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Background and Purpose: Poststroke tissue repair, comprised of macrophage-mediated clearance of myelin debris and pericyte-mediated fibrotic response within the infarct area, is an important process for functional recovery. Herein, we investigated the reciprocal interaction between pericytes and macrophages during poststroke repair and functional recovery., Methods: We performed a permanent middle cerebral artery occlusion in both wild-type and pericyte-deficient PDGFRβ (platelet-derived growth factor receptor β) heterozygous knockout ( Pdgfrb +/- ) mice and compared histological changes and neurological functions between the 2 groups. We also examined the effects of conditioned medium harvested from cultured pericytes, or bone marrow-derived macrophages, on the functions of other cell types., Results: Localization of PDGFRβ-positive pericytes and F4/80-positive macrophages was temporally and spatially very similar following permanent middle cerebral artery occlusion. Intrainfarct accumulation of macrophages was significantly attenuated in Pdgfrb +/- mice. Intrainfarct pericytes expressed CCL2 (C-C motif ligand 2) and CSF1 (colony stimulating factor 1), both of which were significantly lower in Pdgfrb +/- mice. Cultured pericytes expressed Ccl2 and Csf1 , both of which were significantly increased by PDGF-BB and suppressed by a PDGFRβ inhibitor. Pericyte conditioned medium significantly enhanced migration and proliferation of bone marrow-derived macrophages. Poststroke clearance of myelin debris was significantly attenuated in Pdgfrb +/- mice. Pericyte conditioned medium promoted phagocytic activity in bone marrow-derived macrophages, also enhancing both STAT3 (signal transducer and activator of transcription 3) phosphorylation and expression of scavenger receptors, Msr1 and Lrp1 . Macrophages processing myelin debris produced trophic factors, enhancing PDGFRβ signaling in pericytes leading to the production of ECM (extracellular matrix) proteins and oligodendrogenesis. Functional recovery was significantly attenuated in Pdgfrb +/- mice, parallel with the extent of tissue repair., Conclusions: A reciprocal interaction between pericytes and macrophages is important for poststroke tissue repair and functional recovery.

Background and Purpose- Smoking is an established risk factor for stroke; however, it is uncertain whether prestroke smoking status affects clinical outcomes of acute ischemic stroke. This study aimed to elucidate the association between smoking status and functional outcomes after acute ischemic stroke. Methods- Using a multicenter hospital-based stroke registry in Japan, we investigated 10 825 patients with acute ischemic stroke hospitalized between July 2007 and December 2017 who had been independent before stroke onset. Smoking status was categorized into those who had never smoked (nonsmokers), former smokers, and current smokers. Clinical outcomes included poor functional outcome (modified Rankin Scale score ≥2) and functional dependence (modified Rankin Scale score 2-5) at 3 months. We adjusted for potential confounding factors using a logistic regression analysis. Results- The mean age of patients was 70.2±12.2 years, and 37.0% were women. There were 4396 (42.7%) nonsmokers, 3328 (32.4%) former smokers, and 2561 (24.9%) current smokers. The odds ratio (95% CI) for poor functional outcome after adjusting for confounders increased in current smokers (1.29 [1.11-1.49] versus nonsmokers) but not in former smokers (1.05 [0.92-1.21] versus nonsmokers). However, among the former smokers, the odds ratio of poor functional outcome was higher in those who quit smoking within 2 years of stroke onset (1.75 [1.15-2.66] versus nonsmokers). The risk of poor functional outcome tended to increase as the number of daily cigarettes increased in current smokers ( P for trend=0.002). All these associations were maintained for functional dependence. Conclusions- Current and recent smoking is associated with an increased risk of unfavorable functional outcomes at 3 months after acute ischemic stroke. Registration- URL: http://www.fukuoka-stroke.net/english/index.html. Unique identifier: 000000800.

Background: Acute graft-vs-host disease (aGVHD) is a common complication of allogenic hematopoietic stem-cell transplantation (allo-HSCT) and skin is the most common and often the 1st site at which aGVHD develops. Cutaneous aGVHD is usually treated with oral and/or topical corticosteroids as the 1st-line treatment; however, steroid-refractory aGVHD not only impairs patients' quality of life but also causes significant morbidity and mortality after allo-HSCT. Narrow-band ultraviolet B (NB-UVB) phototherapy has been utilized for a wide range of immunologic inflammatory skin diseases, but there is limited information on the efficacy, safety, and biomarkers for response prediction of NB-UVB for cutaneous aGVHD., Aims: The purpose of this study is to investigate the efficacy and safety of NB-UVB phototherapy for steroid-refractory cutaneous aGVHD., Patients and Methods: A total of 40 subjects aged from 16 to 70 years with steroid-refractory cutaneous aGVHD after allo-HSCT will be included in the trial. Patients with worse than stage 2 intestine/liver aGVHD will be excluded. Eligible patients will undergo NB-UVB phototherapy until resolution or further worsening of rash or occurrence of an unmanageable adverse event. The primary endpoint is the overall response rate. The secondary outcomes include rates for complete response, partial response, stable disease, progressive disease, duration of response, sparing effect on calcineurin inhibitors and/or corticosteroids, safety, and predictive biomarkers for treatment response., Ethics and Dissemination: The protocol has been approved by the institutional Clinical Research Review Board of Kyoto Prefectural University of Medicine. Written informed consent will be obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals., Trial Registration: Trial registration numbers UMIN000032426 and jRCTs052180005.

Background: This study aimed at determining whether diabetes or glucose metabolism is associated with ischemic stroke in the posterior circulation., Methods: We included 10,245 patients with acute ischemic stroke (mean age 72.7 ± 12.5 years, men 59.5%) who were enrolled in a multicenter hospital-based stroke registry in Fukuoka, Japan, between June 2007 and August 2016. Posterior circulation ischemic stroke (PCIS) was defined as brain infarction in the territory of the posterior cerebral artery and vertebro-basilar arteries. We investigated the associations between diabetes or glycemic parameters, including plasma glucose concentrations, hemoglobin A1c, and the homeostatic model assessment of insulin resistance (HOMA-IR), and PCIS using logistic regression analysis. To improve covariate imbalance, we further evaluated associations after propensity score matching using 1:1 nearest neighbor matching and inverse probability weighting., Results: Diabetes was significantly associated with PCIS even after adjusting for multiple confounding factors (odds ratio-OR [95% confidence interval], 1.37 [1.25-1.50]). Similarly, fasting (1.07 [1.02-1.12]/SD), casual plasma glucose (1.16 [1.11-1.20]/SD) concentrations, and hemoglobin A1c (1.12 [1.08-1.17]/SD), but not HOMA-IR (1.02 [0.97-1.07]/SD), were associated with PCIS. These associations were maintained in patients with ischemic stroke because of thrombotic etiology and were unchanged even after the propensity score matching methods. In patients with diabetes, the ORs of PCIS further increased with an increase in hemoglobin A1c and the presence of microvascular complications., Conclusions: Poor glycemic control may be associated with an increased risk of thrombotic infarction that occurs preferentially in the posterior circulation of the brain.

Objective: In this study, we aimed to determine whether insulin resistance is associated with clinical outcomes after acute ischemic stroke., Methods: We enrolled 4,655 patients with acute ischemic stroke (aged 70.3 ± 12.5 years, 63.5% men) who had been independent before admission; were hospitalized in 7 stroke centers in Fukuoka, Japan, from April 2009 to March 2015; and received no insulin therapy during hospitalization. The homeostasis model assessment of insulin resistance (HOMA-IR) score was calculated using fasting blood glucose and insulin levels measured 8.3 ± 7.8 days after onset. Study outcomes were neurologic improvement (≥4-point decrease in NIH Stroke Scale score or 0 at discharge), poor functional outcome (modified Rankin Scale score of ≥3 at 3 months), and 3-month prognosis (stroke recurrence and all-cause mortality). Logistic regression analysis was used to evaluate the association of the HOMA-IR score with clinical outcomes., Results: The HOMA-IR score was associated with neurologic improvement (odds ratio, 0.68 [95% confidence interval, 0.56-0.83], top vs bottom quintile) and with poor functional outcome (2.02 [1.52-2.68], top vs bottom quintile) after adjusting for potential confounding factors, including diabetes and body mass index. HOMA-IR was not associated with stroke recurrence or mortality within 3 months of onset. The associations were maintained in nondiabetic or nonobese patients. No heterogeneity was observed according to age, sex, stroke subtype, or stroke severity., Conclusions: These findings suggest that insulin resistance is independently associated with poor functional outcome after acute ischemic stroke apart from the risk of short-term stroke recurrence or mortality., (© 2018 American Academy of Neurology.)

Background and Purpose: The role of early hospital arrival in improving poststroke clinical outcomes in patients without reperfusion treatment remains unclear. This study aimed to determine whether early hospital arrival was associated with favorable outcomes in patients without reperfusion treatment or with minor stroke., Methods: This multicenter, hospital-based study included 6780 consecutive patients (aged, 69.9±12.2 years; 63.9% men) with ischemic stroke who were prospectively registered in Fukuoka, Japan, between July 2007 and December 2014. Onset-to-door time was categorized as T 0-1 , ≤1 hour; T 1-2 , >1 and ≤2 hours; T 2-3 , >2 and ≤3 hours; T 3-6 , >3 and ≤6 hours; T 6-12 , >6 and ≤12 hours; T 12-24 , >12 and ≤24 hours; and T 24- , >24 hours. The main outcomes were neurological improvement (decrease in National Institutes of Health Stroke Scale score of ≥4 during hospitalization or 0 at discharge) and good functional outcome (3-month modified Rankin Scale score of 0-1). Associations between onset-to-door time and main outcomes were evaluated after adjusting for potential confounders using logistic regression analysis., Results: Odds ratios (95% confidence intervals) increased significantly with shorter onset-to-door times within 6 hours, for both neurological improvement ( T 0- 1 , 2.79 [2.28-3.42]; T 1-2 , 2.49 [2.02-3.07]; T 2-3 , 1.52 [1.21-1.92]; T 3-6 , 1.72 [1.44-2.05], with reference to T 24- ) and good functional outcome ( T 0-1 , 2.68 [2.05-3.49], T 1-2 2.10 [1.60-2.77], T 2-3 1.53 [1.15-2.03], T 3-6 1.31 [1.05-1.64], with reference to T 24- ), even after adjusting for potential confounding factors including reperfusion treatment and basal National Institutes of Health Stroke Scale. These associations were maintained in 6216 patients without reperfusion treatment and in 4793 patients with minor stroke (National Institutes of Health Stroke Scale ≤4 on hospital arrival)., Conclusions: Early hospital arrival within 6 hours after stroke onset is associated with favorable outcomes after ischemic stroke, regardless of reperfusion treatment or stroke severity., (© 2017 American Heart Association, Inc.)

Background and Purpose: Recent studies show that successful endovascular thrombectomy 6 to 12 hours after stroke onset enhances functional outcomes 3 months later. In this study, we investigated the effects of reperfusion after ischemia on repair processes in the ischemic areas, as well as on functional recovery, using mouse stroke models., Methods: We examined time-dependent histological changes and functional recovery after transient middle cerebral artery occlusion of different durations, including permanent middle cerebral artery occlusion, using the CB-17 (CB-17/lcr-+/+Jcl) mouse strain, which has poor pial collateral blood flow., Results: Large microtubule-associated protein 2-negative areas of neuronal death were produced in mice subjected to ≥60 minutes of ischemia followed by reperfusion on day 1, while restricted microtubule-associated protein 2-negative regions were observed in mice subjected to a 45-minute period of ischemia. A substantial reduction in microtubule-associated protein 2-negative areas was observed on day 7 in mice given early reperfusion and was associated with better functional recovery. Klüver-Barrera staining demonstrated that white matter injury on day 1 was significantly lesser in mice with reperfusion. Immunohistochemistry and electron microscopy revealed that a greater number of endothelial cells were present in the infarct areas in mice with earlier reperfusion and were associated with a more rapid recruitment of platelet-derived growth factor receptor β-positive pericytes and subsequent intrainfarct fibrosis. Early reperfusion also resulted in a greater accumulation of glial fibrillary acidic protein-positive astrocytes in peri-infarct areas. Peri-infarct astrogliosis was attenuated in platelet-derived growth factor receptor β heterozygous knockout mice., Conclusions: Early reperfusion after ischemia enhances the survival of endothelial cells and pericytes within ischemic areas even after the infarct is established, resulting in efficient intrainfarct fibrosis and peri-infarct astrogliosis. These effects might be associated with efficient peri-infarct reorganization and functional recovery., (© 2017 American Heart Association, Inc.)

Background and Purpose: Variable sex differences in clinical outcomes after stroke have been reported worldwide. This study aimed to elucidate whether sex is an independent risk factor of poor functional outcome after acute ischemic stroke., Methods: Using the database of patients with acute stroke registered in the Fukuoka Stroke Registry in Japan from 1999 to 2013, 6236 previously independent patients with first-ever ischemic stroke who were admitted within 24 hours of onset were included in this study. Baseline characteristics were assessed on admission. Study outcomes included neurological improvement, neurological deterioration, and poor functional outcome (modified Rankin Scale score, 3-6 at discharge). Logistic regression analyses were performed to evaluate the association between sex and clinical outcomes., Results: Overall, 2398 patients (38.5%) were women. Severe stroke (National Institutes of Health Stroke Scale score, ≥8) on admission was more prevalent in women than in men. The frequency of neurological improvement or deterioration during hospitalization was not different between the sexes. After adjusting for possible confounders, including age, stroke subtype and severity, risk factors, and poststroke treatments, it was found that female sex was independently associated with poor functional outcome at discharge (odds ratio, 1.30; 95% confidence interval, 1.08-1.57). There was heterogeneity of the association between sex and poor outcome according to age: women had higher risk of poor outcome than men among patients aged ≥70 years, but no clear sex difference was found in patients aged <70 years., Conclusions: Female sex was associated with the risk of poor functional outcome at discharge after acute ischemic stroke., (© 2014 American Heart Association, Inc.)

Background and Purpose: The relationship between the intensity of anticoagulation at the onset of acute cardioembolic stroke and clinical outcome after stroke is unclear. Here, we elucidated the relationship between prothrombin time-international normalized ratio (PT-INR) values on admission and clinical outcomes in patients with acute cardioembolic stroke., Methods: A total of 602 patients from the Fukuoka Stroke Registry in Japan who had been treated with warfarin but developed cardioembolic stroke were enrolled. The patients were classified into 3 groups according to their PT-INR values on admission: PT-INR <1.50, 411 patients; PT-INR 1.50 to 1.99, 146 patients; and PT-INR ≥2.00, 45 patients. The associations between PT-INR categories and severe neurological deficits (National Institutes of Health Stroke Scale ≥10) on admission and poor functional outcome (modified Rankin scale 4-6) at discharge were investigated using a logistic regression analysis., Results: Neurological deficits on admission were less severe, and functional outcome at discharge was more favorable as the PT-INR level on admission increased. The multivariate analysis revealed that severe neurological deficits were inversely associated with PT-INR on admission (PT-INR 1.50-1.99: odds ratio, 0.66; 95% confidence interval, 0.43-1.00; PT-INR ≥2.00: odds ratio, 0.41; 95% confidence interval, 0.20-0.83; compared with a reference group of PT-INR <1.50). Poor functional outcome was less likely in patients with PT-INR ≥2.00 (odds ratio, 0.20; 95% confidence interval, 0.06-0.55) after adjustment for confounders., Conclusions: Prestroke PT-INR ≥2.0 is associated with favorable clinical outcomes after acute cardioembolic stroke.

Rationale: NADPH oxidase (Nox) 2 and Nox4 are major components of the Nox family which purposefully produce reactive oxidative species, namely O2(-) and H2O2, in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood., Objective: We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss-of-function mouse models., Methods and Results: Systemic (s) Nox2 knockout (KO), sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I/R (30 minutes/24 hours, respectively). Both myocardial infarct size/area at risk and O2(-) production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type mice. Unexpectedly, however, the myocardial infarct size/area at risk was greater, despite less O2(-) production, in sNox2 KO+cNox4 KO (double-KO) mice and transgenic mice (Tg) with cardiac-specific expression of dominant-negative Nox, which suppresses both Nox2 and Nox4, than in wild-type or single KO mice. Hypoxia-inducible factor-1α was downregulated whereas peroxisome proliferator-activated receptor-α was upregulated in Tg-dominant-negative Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates hypoxia-inducible factor-1α, rescued the I/R injury and prevented upregulation of peroxisome proliferator-activated receptor-α in Tg-dominant-negative Nox mice. A cross with peroxisome proliferator-activated receptor-α KO mice also attenuated the injury in Tg- dominant-negative Nox mice., Conclusions: Both Nox2 and Nox4 contribute to the increase in reactive oxidative species and injury by I/R. However, low levels of reactive oxidative species produced by either Nox2 or Nox4 regulate hypoxia-inducible factor-1α and peroxisome proliferator-activated receptor-α, thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.

Rationale: Oxidation of cysteine residues in class II histone deacetylases (HDACs), including HDAC4, causes nuclear exit, thereby inducing cardiac hypertrophy. The cellular source of reactive oxygen species responsible for oxidation of HDAC4 remains unknown., Objective: We investigated whether nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4), a major nicotinamide adenine dinucleotide phosphate oxidase, mediates cysteine oxidation of HDAC4., Methods and Results: Phenylephrine (100 μmol/L), an α1 adrenergic agonist, induced upregulation of Nox4 (1.5-fold; P<0.05) within 5 minutes, accompanied by increases in O(2)(-) (3.5-fold; P<0.01) from the nuclear membrane and nuclear exit of HDAC4 in cardiomyocytes. Knockdown of Nox4, but not Nox2, attenuated O(2)(-) production in the nucleus and prevented phenylephrine-induced oxidation and nuclear exit of HDAC4. After continuous infusion of phenylephrine (20 mg/kg per day) for 14 days, wild-type and cardiac-specific Nox4 knockout mice exhibited similar aortic pressures. Left ventricular weight/tibial length (5.7±0.2 versus 6.4±0.2 mg/mm; P<0.05) and cardiomyocytes cross-sectional area (223±13 versus 258±12 μm(2); P<0.05) were significantly smaller in cardiac-specific Nox4 knockout than in wild-type mice. Nuclear O(2)(-)production in the heart was significantly lower in cardiac-specific Nox4 knockout than in wild-type mice (4116±314 versus 7057±1710 relative light unit; P<0.05), and cysteine oxidation of HDAC4 was decreased. HDAC4 oxidation and cardiac hypertrophy were also attenuated in cardiac-specific Nox4 knockout mice 2 weeks after transverse aortic constriction., Conclusions: Nox4 plays an essential role in mediating cysteine oxidation and nuclear exit of HDAC4, thereby mediating cardiac hypertrophy in response to phenylephrine and pressure overload.

Objective: We have previously shown that Na(+)/H(+) exchanger isoform 1 (NHE1) plays an important role in Ca(2+) signaling and cell proliferation in human central nervous system (CNS) pericytes. The aims of the present study were to elucidate how NHE1-induced Ca(2+) signaling during acidosis is transformed into cellular responses in CNS pericytes., Methods and Results: Human CNS pericytes were cultured, and the activation of cAMP responsive element-binding protein (CREB) was evaluated by Western blotting analysis, immunofluorescence, and luciferase assays. In human CNS pericytes, low extracellular Na(+) or low pH generated Ca(2+) oscillation and subsequently phosphorylated Ca(2+)/calmodulin-dependent kinase II (CaMKII) and CREB in a time-dependent manner. Focal cerebral ischemia was applied using photothrombotic distal middle cerebral artery occlusion in mice, and the phosphorylation of CREB and the production of interleukin-6 were observed in pericytes migrating into the peri-infarct penumbra during the early phase after ischemic insult., Conclusions: Our results indicate that extracellular acidosis induces Ca(2+) oscillation via NHE1, leading to Ca(2+)/CaMKII-dependent CREB activation in human CNS pericytes. Acidosis may upregulate a variety of proteins, such as interleukin-6, through the NHE1-Ca2+/CaMKII-CREB pathway in brain pericytes and may thus modulate brain ischemic insult.

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: 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.

Pancreatic acinar cells within the islets of Langerhans in human pancreas were investigated both histopathologically and electron microscopically. Acinar cells inside the islets of Langerhans were observed in all 50 adult cases investigated. Between such acinar cells and endocrine tissues, no reticular fiber was observed; they were encapsulated together instead. The acinar cells were immunostained with antiamylase similar to the "extrainsular" acinar cells and were also connected to the small pancreatic duct. Electron microscopically, intercellular junctions of the desmosomal type were found between the acinar cells and the adjacent endocrine cells. Therefore, the acinar cells within the islets of Langerhans were true acinar cells, based on the incomplete demarcation between the islets and the acinar tissue.