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Background: Despite recent progress in treatments for secondary prevention, ischemic stroke recurs in 8% to 12% of stroke survivors. We investigated the predisposing factors associated with recurrence within the first 12 months after an ischemic event to explore more effective preventive strategies., Methods: Between June 2007 and April 2008, acute (within 7 days of onset) ischemic stroke patients were registered in the Fukuoka Stroke Registry (FSR), a multicenter, prospective, observational database. The clinical characteristics on admission were analyzed, and the patients were followed for 12 months., Results: Two hundred sixty patients (151 males and 109 females, 71 ± 11 years of age) were registered; 25 (9.6%) had recurrence of ischemic stroke during the follow-up period. Kaplan-Meier curve analysis revealed a significant difference in recurrence-free survival between patients with high-density lipoprotein (HDL) cholesterol <40 mg/dL on admission and those with HDL cholesterol ≥ 40 mg/dL (P = .042). Adjusted multivariate logistic regression analysis showed that age (odds ratio 1.06; 95% CI, 1.00-1.11; P = .035) and HDL cholesterol <40 mg/dL (odds ratio 2.73; 95% CI, 1.01-7.38; P = .048) on admission were independently associated with a recurrence of ischemic stroke within 12 months of the initial onset., Conclusions: Aging and low HDL cholesterol levels are considered independent risk factors for a recurrence of ischemic stroke., (Copyright © 2012 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Neurotrophins are crucial regulators of neuronal survival and death. Evidence suggests that cells comprising the neurovascular unit (NVU) cooperatively mediate neuronal development, survival and regeneration. The aim of this study was to test whether cerebrovascular cells, endothelial cells and pericytes, produce neurotrophins and play neuroprotective roles during hypoxic insults. We examined the expression of neurotrophins and their receptors in cultured human cerebral microvascular endothelial cells and pericytes, astrocytes and the rat neuronal cell line PC12. Differentiated PC12 cells expressed TrkA, the NGF receptor, which was significantly upregulated by hypoxia at 1% O(2) and regulated neuronal survival. Both pericytes and astrocytes expressed three neurotrophins, i.e. NGF, BDNF and NT-3, while TrkB and TrkC, specific receptors for BDNF and NT-3, were expressed in astrocytes, but not pericytes. In response to hypoxia, among the neurotrophins expressed in pericytes and astrocytes only NT-3 expression was significantly upregulated in pericytes. Treatment of astrocytes with NT-3 significantly activated Erk1/2 and increased the expression of NGF both at mRNA and protein levels. The MEK1 inhibitor U0126 or siRNA-mediated knockdown of TrkC abolished the NT-3-induced upregulation of NGF in astrocytes. Taken together, cerebral microvascular pericytes and astrocytes are potent producers of neurotrophins in the NVU. In response to hypoxia, pericytes increase NT-3 production, which induces astrocytes to increase NGF production through the TrkC-Erk1/2 pathway. The interplay between pericytes and astrocytes through neurotrophins in the NVU may play an important role in neuronal survival under hypoxic conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Brain pericytes regulate a variety of functions, such as microcirculation, angiogenesis, and the blood brain barrier in the brain. Recent studies have also shown that they are pluripotent in a manner similar to mesenchymal stem cells. Since, brain pericytes actively control these functions, these cells probably play an important role not only during brain ischemia, but also in the post-stroke period.

Background: The prognosis in ischemic stroke is poor in diabetic patients. However, scant research has so far been done on the predisposing factors associated with poor outcomes., Methods: We prospectively investigated the background characteristics and prognosis at 3 months in 241 consecutive diabetic patients having their first ischemic stroke (153 males, 88 females, mean age ± SD, 71 ± 10 years). Poor functional outcome was defined as modified Rankin scale ≥ 3 at 3 months after onset., Results: Univariate analysis showed that age, dementia, initial National Institutes of Health Stroke Scale (NIHSS) score, systolic blood pressure on admission, proteinuria, stroke subtype, and prior use of angiotensin receptor blocker (ARB) were significantly related to an outcome at 3 months after onset. A multivariate logistic regression analysis showed that age (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.01 to 1.13, p=0.017, per 1-year increase), NIHSS score (OR 1.22, 95% CI 1.12 to 1.35, p<0.001, per 1-score increase), and proteinuria (OR 4.22, 95% CI 1.71 to 10.92, p=0.002) were significantly and independently associated with poor clinical outcome after ischemic stroke in diabetic patients. Conversely, prior use of ARB (OR 0.28, 95% CI 0.09 to 0.79, p=0.023) was associated with a better outcome., Conclusions: In diabetic patients, proteinuria was independently associated with a poor clinical outcome after ischemic stroke, whereas the prior use of ARB appeared to be beneficial., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Platelet derived growth factor (PDGF)-B plays a neuroprotective role in brain damages, including ischemic stroke. It has been suggested recently that PDGF receptor β (PDGFRβ) expressed in brain pericytes as well as in neurons and astrocytes may mediate the neuroprotective role of PDGF-B. The aims of this study were to elucidate the roles of PDGFRβ signaling in brain pericytes after ischemic stroke. In a rat middle cerebral artery occlusion (MCAO) model, PDGFRβ expression was induced specifically in the pericytes in peri-infarct areas and its level was gradually increased. PDGF-B induced marked phosphorylation of Akt in cultured brain pericytes. Consistently, PDGF-B was upregulated in endothelial cells in per-infarct areas and Akt was strongly phosphorylated in the PDGFRβ-expressing pericytes in periinfarct areas after MCAO. In the cultured pericytes, PDGF-B induced cell growth and anti-apoptotic responses through Akt. Furthermore, PDGF-B significantly increased the expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3) through Akt in the pericytes. Thus, the PDGFRβ-Akt signaling in brain pericytes may play various important roles leading to neuroprotection after ischemic stroke.

Background: Recurrent strokes occur more frequently during the first year after an ischemic stroke. We investigated the relationship between patient clinical characteristics and stroke recurrence according to the non-cardioembolic type of ischemic stroke, and determined the predisposing factors associated with a recurrence within the first year., Methods: From June 2007 to December 2008, 1,106 consecutive ischemic stroke patients who were hospitalized in the 7 FSR stroke centers within 7 days after the onset of their stroke were enrolled in this study. We assessed the clinical characteristics of the patients on admission, and followed their clinical courses for one year., Results: Of all patients, 876 (537 males and 339 females, 70 ± 12 years of age) who had suffered from a non-cardioembolic stroke were investigated. Seventy-one patients (8.1%) suffered from a recurrence of ischemic stroke during the follow-up period of one year. On multivariate Cox hazard regression analyses, age (HR 1.03, 95% CI 1.00-1.05, p = 0.030, per 1-year increase), high-density lipoprotein (HDL) cholesterol <40 mg/dl (HR 1.89, 95% CI 1.10-3.24, p = 0.021), and chronic kidney disease (CKD) (HR 1.73, 95% CI 1.03-2.90, p = 0.038) were independent predictors of a recurrence within one year after the non-cardioembolic stroke., Conclusions: In the patients demonstrating non-cardioembolic ischemic stroke, low HDL cholesterol levels and CKD in addition to aging were independent risk factors for a recurrence within one year after the onset., (Copyright © 2011 S. Karger AG, Basel.)

Background and Purpose: Diabetes mellitus is an established risk factor for stroke. However, it is uncertain whether prestroke glycemic control (PSGC) status affects clinical outcomes of acute ischemic stroke. The aim of this study was to elucidate the association between PSGC status and neurological or functional outcomes in patients with acute ischemic stroke., Methods: From the Fukuoka Stroke Registry (FSR), a multicenter stroke registry in Japan, 3627 patients with first-ever ischemic stroke within 24 hours after onset were included in the present analysis. The patients were categorized into 4 groups based on their PSGC status: excellent (hemoglobin [Hb] A1c on admission<6.2%), good (6.2-6.8%), fair (6.9-8.3%) and poor (≥8.4%). Study outcomes were neurological improvement (≥4 points decrease in the National Institutes of Health Stroke Scale [NIHSS] score during hospitalization or 0 points on NIHSS score at discharge), neurological deterioration (≥1 point increase in NIHSS score) and poor functional outcome (death or dependency at discharge, modified Rankin Scale 2-6)., Results: The age- and sex-adjusted ORs for neurological improvement were lower, and those for neurological deterioration and a poor functional outcome were higher in patients with poorer PSGC status. After adjusting for multiple confounding factors, these trends were unchanged (all probability values for trends were <0.002). These findings were comparable in patients with noncardioembolic and cardioembolic infarctions., Conclusions: In ischemic stroke patients, HbA1c on admission was an independent significant predictor for neurological and functional outcomes.

Brain pericytes are an important constituent of neurovascular unit. They encircle endothelial cells and contribute to the maturation and stabilization of the capillaries in the brain. Recent studies have revealed that brain pericytes play pivotal roles in a variety of brain functions, such as regulation of capillary flow, angiogenesis, blood brain barrier, immune responses, and hemostasis. In addition, brain pericytes are pluripotent and can differentiate into different lineages similar to mesenchymal stem cells. The brain pericytes are revisited as a key player to maintain brain function and repair brain damage.

The NADPH oxidases (Nox) are transmembrane proteins dedicated to producing reactive oxygen species (ROS), including superoxide and hydrogen peroxide, by transferring electrons from NAD(P)H to molecular oxygen. Nox2 and Nox4 are expressed in the heart and play an important role in mediating oxidative stress at baseline and under stress. Nox2 is primarily localized on the plasma membrane, whereas Nox4 is found primarily on intracellular membranes, on mitochondria, the endoplasmic reticulum or the nucleus. Although Nox2 plays an important role in mediating angiotensin II-induced cardiac hypertrophy, Nox4 mediates cardiac hypertrophy and heart failure in response to pressure overload. Expression of Nox4 is upregulated by hypertrophic stimuli, and Nox4 in mitochondria plays an essential role in mediating oxidative stress during pressure overload-induced cardiac hypertrophy. Upregulation of Nox4 induces oxidation of mitochondrial proteins, including aconitase, thereby causing mitochondrial dysfunction and myocardial cell death. On the other hand, Noxs also appear to mediate physiological functions, such as erythropoiesis and angiogenesis. In this review, we discuss the role of Noxs in mediating oxidative stress and both pathological and physiological functions of Noxs in the heart., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rationale: Thioredoxin (Trx)1 inhibits pathological cardiac hypertrophy. MicroRNAs (miRNAs) are small noncoding RNAs that downregulate posttranscriptional expression of target molecules., Objectives: We investigated the role of miRNAs in mediating the antihypertrophic effect of Trx1 on angiotensin II (Ang II)-induced cardiac hypertrophy., Methods and Results: Microarray analyses of mature rodent microRNAs and quantitative RT-PCR/Northern blot analyses showed that Trx1 upregulates members of the let-7 family, including miR-98, in the heart and the cardiomyocytes therein. Adenovirus-mediated expression of miR-98 in cardiomyocytes reduced cell size both at baseline and in response to Ang II. Knockdown of miR-98, and of other members of the let-7 family, augmented Ang II-induced cardiac hypertrophy, and attenuated Trx1-mediated inhibition of Ang II-induced cardiac hypertrophy, suggesting that endogenous miR-98/let-7 mediates the antihypertrophic effect of Trx1. Cyclin D2 is one of the predicted targets of miR-98. Ang II significantly upregulated cyclin D2, which in turn plays an essential role in mediating Ang II-induced cardiac hypertrophy, whereas overexpression of Trx1 inhibited Ang II-induced upregulation of cyclin D2. miR-98 decreased both expression of cyclin D2 and the activity of a cyclin D2 3'UTR luciferase reporter, suggesting that both Trx1 and miR-98 negatively regulate cyclin D2. Overexpression of cyclin D2 attenuated the suppression of Ang II-induced cardiac hypertrophy by miR-98, suggesting that the antihypertrophic actions of miR-98 are mediated in part by downregulation of cyclin D2., Conclusions: These results suggest that Trx1 upregulates expression of the let-7 family, including miR-98, which in turn inhibits cardiac hypertrophy, in part through downregulation of cyclin D2.

It has been established that oxidative stress plays a crucial role in the development and progression of vascular diseases. Besides the mitochondria, the NADPH oxidase/Nox family proteins are now thought to be important origins of the reactive oxygen species that underlie various vascular disease states, such as hypertension, atherosclerosis, angiogenesis, and ischemia/reperfusion injury. This review summarizes the basis of vascular Nox proteins and discusses their pathophysiological roles in the vascular system.

We report a 50-year-old woman with systemic lupus erythematosus (SLE) who developed stroke-like symptoms, including disturbance of consciousness and severe left hemiparesis. Despite the stroke-like symptoms, MR imaging showed quite atypical findings for stroke; broad high-intensity areas in the right fronto-parietal lobes on diffusion-weighted images were shown mainly as iso-intensity areas on the ADC map, without any findings of stenosis of the cerebral large arteries. The cerebral blood flow in these areas was significantly decreased, as evaluated by single photon emission computed tomography. The present case suggests that small vessel vasculopathies localized in the unilateral hemisphere could cause "stroke-like" symptoms in SLE., (© 2011 The Japanese Society of Internal Medicine)

Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.

NAD(P)H oxidases (Noxs) produce O(2)(-) and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4(-/-) (c-Nox4(-/-)) mice. Nox4 expression was inhibited in c-Nox4(-/-) mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O(2)(-) in the heart, indicating that Nox4 is a significant source of O(2)(-) in cardiac myocytes. The baseline cardiac phenotype was normal in young c-Nox4(-/-) mice. In response to pressure overload (PO), however, increases in Nox4 expression and O(2)(-) production in mitochondria were abolished in c-Nox4(-/-) mice, and c-Nox4(-/-) mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c-Nox4(-/-) mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

Pathological hypertrophy is commonly induced by activation of protein kinases phosphorylating class II histone deacetylases (HDACs) and desuppression of transcription factors, such as nuclear factor of activated T cell (NFAT). We hypothesized that nifedipine, an L-type Ca(2+) channel blocker, inhibits Ca(2+) calmodulin-dependent kinase II (CaMKII) and NFAT, thereby inhibiting pathological hypertrophy. Mice were subjected to sham operation or transverse aortic constriction (TAC) for 2 weeks with or without nifedipine (10 mg/kg/day). Nifedipine did not significantly alter blood pressure or the pressure gradient across the TAC. Nifedipine significantly suppressed TAC-induced increases in left ventricular (LV) weight/body weight (BW; 5.09 +/- 0.80 vs. 4.16 +/- 0.29 mg/g, TAC without and with nifedipine, n = 6,6, p < 0.05), myocyte cross-sectional area (1,681 +/- 285 vs. 1,434 +/- 197 arbitrary units, p < 0.05), and expression of fetal-type genes, including atrial natriuretic factor (35. 9 +/- 6.4 vs. 8.6 +/- 3.3 arbitrary units, p < 0.05). TAC-induced increases in lung weight/BW (7.7 +/- 0.9 vs. 5.5 +/- 0.5 mg/g, p < 0.05) and decreases in LV ejection fraction (65.5 +/- 3.1% vs. 75.7 +/- 3.3%, p < 0.05) were attenuated by nifedipine. Nifedipine caused significant inhibition of TAC-induced activation of NFAT-mediated transcription, which was accompanied by suppression of Thr 286 phosphorylation in CaMKII. Nifedipine inhibited activation of CaMKII and NFAT by phenylephrine, accompanied by suppression of Ser 632 phosphorylation and nuclear exit of HDAC4 in cardiac myocytes. These results suggest that a subpressor dose of nifedipine inhibits pathological hypertrophy in the heart by inhibiting activation of CaMKII and NFAT, a signaling mechanism commonly activated in pathological hypertrophy.

Rationale: NADPH oxidases are a major source of superoxide (O(2)(-)) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood., Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart., Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging. Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O(2)(-) production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone- and diphenyleneiodonium-sensitive O(2)(-) production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4., Conclusions: Upregulation of Nox4 by hypertrophic stimuli and aging induces oxidative stress, apoptosis and LV dysfunction, in part because of mitochondrial insufficiency caused by increased O(2)(-) production and consequent cysteine oxidation in mitochondrial proteins.

Background and Purpose: Midkine is a heparin-binding growth factor having various biological activities including chemotaxis of inflammatory cells, angiogenesis and migration of neuronal cells. These biological activities are expected to have a great impact on the pathology of brain infarction in subacute phase. Therefore, we investigated the effect of post-ischemic gene transfer of midkine in the phase., Methods: Brain ischemia was produced by the photothrombotic distal middle cerebral artery occlusion in spontaneously hypertensive rats. We measured cerebral blood flow by laser Doppler flowmetry. At 90 min after induction of brain ischemia, adenovirus vectors encoding mouse midkine (AdMK) or enhanced green fluorescence protein (AdGFP) were injected into the lateral ventricle. At 7 days after brain ischemia, the infarct volume, angiogenesis, inflammation and neuronal regeneration were evaluated., Results: There were no differences in cerebral blood flow changes between AdMK and AdGFP groups. However, infarct volume of AdMK group was significantly smaller than AdGFP group by 33%. The vascular density, the numbers of leukocytes in blood vessels, infiltrated macrophages and proliferated neuronal precursor cells were not significantly different between both groups. Contrastingly the numbers of migrating neuronal precursor cells toward the brain infarction were significantly increased in AdMK group than AdGFP group., Conclusions: Neuroprotective effect of midkine gene transfer persisted until the subacute phase of brain infarction. Midkine may contribute to neuronal regeneration. These results suggest the usefulness of midkine gene transfer for treatment of brain infarction.

Many biological functions in cells are regulated by the effects of the redox state on cellular signaling pathways. In the heart, pathological hypertrophy caused by a wide variety of stimuli is commonly mediated by nucleo-cytoplasmic translocation of class II histone deacetylases (HDACs) and subsequent de-suppression of transcription factors, including nuclear factor of activated T-cells and MEF2. One of the primary triggers of class II HDAC nuclear export is phosphorylation by HDAC kinases activated by hypertrophic stimuli. However, oxidative modification of conserved cysteine residues can also potentially induce nuclear export of class II HDACs. Thioredoxin 1 (Trx1), a 12 kDa anti-oxidant, inhibits pathological hypertrophy through reduction of cysteine residues in class II HDACs. In this review, we discuss the role of posttranslational modification of class II HDACs in mediating cardiac hypertrophy and the molecular mechanism by which Trx1 inhibits pathological cardiac hypertrophy.

Thioredoxin 1 (Trx1) is a key redox modulator that is functionally conserved across a wide range of species, including plants, bacteria, and mammals. Using a conserved CXXC motif, Trx1 catalyzes the reduction of cysteine disulfides and S-nitrosothiols. In contrast to small molecular reductants such as glutathione and cysteine that can reduce a wide range of oxidized proteins, Trx1 reduces only selected proteins via specific protein-protein interaction. Trx1 has been shown to regulate numerous signal transduction pathways, and its dysfunctions have been implicated in several diseases, including cancer, inflammation, and neurodegenerative and cardiovascular diseases. Identification of Trx1 target proteins may help to identify novel signaling mechanisms that are important for Trx1 antistress responses. In this study, we performed an ICAT proteomics study for the identification of Trx1 target proteins from the hearts of a cardiac specific Trx1-overexpressing transgenic mouse model (Tg-Trx1). Trx1-reduced proteins were distinguished from Trx1-induced proteins by comparison of the ICAT results with those obtained using a parallel iTRAQ (isobaric tags for relative and absolute quantitation) protein expression analysis. We were able to identify 78 putative Trx1 reductive sites in 55 proteins. Interestingly we identified a few protein functional networks that had not been shown previously to be regulated by Trx1, including the creatine-phosphocreatine shuttle, the mitochondrial permeability transition pore complex, and the cardiac contractile apparatus. The results presented here suggest that in addition to a general antioxidant function, Trx1 may be involved in the coordination of a wide array of cellular functions for maintaining proper cardiac energy dynamics and facilitating muscle contraction.

The aims of the present study were to investigate the mechanisms of Ca(2+) signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca(2+) concentration was measured by means of fura-2 fluorescence. Reverse transcription and polymerase chain reaction was performed to examine the expression of mRNA. Knockdown of Na(+)/H(+) exchanger (NHE) was done by transfecting the cells with specific double-strand siRNAs for NHE. Externally applied hydrogen peroxide dose-dependently (100 microM-10 mM) increased cytosolic Ca(2+) in human CNS pericytes. Cytosolic Ca(2+) remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca(2+) to the resting level. The hydrogen peroxide-induced Ca(2+) increase was not inhibited by nicardipine, Gd(3+), La(3+), or omission of external Ca(2+). Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca(2+) rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca(2+) increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na(+)/Ca(2+) exchanger (NCX) 1, Na(+)/H(+) exchanger (NHE) 1, and NHE7. However, the removal of external Na(+), treatment with KB-R 7943 and mibefradil, or knockdown of NHE1 and NHE7 did not affect the hydrogen peroxide-induced Ca(2+) increase. Hydrogen peroxide releases Ca(2+) from intracellular Ca(2+) pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.

Oxidative modifications of protein thiols are important mechanisms for regulating protein functions. The present study aimed to compare the relative effectiveness of two thiol-specific quantitative proteomic techniques, difference gel electrophoresis (DIGE) and isotope coded affinity tag (ICAT), for the discovery of redox-sensitive proteins in heart tissues. We found that these two methods were largely complementary; each could be used to reveal a set of unique redox-sensitive proteins. Some of these proteins are low-abundant signaling proteins and membrane proteins. From DIGE analysis, we found that both NF-kappaB-repressing protein and epoxide hydrolase were sensitive to H 2O 2 oxidation. In ICAT analysis, we found that specific cysteines within sacroplasmic endoplamic reticulum calcium ATPase 2 and voltage-dependent anion-selective channel protein 1 were sensitive to H 2O 2 oxidation. From these analyses, we conclude that both methods should be employed for proteome-wide studies, to maximize the possibility of identifying proteins containing redox-sensitive cysteinyl thiols in complex biological systems.

Thioredoxin 1 (Trx1) facilitates the reduction of signaling molecules and transcription factors by cysteine thiol-disulfide exchange, thereby regulating cell growth and death. Here we studied the molecular mechanism by which Trx1 attenuates cardiac hypertrophy. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II histone deacetylases (HDACs), master negative regulators of cardiac hypertrophy. Both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 are oxidized and form intramolecular disulfide bonds in response to reactive oxygen species (ROS)-generating hypertrophic stimuli, such as phenylephrine, whereas they are reduced by Trx1. Whereas reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export, independently of its phosphorylation status. Our study reveals a novel regulatory mechanism of cardiac hypertrophy through which the nucleocytoplasmic shuttling of class II HDACs is modulated by their redox modification in a Trx1-sensitive manner.

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.

Angiotensin II type 1 receptor (AT1R) blocker (ARB) has been reported to modify hypertensive cerebrovascular changes; however, it is not clear whether its protective effects are independent of blood pressure. The aim of this study was to clarify the role of AT1R-mediated signals in cerebral circulation by the chronic treatment with telmisartan, an ARB, at a dose that did not lower the blood pressure. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were treated for 4 weeks from 16 weeks of ages with telmisartan (SHR-L: 0.3 mg/kg/day, SHR-H: 3 mg/kg/day, WKY-H: 3 mg/kg/day) or vehicle (SHR-V, WKY-V). Superoxide measured by a chemiluminescent assay or dihydroethidium fluorescence and vascular morphology were examined for the thoracic aorta (Ao), common carotid (CCA), middle cerebral (MCA) and basilar arteries (BA). After 4 weeks of treatment, the blood pressure significantly declined in SHR-H but not in SHR-L in comparison to SHR-V. The lower limit of cerebral blood flow (CBF) autoregulation, evaluated by hemorrhagic hypotension, was significantly lower in SHR-L and SHR-H than SHR-V. In both SHR and WKY, the superoxide levels in the arteries were significantly attenuated by both doses of ARB. ARB also reversed vascular hypertrophy in Ao, CCA and BA and the inward remodeling in MCA. These results suggest that chronic treatment with telmisartan may therefore improve CBF autoregulation with a restoration of the vascular structure and an attenuation of superoxide generation, even at a dose that does not lower the blood pressure.

Background and Purpose: Lymphotoxin alpha (LTA), one of the tumor necrosis factor family proteins, is an important proinflammatory cytokine and appears to play a putative role in the inflammatory process of atherosclerosis. Recent genetic studies have suggested that variations in the gene encoding LTA, which affect its expression and biological function, may contribute to the development of vascular diseases. We conducted a case-control study to clarify the association of LTA gene polymorphisms with ischemic stroke in a large Japanese population., Methods: Genotyping for LTA A252G and C804A polymorphisms was achieved by a rapid-cycle polymerase chain reaction and melting curve analysis using fluorescent probes in 1,044 incident cases of ischemic stroke recruited from the Fukuoka Stroke Registry and 1,044 age- and sex-matched control subjects recruited from the Hisayama Study., Results: The overall distribution of allele and genotype for each polymorphism was similar between stroke patients and control subjects. The allele frequencies of 252G and 804A were slightly lower in stroke patients than in control subjects; however, conditional logistic regression analysis adjusted for potential risk factors found no association between the risk of ischemic stroke and either polymorphism. In terms of stroke subtype, we also found no association of these polymorphisms with any subtypes of ischemic stroke., Conclusions: Neither the A252G nor C804A polymorphism of the LTA gene was associated with stroke overall and any subtypes of ischemic stroke in the Japanese population., ((c) 2008 S. Karger AG, Basel.)

Excessive reactive oxygen species (ROS) play an important role in the development of cardiac hypertrophy. In contrast, antioxidants scavenge ROS, thereby maintaining the reduced environment of cells and inhibiting hypertrophy in the heart. Thioredoxin1 (Trx1) not only functions as a major antioxidant in the heart but also interacts with important signaling molecules and transcription factors, thereby attenuating cardiac hypertrophy. This review will discuss the molecular mechanisms by which Trx1 exerts antihypertrophic effects in the heart.

Objective: The aims of the present study were to elucidate the interaction of reactive oxygen species (ROS) and Ca(2+) response in central nervous system (CNS) pericytes., Methods: The intracellular Ca(2+) concentration was measured using fluorescent Ca(2+) indicator, fura-2, in cultured CNS pericytes., Results: Hydrogen peroxide evoked a dose-dependent increase in cytosolic Ca(2+), which was completely inhibited by catalase. Removal of external Ca(2+) or addition of nicardipine (1 microM) during application of hydrogen peroxide did not affect Ca(2+) response. Incubation of the cells in Ca(2+) free solution did not abolish but slightly reduced Ca(2+) response by hydrogen peroxide. Ca(2+) response to hydrogen peroxide was not altered by the depletion of intracellular Ca(2+) by thapsigargin (1 microM). Pretreatment of the cells with tyrosine kinase inhibitor genistein (100 microM) or tyrphostin A47 (30 microM) significantly reduced Ca(2+) increase by hydrogen peroxide., Conclusions: These results indicate that hydrogen peroxide evokes Ca(2+) increase predominantly by release from intracellular Ca(2+) store, which may be regulated by tyrosine kinases.

Increasing bodies of evidence indicate that reactive oxygen species (ROS) produced by mitochondria and other sources play an essential role in mediating ventricular remodeling after myocardial infarction and the development of heart failure. Antioxidants scavenge ROS, thereby maintaining the reduced environment of cells and inhibiting ventricular remodeling in the heart. Thioredoxin not only functions as a major antioxidant in the heart but also interacts with important signaling molecules and transcription factors, thereby modulating various cellular functions. The activity of thioredoxin is regulated by a variety of mechanisms, such as transcription, localization, protein-protein interaction, and post-translational modification. In this review, we will summarize the cardiac effects of thioredoxin and the mechanisms by which thioredoxin mediates inhibition of ventricular remodeling.

Thioredoxin1 (Trx1) inhibits hypertrophy and exhibits protective functions in the heart. To elucidate further the cardiac functions of Trx1, we used a DNA microarray analysis, with hearts from transgenic mice with cardiac- specific overexpression of Trx1 (Tg-Trx1, n = 4) and nontransgenic controls (n = 4). Expression of a large number of genes is regulated in Tg-Trx1, with a greater number of genes downregulated, versus upregulated, at high-fold changes. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1gamma) gene was among the top 50 significantly upregulated genes. By pathway analyses, we found that genes involved in both mitochondrial oxidative phosphorylation and the TCA cycle were upregulated in Tg-Trx1. We confirmed upregulation of cytochrome c oxidase (COX) components and mitochondrial transcription factor A in Tg-Trx1. The activity of citrate synthase and COX and the cardiac ATP content were significantly higher in Tg-Trx1. A transcription factor binding-site analysis showed that upregulated genes frequently contained binding sites for nuclear respiratory factor 1 (NRF1). Expression of NRF1 and PGC-1gamma was upregulated in Tg-Trx1, and Trx1 stimulated the transcriptional activity of NRF1 and NRF2 in cardiac myocytes. These results suggest that, in cardiac myocytes, Trx1 upregulates mitochondrial proteins and enhances mitochondrial functions, possibly through PGC-1alpha and NRFs.

The effects of angiotensin II type 1 receptor blockers (ARBs) on cerebral blood flow (CBF) autoregulation have not been fully clarified. Thus, we examined the acute effect of valsartan, the most selective ARB, on CBF autoregulation in spontaneously hypertensive rats. Intravenous administration of valsartan (0.3 mg/kg) reduced the mean arterial pressure (MAP) from 184+/-5 (mean+/-SEM) to 174+/-5 mmHg (p<0.001) without affecting CBF as measured by laser-Doppler flowmetry. The lower limit of CBF autoregulation (the MAP at which the CBF was 80% of the baseline value) in the valsartan-treated group (122+/-3 mmHg) was significantly lower than that in the control group (135+/-4 mmHg, p<0.05). Reverse transcribed-polymerase chain reaction and immunohistochemical staining demonstrated that both angiotensin II type 2 receptors and angiotensin II type 1 receptors (AT1Rs) were expressed in endothelial and smooth muscle cells of the rat cerebral arteries. These results suggest that specific inhibition of AT1Rs in the cerebral circulation causes the leftward shift of the lower limit of autoregulation.

Background and Purpose: Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries., Methods: We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining., Results: RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production., Conclusions: Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.

Background: Gene therapy may be a promising approach for treatment of brain ischemia, although the efficiency of postischemic gene therapy is not established. Our goal in this study was to examine the effects of gene transfer of interleukin-10 (IL-10), an antiinflammatory cytokine, after induction of brain ischemia., Methods and Results: Brain ischemia was produced by either photochemical occlusion of distal middle cerebral artery for focal ischemia or bilateral carotid occlusion for global ischemia in spontaneously hypertensive rats. Adenoviral vectors encoding human IL-10 (AdIL10) or beta-galactosidase (control) were injected into the lateral ventricle 90 or 60 minutes after focal or global ischemia. Five days after ischemia, IL-10, IL-1beta, or tissue necrosis factor-alpha in the cerebrospinal fluid, infarct volume, infiltrations of leukocytes/macrophages in the infarct area, or hippocampal neuronal damages were determined. The transduced IL-10 was released to the cerebrospinal fluid from the ventricular wall and increased to 7623+/-2965 pg/mL 5 days after AdIL10 transfection. Cerebral blood flow during ischemia was not different between treatments in either focal or global ischemia. Brain infarction of the AdIL10 group was significantly smaller and infiltrations of leukocytes and macrophages were fewer in the IL-10 treatment than control. Hippocampal neurons after global ischemia were more preserved, and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling-positive cells were diminished by the IL-10 gene transfer with attenuated IL-1beta and augmented tissue necrosis factor-alpha., Conclusions: Postischemic gene transfer of IL-10 into the lateral ventricle attenuated brain infarction and hippocampal damages, suggesting the promise for treatment of brain ischemia.

In central nervous system (CNS), pericytes have been proposed to play a role in broad functional activities including blood-brain barrier, microcirculation, and macrophage activity. However, contractile responses and Ca2+ signaling in CNS pericytes have not been elucidated. The aim of the present study is to investigate contractility and Ca2+ influx pathway in CNS pericytes. CNS pericytes were cultured from rat brain. Contraction of the pericytes in response to various stimuli was evaluated by the change in surface area measured by a light microscope with a digital camera. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to examine the expression of mRNA of alpha-smooth muscle actin. Intracellular Ca2+ was measured using fura-2 fluorescence spectroscopy. A23187 (Ca2+ ionophore), high external K+ (4 x 10(-2) mol/l), endothelin-1, and serotonin induced contraction of CNS pericytes. RT-PCR analysis revealed the expression of alpha-smooth muscle actin in CNS pericytes. Cytosolic Ca2+ ([Ca2+]i) increased after application of high concentration of external K+, tetraethylammonium, and charybdotoxin, which was inhibited by nicardipine and removal of external Ca2+. Angiotensin-II, serotonin, acetylcholine, ATP, and endothelin-1 caused biphasic response in [Ca2+]i. In response to these agents, [Ca2+]i rapidly increased and then decayed to a relatively constant Ca2+ plateau. The Ca2+ plateau was partially inhibited by nicardipine and completely abolished by omission of external Ca2+. After intracellular Ca2+ store was depleted by the removal of external Ca2+ and addition of thapsigargin, reapplication of external Ca2+ evoked increases in [Ca2+]i. These results indicate that CNS pericytes express mRNA of alpha-smooth muscle actin and possess contractile ability. In CNS pericytes, resting membrane potential is regulated by large conductance Ca2+-activated K+ channels and Ca2+ enters into the cells via L-type voltage-dependent Ca2+ channels, agonist-activated Ca2+ permeable channels, and capacitative Ca2+ entry pathways.

Background: To elucidate the regulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and its roles in mesangial cells, we examined the expression of PPARgamma1 and effects of its ligands on cell phenotypes and angiotensin II-induced contractile response in cultured rat mesangial cells under a high (20 mmol/L) glucose condition., Methods: The effects of tumor necrosis factor alpha (TNFalpha), protein kinase C (PKC) activation, antisense DNA for PPARgamma1, PPARgamma ligands and PD98059 were examined in mesangial cells cultured in either 5 mmol/L or 20 mmol/L glucose. The expressions of PPARgamma1 protein and alpha-smooth muscle actin (alphaSMA) as a marker of phenotype of cells were determined by Western blot. The expression of PPARgamma1 mRNA was determined by a reverse transcription-polymerase chain reaction method. The reduction of cell surface area in response to angiotensin II was measured by microscope to determine cellular contraction., Results: PKC activation, TNFalpha, and 20 mmol/L glucose decreased PPARgamma1 at both protein and mRNA levels, which was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase (MAPK). Decreases of PPARgamma1 protein and contractile response and an increase of alphaSMA occurred simultaneously in the cells treated with 20 mmol/L glucose after 5 days, which were attenuated to the normal levels by PPARgamma ligands. The antisense DNA also induced the decrease of PPARgamma1 protein, contractile dysfunction, and increase of alphaSMA., Conclusion: MAPK suppresses PPARgamma1 at the transcriptional level, and the reduction of PPARgamma1 in cultured rat mesangial cells under the high glucose condition induces phenotypic change and loss of contractile function. PPARgamma ligands recover both reductions of PPARgamma 1 protein and contractile response.

Background: Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood., Methods and Results: Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro., Conclusions: These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.

Background and Purpose: During cerebral ischemia, both hypoxia and hypercapnia appear to produce marked dilatation of the cerebral arteries. Hypercapnia and hypoxia may be accompanied by extracellular and intracellular acidosis, which is another potent dilator of cerebral arteries. However, the precise mechanism by which acidosis produces dilatation of the cerebral arteries is not fully understood. The objective of the present study was to examine the mechanisms by which intracellular acidosis produces dilatation of the basilar artery in vivo., Methods: Using a cranial window in anesthetized rats, we examined responses of the basilar artery to sodium propionate, which was used to cause intracellular acidosis specifically. Expression of subunits of potassium channels was determined by reverse transcription and polymerase chain reaction (RT-PCR)., Results: Topical application of propionate increased diameter of the basilar artery in a concentration-related manner. Propionate-induced dilatation of the artery was attenuated by glibenclamide, an inhibitor of ATP-sensitive potassium channels. However, inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine), large-conductance calcium-activated potassium channels (iberiotoxin), and cyclooxygenase (indomethacin) did not affect the vasodilatation. Expression of mRNA for SUR2B and Kir6.1 was detected, with the use of RT-PCR, in the cultured basilar arterial muscle cells., Conclusions: The findings suggest that intracellular acidification may produce dilatation of the basilar artery through activation of ATP-sensitive potassium channels in vivo. Kir6.1/SUR2B may be the major potassium channels that mediate propionate-induced dilatation of the artery.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47(phox), a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47(phox) with phosphoinositides. Although the isolated phox homology (PX) domain of p47(phox) can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47(phox) is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47(phox) to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47(phox), because protein kinase C treatment of the wild-type p47(phox) but not of a mutant protein with the S303304328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47(phox) translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47(phox) nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47(phox) enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47(phox) from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Objective: Activation of tyrosine kinase appears to play an important role in pathogenesis of cardiovascular disease during chronic hypertension. In the present study, we tested the hypothesis that long-term treatment with an inhibitor of tyrosine kinase would have beneficial effects on hypertension-induced morphological and functional changes of the cerebral artery., Methods: Male spontaneously hypertensive rats (SHR; 4 months old) were fed normal rat chow, or that containing an inhibitor of tyrosine kinase, genistein (1 mg/kg chow). Normotensive Wistar-Kyoto (WKY) rats were also fed either of the chows. After feeding the rats for 2 months, we measured wall thickness, diameter of the basilar artery and its dilator responses to acetylcholine (ACh); Y-26763, an opener of ATP-sensitive potassium channels; and Y-27632, an inhibitor of Rho-associated kinase., Results: Treatment with genistein did not cause significant changes in physiological variables, including mean arterial pressure in either strain. In control SHR, the wall thickness of the basilar artery was greater than that of WKY rats. Genistein treatment reduced the wall thickness significantly in SHR. Vasodilator responses induced by ACh and Y-26763 were markedly attenuated in SHR compared to WKY rats, and treatment of SHR with genistein significantly improved the vasodilatation. Dilatation of the artery in response to Y-27632 was enhanced in SHR compared to WKY rats and treatment of SHR with genistein did not affect the enhanced vasodilator responses to Y-27632., Conclusions: Chronic treatment with genistein may be a novel approach to prevent cerebrovascular disorders.

Objectives: Several factors mediating vascular responses appear to play an important role in the increased resistance of cerebral blood vessels during hypertension. The objective of this study was to elucidate the mechanisms by which hypertension increases the basal tone of the basilar artery in vivo., Methods: Using a cranial window, we examined effects of inhibitors of L-type voltage-dependent calcium channels (nicardipine) and Rho-associated kinase (Y-27632) on the baseline diameter of the basilar artery in spontaneously hypertensive rats (SHR) and compared to the responses in normotensive Wistar-Kyoto (WKY) rats., Results: Topical application of nicardipine (10(-8), 10(-7) and 10(-6) mol/l) produced dilatation of the basilar artery. Nicardipine-induced vasodilatation was enhanced in SHR compared to WKY rats. Nicardipine (10-6 mol/l) dilated the artery in WKY rats and SHR by 9 +/- 2 and 24 +/- 4%, respectively. Topical application of Y-27632 (10(-7), 10(-6) and 10(-5) mol/l) produced dilatation of the basilar artery in WKY rats in a concentration-related manner. The vasodilatation produced by Y-27632 was markedly enhanced in SHR compared to WKY rats. Y-27632 (10(-5) mol/l) dilated the artery in WKY rats and SHR by 14 +/- 2 and 45 +/- 6%, respectively. We also tested the effects of inhibitors of nitric oxide synthase (NG-nitro-l-arginine methyl ester, l-NAME), ATP-sensitive potassium channels (glibenclamide) and large-conductance calcium-activated potassium channels (tetraetyhlammonium, TEA). l-NAME and, to a lesser extent, glibenclamide produced similar constriction of the basilar artery in both strains. TEA did not affect the baseline diameter of the basilar artery in WKY rats and produced small but significant vasoconstriction in SHR., Conclusions: These results suggest that the activity of L-type calcium channels and Rho-associated kinase is enhanced in the basilar artery during hypertensionin vivo. The enhanced contractility may contribute to the increased resistance of the basilar artery during chronic hypertension in vivo.