trinucleotides being the dominant repeat motif, and 1438 (72.6%) SSR primers were designed. Eighteen randomly selected primer pairs were used to genotype 24 I. sinensis accessions, which confirmed the suitability of these novel markers for molecular studies of I. sinensis. The heterozygosity index value ranged between 0.0799 and 0.9106, while the Shannon-Wiener diversity index value ranged between 0.1732 and 2.5589. The EST-SSRs reported in this study are linked to genic sequences, and are therefore ideal for investigating the evolutionary history of I. sinensis. These markers, together with the large EST dataset generated in this study, will greatly facilitate conservation genetic studies of I. sinensis.

Background: Left ventricular hypertrophy (LVH) is an independent risk factor for heart failure and sudden death. In addition, LVH is also a compensatory mechanism that helps the heart cope with pressure overload. Stress is considered one factor that is related to cardiac outcomes. Glucocorticoids are primary stress hormones, whose role in the heart is poorly understood. Here, we hypothesize that a reduction in the expression of the glucocorticoid receptor (GR) would decrease cardiac hypertrophy in response to pressure overload. Methods and Results: The GR homozygous mutation (GR-/-) is embryonic lethal. However, GR heterozygous mice (GR+/-) show a normal phenotype. We subjected GR+/- mice to transverse aortic constriction (TAC). At four weeks after TAC, the ratio of heart weight to tibia length increased significantly in wild-type mice (control) littermates compared with GR+/- mice. Cardiac myocyte size was also smaller in GR+/- mice vs controls, suggesting an attenuated cardiac growth response in these mice. In addition, GR+/- hearts displayed increased cell death and enhanced fibrosis in response to TAC. Cardiac function, determined by EF% and FS% (measured using the Vevo2100 imaging system), was significantly reduced in GR+/- mice compared with controls at eight weeks post-operation, while LVEDD was increased. Together, with the increased ratio of lung weight to body weight in GR+/- mice at eight weeks following TAC, this suggests an exaggerated heart failure in GR+/- mice. In vitro, hydrocortisone-induced cell growth in H9c2 cells was abolished by GR knockdown using siRNA. Finally, we looked at the mechanisms by which GR may play a role in the development of hypertrophy. We found reduced ERK-JNK activity in GR+/- hearts, suggesting that the reduced hypertrophic response in GR+/- mice occurs, at least partially, through abolished JNK and ERK activity. Conclusion: The glucocorticoid receptor is required for cardiac hypertrophy and protects the heart from heart failure during cardiac pressure overload.

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.

Davidia involucrata, reputed to be a "living fossil" in the plant kingdom, is a relict tree endemic to China. Extant natural populations are diminishing due to anthropogenic disturbance. In order to understand its ability to survive in a range of climatic conditions and to design conservation strategies for this endangered species, we developed genic simple sequence repeats (SSRs) from mRNA transcripts. In total, 142,950 contigs were assembled. Of these, 30,411 genic SSR loci were discovered and 12,208 primer pairs were designed. Dinucleotides were the most common (77.31%) followed by trinucleotides (16.44%). Thirteen randomly selected primers were synthesized and validated using 24 individuals of D. involucrata. The markers displayed high polymorphism with the number of alleles per locus ranging from 3 to 12 and the observed and expected heterozygosities ranging from 0.083 to 1.0 and 0.102 to 0.69, respectively. This large expressed sequence tag dataset and the novel SSR markers will be key tools in comparative studies that may reveal the adaptive evolution, population structure, and resolve the genetic diversity in this endangered species.

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.

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.

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.

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.

Increased plasma total homocysteine (tHcy) and high sensitivity C-reactive protein (hsCRP) levels are independent risk factors for cardiovascular disease. However, the predictive value of tHcy in combination with hsCRP in patients with stroke is not known. To determine the relationship between tHcy and hsCRP, we enrolled 291 patients with first-onset stroke (196 ischemic and 95 hemorrhagic). Plasma tHcy and hsCRP levels were measured and subsequent vascular events and deaths were determined over a 5-year period. Using the arbitrary cutoff for tHcy (3 mg/L), the patients were divided into 6 groups. Survival analysis showed that the probability of death or new vascular events during a 5-year follow-up increased according to tHcy and hsCRP levels (P3 mg/L) compared with those with low tHcy (3 mg/L) was still significantly associated with the risk of death or new vascular events (RR, 4.10, 95% CI, 1.61 to 10.45, P=0.003) even when adjusted for other risk factors at inclusion. The combination of increased tHcy and hsCRP levels had a stronger predictive value than increased hsCRP alone or increased tHcy level alone. Further studies are required to evaluate the potential decrease in risks associated with lowering both Hcy and hsCRP levels in patients that present with both increased tHcy and hsCRP.

Acute cerebral ischemia elicits an innate immune response that leads to a cascade of events that culminates in necrotic death of neurons and injury to their supportive structures in the neurovascular unit. Indeed, clinical studies have shown a close relationship between elevated levels of inflammatory markers and the risk for ischemic stroke. However, the signaling pathways that link these events are not well understood. A central regulator of inflammatory response is the transcription factor, nuclear factor-kappa B (NF-κB). The activation of NF-κB is required for the transcriptional induction of many proinflammatory mediators involved in innate immunity, such as cellular adhesion molecules, cytokines, and growth factors. Therefore, factors that modulate the activity of NF-κB could potentially regulate inflammatory processes in ischemic stroke. Here, we review the relationship between NF-κB and ischemic stroke, its role in the neurovascular unit, and discuss some animal models that suggest that this relationship is causal.

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.

Background Increased sympathetic nervous system activity is commonly observed in heart failure with reduced ejection fraction and is one cause of pathological structural remodeling of the left ventricle. Sacubitril/valsartan reduces mortality and heart failure hospitalizations, but its effect on sympathetic nervous system activity is unknown. Methods Patients enrolled in the prospective, longitudinal REMODEL study underwent 123-meta-iodobenzylguanidine scintigraphy prior to and three months following conversion from angiotensin converting enzyme inhibitor or angiotensin receptor blocker therapy to sacubitril/valsartan. The early heart-to-mediastinum (H/M) ratio, late H/M ratio and washout rate were calculated. Baseline and on-therapy values were compared using a paired t-test analysis. Results 40 patients were enrolled in this study. The mean age was 55 ± 12 years, 25 (63%) were male, and 30 (75%) had a non-ischemic heart failure etiology. Following transition to sacubitril/valsartan therapy, the late H/M ratio increased from 1.39 ± 0.19 to 1.52 ± 0.19 (p Conclusions Addition of sacubitril/valsartan to baseline heart failure therapy resulted in a decrease in cardiac sympathetic activity, as shown by a decrease in both the early and late H/M ratio and an increase in the washout rate on MIBG imaging. The decrease in sympathetic nervous system activity may underlie the improvement in survival seen with sacubitril/valsartan and can serve as a good measure of medication response.

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.

Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are widely used to lower serum cholesterol levels in the primary and secondary prevention of cardiovascular disease. Recent experimental and clinical evidence suggests that the beneficial effects of statins may extend beyond their cholesterol-lowering effects, to include so-called pleiotropic effects. These cholesterol-independent effects include improving endothelial function, attenuating vascular and myocardial remodeling, inhibiting vascular inflammation and oxidation, and stabilizing atherosclerotic plaques. The mechanism underlying some of these pleiotropic effects is the inhibition of isoprenoid synthesis by statins, which leads to the inhibition of intracellular signaling molecules Rho, Rac and Cdc42. In particular, inhibition of Rho and one of its downstream targets, Rho kinase, may be a predominant mechanism contributing to the pleiotropic effects of statins. The aim of the present review is to provide an update on the non-cholesterol-dependent statin effects in the cardiovascular system and highlight some of the recent findings from bench to bedside to support the concept of statin pleiotropy.

Background: In addition to inhibiting cholesterol synthesis, statins (HMG-CoA reductase inhibitors) decrease the formation of isoprenoid intermediates required for the activation of key signaling pathways, including Rho/Rho kinase (ROCK). In experimental settings, statins inhibit ROCK and reverse vascular dysfunctions in atherosclerosis, independent of cholesterol reduction. It is not known whether statins inhibit ROCK activity in humans with atherosclerosis. Methods: We investigated 35 patients with stable atherosclerosis in a randomized, double-blind study comparing treatment with high-dose (80 mg/d) or low-dose (10 mg/d) atorvastatin to placebo for 28 days. Blood samples for leukocyte ROCK activity, fasting lipids, and high-sensitivity C-reactive protein (hs-CRP) were obtained on days 0, 7, 14, and 28 after randomization and change over time with the two statin treatments relative to placebo was examined. Results: Atorvastatin 80 mg/d reduced ROCK activity (p = 0.002 vs. placebo). This decline was rapid and significant within 2 weeks of treatment. The inhibition of ROCK by atorvastatin (80 mg/d) remained significant even after controlling for changes in low-density lipoprotein cholesterol (LDL-C) and triglycerides (p = 0.01). Furthermore, there was no correlation between changes in ROCK activity and changes in LDL-C (r = 0.2, p = 0.25) or triglycerides (r =0 .1,p = 0.55). There was a modest correlation between ROCK inhibition and change in hs-CRP among patients randomized to atorvastatin 80 mg/d (r = 0.6, p = 0.07). Conclusions: These first-in-man findings demonstrate that high-dose atorvastatin rapidly inhibits the proatherogenic Rho/ROCK pathway, independent of cholesterol reduction. This inhibition may contribute to the clinical benefits of statins. Rho/ROCK may provide a useful therapeutic target in patients with

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.

Wild European plum (Prunus xdomestica L.) is a rare and endangered species in China. To conserve this germplasm, a protocol for in vitro propagation of this species was developed. Nodal segments were used as the explants in our study. For the initiation of culture, B5 medium was better than Murashige and Skoog (MS) medium, which showed rapid growth of axillary bud within 3 days. Shoot organogenesis could be induced from both 6-benzylaminopurine (BAP) with α-naphthalene acetic acid (NAA) and BAP with indole-3-butyric acid (IBA) in B5 medium, but the regeneration efficiency depended on the ratio and concentration used. The higher regeneration efficiency existed in BAP:NAA=3-5:1 and BAP:IBA=1-3:1, when BAP concentration was in 0.5-1.0 mg/L. Rooting efficiency could be greatly increased with the addition of 0.4 mg/L IBA and no callus was observed on shoots in rooting media. Rooted plantlets were successfully acclimatized and grown in different transplanting medium. Plantlets in sawdust show higher survival rate than in soil and in vermiculite. This new system proved to be a practical way for germplasm conservation and for further investigation of wild European plum.

Background— Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. Methods and Results— We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1 +/− ), SMC-specific Notch1 heterozygous deficient (smN1 +/− ), and general Notch3 homozygous deficient (N3 −/− ) mice. Compared with wild-type or control mice, N1 +/− and smN1 +/− mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3 −/− mice. Indeed, SMCs derived from explanted aortas of either N1 +/− - or smN1 +/− mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3 −/− mice. This correlated with decreased staining of proliferating cell nuclear antigen–positive cells and increased staining of cleaved caspase-3 in the intima of N1 +/− - or smN1 +/− mice. In SMCs derived from CHF1/Hey2 −/− mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. Conclusions— These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases.

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.

Bradykinin 2 receptor (B2R) deficiency predisposes to cardiac hypertrophy and hypertension. The pathways mediating these effects are not known. Two-month-old B2R knockout (KO) and wild-type (WT) mice were assigned to 4 treatment groups (n = 12–14/group): control (vehicle); nitro-l-arginine methyl ester (l-NAME) an NO synthase inhibitor; simvastatin (SIM), an NO synthase activator; and SIM+l-NAME. Serial echocardiography was performed and blood pressure (BP) at 6 weeks was recorded using a micromanometer. Myocardial eNOS and mitogen-activated protein kinase (MAPK, including ERK, p38, and JNK) protein expression were measured. Results showed that (i) B2RKO mice had significantly lower ejection fraction than did WT mice (61% ± 1% vs. 73% ± 1%), lower myocardial eNOS and phospho-eNOS, normal systolic BP, and higher LV mass, phospho-p38, and JNK; (ii) l-NAME increased systolic BP in KO mice (117 ± 19 mm Hg) but not in WT mice and exacerbated LV hypertrophy and dysfunction; and (iii) in KO mice, SIM decreased hypertrophy, p38, and JNK, improved function, increased capillary eNOS and phospho-eNOS, and prevented l-NAME-induced LV hypertrophy without lowering BP. We conclude that disruption of the B2R causes maladaptive cardiac hypertrophy with myocardial eNOS downregulation and MAPK upregulation. SIM reverses these abnormalities and prevents the development of primary cardiac hypertrophy as well as hypertrophy secondary to l-NAME-induced hypertension.