Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Kuniyuki Nakamura Last modified date:2023.12.07

Assistant Professor / Department of Nephrology,Hypertension,and Strokology / Kyushu University Hospital

1. Kuniyuki Nakamura, Kana Ueki, Ryu Matsuo, Takuya Kiyohara, Fumi Irie, Yoshinobu Wakisaka, Tetsuro Ago, Masahiro Kamouchi, Takanari Kitazono, Association between decreases in serum uric acid levels and unfavorable outcomes after ischemic stroke: A multicenter hospital-based observational study., PloS one, 10.1371/journal.pone.0287721, 18, 6, e0287721, 2023.06, BACKGROUND: The association between clinical outcomes in ischemic stroke patients and decreases in serum uric acid levels, which often occur during the acute phase, remains unknown. Herein, we aimed to investigate the association using a large-scale, multicenter stroke registry. METHODS: We analyzed 4,621 acute ischemic stroke patients enrolled in the Fukuoka Stroke Registry between June 2007 and September 2019 whose uric acid levels were measured at least twice during hospitalization (including on admission). The study outcomes were poor functional outcome (modified Rankin Scale score ≥3) and functional dependence (modified Rankin Scale score 3-5) at 3 months after stroke onset. Changes in uric acid levels after admission were evaluated using a decrease rate that was classified into 4 sex-specific grades ranging from G1 (no change/increase after admission) to G4 (most decreased). Multivariable logistic regression analyses were used to assess the associations between decreases in uric acid levels and the outcomes. RESULTS: The frequencies of the poor functional outcome and functional dependence were lowest in G1 and highest in G4. The odds ratios (95% confidence intervals) of G4 were significantly higher for poor functional outcome (2.66 [2.05-3.44]) and functional dependence (2.61 [2.00-3.42]) when compared with G1 after adjusting for confounding factors. We observed no heterogeneity in results for subgroups categorized according to age, sex, stroke subtype, neurological severity, chronic kidney disease, or uric acid level on admission. CONCLUSIONS: Decreases in serum uric acid levels were independently associated with unfavorable outcomes after acute ischemic stroke..
2. Kuniyuki Nakamura, Tetsuro Ago, Pericyte-Mediated Molecular Mechanisms Underlying Tissue Repair and Functional Recovery after Ischemic Stroke., Journal of atherosclerosis and thrombosis, 10.5551/jat.RV22007, 30, 9, 1085-1094, 2023.09, There are still many patients suffering from ischemic stroke and related disabilities worldwide. To develop a treatment that promotes functional recovery after acute ischemic stroke, we need to elucidate endogenous tissue repair mechanisms. The concept of a neurovascular unit (NVU) indicates the importance of a complex orchestration of cell-cell interactions and their microenvironment in the physiology and pathophysiology of various central nervous system diseases, particularly ischemic stroke. In this concept, microvascular pericytes play a crucial role in regulating the blood-brain barrier integrity, cerebral blood flow (CBF), and vascular stability. Recent evidence suggests that pericytes are also involved in the tissue repair leading to functional recovery following acute ischemic stroke through the interaction with other cell types constituting the NVU; pericytes may organize CBF recovery, macrophage-mediated clearance of myelin debris, intrainfarct fibrosis, and periinfarct astrogliosis and remyelination. In this review, we will discuss the physiological and pathophysiological functions of pericytes, their involvement in the molecular mechanisms underlying tissue repair and functional recovery after ischemic stroke, and a therapeutic strategy to promote endogenous regeneration..
3. Fumi Irie, Ryu Matsuo, Kuniyuki Nakamura, Yoshinobu Wakisaka, Tetsuro Ago, Takanari Kitazono, Masahiro Kamouchi; Fukuoka Stroke Registry Investigators, Sex Differences in Long-Term Functional Decline after Ischemic Stroke: A Longitudinal Observational Study from the Fukuoka Stroke Registry, Cerebrovasc Dis ., 10.1159/000526940, 1-8, 2023.02, INTRODUCTION: Data on sex differences in poststroke functional status for a period longer than 1 year based on large cohorts are sparse. This study aimed to determine whether there are sex differences in long-term functional decline after ischemic stroke. METHODS: We tracked functional status for 5 years among 3-month survivors of acute ischemic stroke and compared outcomes between women and men using a large-scale hospital-based stroke registry in Fukuoka, Japan. Functional status was assessed using the modified Rankin Scale (mRS). Functional dependency was defined as an mRS score of 3, 4, or 5. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% confidence intervals of outcomes after adjusting for possible confounders. RESULTS: A total of 8,446 patients (71.9 ± 12.5 years, 3,377 (40.0%) female patients) were enrolled in this study. Female sex was associated with a higher risk of functional dependency at 5 years poststroke even when adjusting for age, 3-month mRS score, and other confounding factors (multivariable-adjusted OR vs. men, 1.56 [95% confidence interval, 1.26-1.93]). This significant association of female sex with higher dependency at 5 years was also found among patients who were independent at 3 months poststroke. Subgroup analysis showed that increased risk of functional dependency in female patients was more marked in patients aged ≥75 years than in those aged
4. Kei Yamanaka, Kuniyuki Nakamura, Tomoya Shibahara, Masamitsu Takashima, Hayato Takaki, Masaoki Hidaka, Motohiro Komori, Yoji Yoshikawa, Yoshinobu Wakisaka, Tetsuro Ago, Takanari Kitazono, Deletion of Nox4 enhances remyelination following cuprizone-induced demyelination by increasing phagocytic capacity of microglia and macrophages in mice., Glia, 10.1002/glia.24292, 2022.11, NOX4 is a major reactive oxygen species-producing enzyme that modulates cell stress responses. We here examined the effect of Nox4 deletion on demyelination-remyelination, the most common pathological change in the brain. We used a model of cuprizone (CPZ)-associated demyelination-remyelination in wild-type and Nox4-deficient (Nox4-/- ) mice. While the CPZ-induced demyelination in the corpus callosum after 4 weeks of CPZ intoxication was slightly less pronounced in Nox4-/- mice than that in wild-type mice, remyelination following CPZ withdrawal was significantly enhanced in Nox4-/- mice with an increased accumulation of IBA1-positive microglia/macrophages in the demyelinating corpus callosum. Consistently, locomotor function, as assessed by the beam walking test, was significantly better during the remyelination phase in Nox4-/- mice. Nox4 deletion did not affect autonomous growth of primary-culture oligodendrocyte precursor cells. Although Nox4 expression was higher in cultured macrophages than in microglia, Nox4-/- microglia and macrophages both showed enhanced phagocytic capacity of myelin debris and produced increased amounts of trophic factors upon phagocytosis. The expression of trophic factors was higher, in parallel with the accumulation of IBA1-positive cells, in the corpus callosum in Nox4-/- mice than that in wild-type mice. Nox4 deletion suppressed phagocytosis-induced increase in mitochondrial membrane potential, enhancing phagocytic capacity of macrophages. Treatment with culture medium of Nox4-/- macrophages engulfing myelin debris, but not that of Nox4-/- astrocytes, enhanced cell growth and expression of myelin-associated proteins in cultured oligodendrocyte precursor cells. Collectively, Nox4 deletion promoted remyelination after CPZ-induced demyelination by enhancing microglia/macrophage-mediated clearance of myelin debris and the production of trophic factors leading to oligodendrogenesis..
5. Masanori Wakisaka, Kuniyuki Nakamura, Takanari Kitazono, The presence of sodium glucose co-transporter 2 in mesangial cells and pericytes and its roles in mesangial lesions and in capillaries under diabetic and ischemic conditions., Diabetes research and clinical practice, 10.1016/j.diabres.2022.110096, 192, 110096-110096, 2022.10, SGLT2 is expressed in mesangial cells and pericytes, and is upregulated byhigh glucose and ischemia. Upregulated SGLT2 in both cells might directly worsen ischemia in kidney interstitial legion, heart and brain. The overexpression of SGLT2 in these cells could induce various organ failures via damages or loss of capillaries and dysfunctions of mesangial cells, which are attenuated by SGLT2 inhibitors..
6. Tomoya Shibahara, Fumitaka Yoshino, Mikiaki Matsuoka, Masaki Tachibana, Kuniyuki Nakamura, Tetsuro Ago, Junya Kuroda, Hiroshi Nakane, Neurosarcoidosis Presenting with Prominent Periventricular White-Matter Lesions during Steroid Treatment for Autoimmune Hepatitis, Case Reports in Neurology, 10.1159/000526223, 334-340, 2022.08.
7. Masamitsu Takashima, Kuniyuki Nakamura, Takuya Kiyohara, Yoshinobu Wakisaka, Masaoki Hidaka, Hayato Takaki, Kei Yamanaka, Tomoya Shibahara, Masanori Wakisaka, Tetsuro Ago, Takanari Kitazono, Low-dose sodium-glucose cotransporter 2 inhibitor ameliorates ischemic brain injury in mice through pericyte protection without glucose-lowering effects., Communications biology, 10.1038/s42003-022-03605-4, 5, 1, 653-653, 2022.07, Antidiabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors have attracted attention for their cardiorenal-protective properties beyond their glucose-lowering effect. However, their benefits in ischemic stroke remain controversial. Here we show the effects of luseogliflozin, a selective SGLT2 inhibitor, in acute ischemic stroke, using a permanent middle cerebral artery occlusion (pMCAO) model in non-diabetic mice. Pretreatment with low-dose luseogliflozin, which does not affect blood glucose levels, significantly attenuated infarct volume, blood-brain barrier disruption, and motor dysfunction after pMCAO. SGLT2 was expressed predominantly in brain pericytes and was upregulated in peri- and intra-infarct areas. Notably, luseogliflozin pretreatment reduced pericyte loss in ischemic areas. In cultured pericytes, luseogliflozin activated AMP-activated protein kinase α and increased mitochondrial transcription factor A expression and number of mitochondria, conferring resistance to oxygen-glucose deprivation. Collectively, pre-stroke inhibition of SGLT2 induces ischemic tolerance in brain pericytes independent of the glucose-lowering effect, contributing to the attenuation of ischemic brain injury..
8. Yuichiro Ohya, Ryu Matsuo, Noriko Sato, Fumi Irie, Kuniyuki Nakamura, Yoshinobu Wakisaka, Tetsuro Ago, Masahiro Kamouchi, Takanari Kitazono, Causes of ischemic stroke in young adults versus non-young adults: A multicenter hospital-based observational study., PloS one, 10.1371/journal.pone.0268481, 17, 7, e0268481, 2022.07, BACKGROUND: Very few comparative studies have focused on the differences in the causes of ischemic stroke between young adults and non-young adults. This study was performed to determine what causes of ischemic stroke are more important in young adults than in non-young adults using a large-scale multicenter hospital-based stroke registry in Fukuoka, Japan. METHODS AND RESULTS: We investigated data on 15,860 consecutive patients aged ≥18 years with acute ischemic stroke (mean age: 73.5 ± 12.4 years, 58.2% men) who were hospitalized between 2007 and 2019. In total, 779 patients were categorized as young adults (≤50 years of age). Although vascular risk factors, including hypertension, diabetes mellitus, and dyslipidemia, were less frequent in young adults than in non-young adults, the prevalence of diabetes mellitus and dyslipidemia in young adults aged >40 years were comparable to those of non-young adults. Lifestyle-related risk factors such as smoking, drinking, and obesity were more frequent in young adults than in non-young adults. As young adults became older, the proportions of cardioembolism and stroke of other determined etiologies decreased, but those of large-artery atherosclerosis and small-vessel occlusion increased. Some embolic sources (high-risk sources: arterial myxoma, dilated cardiomyopathy, and intracardiac thrombus; medium-risk sources: atrial septal defect, nonbacterial thrombotic endocarditis, patent foramen ovale, and left ventricular hypokinesis) and uncommon causes (vascular diseases: reversible cerebral vasoconstriction syndrome, moyamoya disease, other vascular causes, arterial dissection, and cerebral venous thrombosis; hematologic diseases: antiphospholipid syndrome and protein S deficiency) were more prevalent in young adults than in non-young adults, and these trends decreased with age. CONCLUSIONS: Certain embolic sources and uncommon causes may be etiologically important causes of ischemic stroke in young adults. However, the contribution of conventional vascular risk factors and lifestyle-related risk factors is not negligible with advancing age, even in young adults..
9. Tomoya Shibahara, Kuniyuki Nakamura, Daisuke Abe, Naoki Tagawa, Yoshinobu Wakisaka, Takanari Kitazono, Tetsuro Ago, Progressive Small-Vessel Strokes Following Antiretroviral Therapy in a Patient with Acquired Immunodeficiency Syndrome., Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association, 10.1016/j.jstrokecerebrovasdis.2022.106409, 31, 5, 106409-106409, 2022.05, We report a case of a 59-year-old man with human immunodeficiency virus (HIV)/ acquired immunodeficiency syndrome (AIDS) who developed multiple small-vessel strokes during the immune reconstitution phase. The patient had been diagnosed with HIV/AIDS with a low CD4 count and high viral load and started combinational antiretroviral therapy (cART) with raltegravir, emtricitabine, and tenofovir alafenamide fumarate seven months before the admission. He was admitted to our hospital with complaints of mild dysarthria and left-sided hemiparesis, but lacking consciousness/cognitive disturbances. Diffusion-weighted images (DWI) revealed multiple areas of hyperintensity in the anterior circulation system of the brain. Because we identified decreased activity of protein S through extensive examinations, we treated him initially with intravenous infusion of heparin sodium and aspirin; however, DWI detected multiple progressive small-vessel strokes after that. We considered that the immune reconstitution accounted for the small-vessel vasculopathy/vasculitis, leading to ischemic stroke. Therefore, we initiated oral administration of prednisolone, which successfully prevented stroke recurrence. This report describes a case of multiple small-vessel strokes following cART for AIDS during the immune reconstitution phase, effectively treated with steroids, which may often go undiagnosed due to their relatively mild symptoms..
10. Fumi Irie, Ryu Matsuo, Kuniyuki Nakamura, Yoshinobu Wakisaka, Tetsuro Ago, Masahiro Kamouchi, Takanari Kitazono, Sex Differences in the Risk of 30-Day Death After Acute Ischemic Stroke., Neurology. Clinical practice, 10.1212/CPJ.0000000000001087, 11, 6, e809-e816, 2021.12, OBJECTIVE: To examine sex differences in early stroke deaths according to cause of death. METHODS: We investigated 30-day deaths in patients with acute ischemic stroke enrolled in a multicenter stroke registry between 2007 and 2019 in Fukuoka, Japan. We estimated the multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of cause-specific deaths for women vs men using Cox proportional hazards models and competing risk models. The risk of acute infections during hospitalization and the associated case fatality rates were also compared between the sexes. RESULTS: Among 17,956 patients with acute ischemic stroke (women: 41.3%), the crude 30-day death rate after stroke was higher in women than men. However, adjusting for age and stroke severity resulted in a lower risk of death among women (HR [95% CI]: 0.76 [0.62-0.92]). Analyses using competing risk models revealed that women were less likely to die of acute infections (subdistribution HR [95% CI]: 0.33 [0.20-0.54]). Further analyses showed that women were associated with a lower risk of acute infections during hospitalization (OR [95% CI]: 0.62 [0.52-0.74]) and a lower risk of death due to these infections (subdistribution HR [95% CI]: 052 [0.33-0.83]). CONCLUSIONS: When adjusting for confounders, the female sex was associated with a lower risk of 30-day death after stroke, which could be explained by a female survival advantage in poststroke infections. Sex-specific strategies are needed to reduce early stroke deaths. CLASSIFICATION OF EVIDENCE: This is a Class I prognostic study because it is a prospective population-based cohort with objective outcomes. Female sex appears to be protective against early stroke deaths and post stroke infections..
11. Kenji Fukuda, Ryu Matsuo, Masahiro Kamouchi, Fumi Kiyuna, Noriko Sato, Kuniyuki Nakamura, Jun Hata, Yoshinobu Wakisaka, Tetsuro Ago, Tsutomu Imaizumi, Hisashi Kai, Takanari Kitazono, FSR Investigators, Day-by-Day Blood Pressure Variability in the Subacute Stage of Ischemic Stroke and Long-Term Recurrence., Stroke, 10.1161/STROKEAHA.120.033751., 53, 1, 70-78, 2021.09, BACKGROUND AND PURPOSE: This study aimed to determine whether variability of day-by-day blood pressure (BP) during the subacute stage of acute ischemic stroke is predictive of long-term stroke recurrence. METHODS: We analyzed 7665 patients (mean±SD age: 72.9±13.1 years; women: 42.4%) hospitalized for first-ever ischemic stroke in 7 stroke centers in Fukuoka, Japan, from June 2007 to November 2018. BP was measured daily during the subacute stage (4-10 days after onset). Its mean and coefficient of variation (CV) values were calculated and divided into 4 groups according to the quartiles of these BP parameters. Patients were prospectively followed up for recurrent stroke or all-cause death. The cumulative event rate was calculated with the Kaplan-Meier method. We estimated the hazard ratios and 95% confidence intervals of the events of interest after adjusting for potential confounders and mean BP values using Cox proportional hazards models. The Fine-Gray model was also used to account for the competing risk of death. RESULTS: With a mean (±SD) follow-up duration of 3.9±3.2 years, the rates of recurrent stroke and all-cause death were 3.9 and 9.9 per 100 patient-years, respectively. The cumulative event rates of recurrent stroke and all-cause death increased with increasing CVs of systolic BP and diastolic BP. The systolic BP CV was significantly associated with an increased risk of recurrent stroke after adjusting for multiple confounders and mean BP (hazard ratio [95% CI] for fourth quartile versus first quartile, 1.26 [1.05-1.50]); the risk of recurrent stroke also increased with an increasing systolic BP CV for nonfatal strokes (1.26 [1.05-1.51]) and when death was regarded as a competing risk (1.21 [1.02-1.45]). Similar associations were observed for the diastolic BP CV. CONCLUSIONS: Day-by-day variability of BP during the subacute stage of acute ischemic stroke was associated with an increased long-term risk of recurrent stroke..
12. Masanori Wakisaka, Kuniyuki Nakamura, Toshiaki Nakano, Takanari Kitazono, Roles of Sodium-Glucose Cotransporter 2 of Mesangial Cells in Diabetic Kidney Disease., Journal of the Endocrine Society, 10.1210/jendso/bvab083, 5, 8, bvab083, 2021.08, We have been studying the presence of sodium-glucose cotransporter 2 (SGLT2) in mesangial cells and pericytes since 1992. Recent large placebo-controlled studies of SGLT2 inhibitors in patients with type 2 diabetes mellitus have reported desirable effects of the inhibitors on the diabetic kidney and the diabetic heart. Most studies have indicated that these effects of SGLT2 inhibitors could be mediated by the tubuloglomerular feedback system. However, a recent study about urine sodium excretion in the presence of an SGLT2 inhibitor did not show any increases in urine sodium excretion. A very small dose of an SGLT2 inhibitor did not inhibit SGLT2 at the S1 segment of proximal tubules. Moreover, SGLT2 inhibition protects against progression in chronic kidney disease with and without type 2 diabetes. In these circumstances, the tubuloglomerular feedback hypothesis involves several theoretical concerns that must be clarified. The presence of SGLT2 in mesangial cells seems to be very important for diabetic nephropathy. We now propose a novel mechanism by which the desirable effects of SGLT2 inhibitors on diabetic nephropathy are derived from the direct effect on SGLT2 expressed in mesangial cells..
13. Takuya Kiyohara, Ryu Matsuo, Jun Hata, Kuniyuki Nakamura, Yoshinobu Wakisaka, Masahiro Kamouchi, Takanari Kitazono, Tetsuro Ago, FSR Investigators, β-Cell Function and Clinical Outcome in Nondiabetic Patients With Acute Ischemic Stroke, Stroke, 10.1161/STROKEAHA.120.031392., 52, 8, 2621-2628, 2021.05, Background and Purpose: Little is known about how β-cell dysfunction affects clinical outcome after ischemic stroke. We examined whether β-cell function is associated with clinical outcome after acute ischemic stroke and if so, whether insulin resistance influences this association in a prospective study of patients with acute stroke. Methods: A total of 3590 nondiabetic patients with acute ischemic stroke (mean age, 71 years) were followed up for 3 months. β-Cell function was assessed using the homeostasis model assessment for β-cell function (HOMA-β). Study outcomes were poor functional outcome (modified Rankin Scale score, 3–6) and stroke recurrence at 3 months after stroke onset and neurological deterioration (≥2-point increase in the National Institutes of Health Stroke Scale score) at discharge. Logistic regression analysis was used to evaluate the association between quintile levels of serum HOMA-β and clinical outcomes. Results: The age- and sex-adjusted odds ratios for poor functional outcome and neurological deterioration increased significantly with decreasing HOMA-β levels (P for trend,
14. Yoshinobu Wakisaka, Ryu Matsuo, Kuniyuki Nakamura, Tetsuro Ago, Masahiro Kamouchi, Takanari Kitazono, Fukuoka Stroke Registry Investigators, Pre-Stroke Cholinesterase Inhibitor Treatment Is Beneficially Associated with Functional Outcome in Patients with Acute Ischemic Stroke and Pre-Stroke Dementia: The Fukuoka Stroke Registry, Cerebrovasc Dis., 10.1159/000514368., 50, 4, 390-396, 2021.03, INTRODUCTION: Pre-stroke dementia is significantly associated with poor stroke outcome. Cholinesterase inhibitors (ChEIs) might reduce the risk of stroke in patients with dementia. However, the association between pre-stroke ChEI treatment and stroke outcome remains unresolved. Therefore, we aimed to determine this association in patients with acute ischemic stroke and pre-stroke dementia. METHODS: We enrolled 805 patients with pre-stroke dementia among 13,167 with ischemic stroke within 7 days of onset who were registered in the Fukuoka Stroke Registry between June 2007 and May 2019 and were independent in basic activities of daily living (ADLs) before admission. Primary and secondary study outcomes were poor functional outcome (modified Rankin Scale [mRS] score: 3-6) at 3 months after stroke onset and neurological deterioration (≥2-point increase in the NIH Stroke Scale [NIHSS] during hospitalization), respectively. Logistic regression analysis was used to evaluate associations between pre-stroke ChEI treatment and study outcomes. To improve covariate imbalance, we further conducted a propensity score (PS)-matched cohort study. RESULTS: Among the participants, 212 (26.3%) had pre-stroke ChEI treatment. Treatment was negatively associated with poor functional outcome (odds ratio: 0.68 [95% confidence interval: 0.46-0.99]) and neurological deterioration (0.52 [0.31-0.88]) after adjusting for potential confounding factors. In the PS-matched cohort study, the same trends were observed between pre-stroke ChEI treatment and poor functional outcome (0.61 [0.40-0.92]) and between the treatment and neurological deterioration (0.47 [0.25-0.86]). CONCLUSIONS: Our findings suggest that pre-stroke ChEI treatment is associated with reduced risks for poor functional outcome and neurological deterioration after acute ischemic stroke in patients with pre-stroke dementia who are independent in basic ADLs before the onset of stroke..
15. Noriko Sato, Ryu Matsuo, Fumi Kiyuna, Kuniyuki Nakamura, Jun Hata, Yoshinobu Wakisaka, Tetsuro Ago, Masahiro Kamouchi, Takanari Kitazono, collab on behalf of the Fukuoka Stroke Registry Investigators, Anticoagulation and Risk of Stroke Recurrence in Patients with Embolic Stroke of Undetermined Source Having No Potential Source of Embolism, Cerebrovasc Dis., 10.1159/000510773., 49, 6, 601-608, 2020.11, BACKGROUND: This study aimed to determine whether use of oral anticoagulants (OACs) was associated with a reduced risk of recurrent stroke compared with use of antiplatelets (APs) in patients with embolic stroke of undetermined source (ESUS) having no potential source of embolism. METHODS: Of 8,790 patients with acute ischemic stroke registered at 7 centers in the Fukuoka Stroke Registry from June 2007 to May 2017, we included 681 patients (mean age 69.7 [SD 14.1] years, 48.3% men) who experienced ESUS without a potential source of embolism and received OAC alone or AP alone. We estimated hazard ratios (HRs) and 95% confidential intervals (CIs) of recurrent ischemic stroke or any stroke after discharge using a Cox proportional hazards model and Fine and Gray model. RESULTS: During a mean follow-up of 3.4 (SD 1.7) years, event rates of recurrent ischemic stroke were 4.4 per 100 person-years in 489 patients treated with AP and 2.0 per 100 person-years in 192 patients treated with OAC. OAC use was associated with a reduced risk of recurrent ischemic stroke, even after adjusting for potential confounding factors (multivariable-adjusted HR [95% CI], 0.42 [0.23-0.80]) and when additionally considering death as a competing risk (0.45 [0.24-0.85]). The reduced risk of recurrent ischemic stroke was still observed in patients treated with OAC (0.32 [0.15-0.67]) in reference to propensity score-matched patients treated with AP. These associations were maintained for all types of stroke, including ischemic and hemorrhagic stroke. CONCLUSIONS: This nonrandomized observational study suggests that anticoagulation therapy might be associated with a reduced risk of recurrent stroke compared with antiplatelet therapy in patients with ESUS in whom no potential source of embolism was identified. Further study should be performed in consideration of a potential source of embolism even in patients with ESUS..
16. Tomoya Shibahara, Tetsuro Ago, Masaki Tachibana, Kuniyuki Nakamura, Kei Yamanaka, Junya Kuroda, Yoshinobu Wakisaka, Takanari Kitazono, Reciprocal Interaction Between Pericytes and Macrophage in Poststroke Tissue Repair and Functional Recovery., Stroke, 10.1161/STROKEAHA.120.029827., 51, 10, 3095-3106, 2020.09, 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..
17. Yuichiro Ohya, Kuniyuki Nakamura, Yoshinobu Wakisaka, Hiroaki Sato, Kayo Wakisaka, Masaya Kumamoto, Yohei Muraya, Junya Kuroda, Hiroshi Nakane, Goichi Yoshimoto, Takanari Kitazono, Tetsuro Ago, Epstein-Barr Virus-Associated Encephalopathy Presenting with Nonconvulsive Status Epilepticus in an Immunosuppressive State., Case Rep Neurol., 10.1159/000507976., 2020.08.
18. Tetsuro Ago, Kana Ueki, Yoshinobu Wakisaka, Kuniyuki Nakamura, Takanari Kitazono, Response to Letter regarding case report, "Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes due to m.3243A > G mutation in a 76-year-old woman"., Journal of the neurological sciences, 10.1016/j.jns.2020.116925, 414, 116925-116925, 2020.07.
19. Kana Ueki, Kuniyuki Nakamura, Yoshinobu Wakisaka, Shinichi Wada, Yoji Yoshikawa, Shinya Matsumoto, Taeko Hotta, Dongchong Kang, Takanari Kitazon, Tetsuro Ago, An Embolic Stroke in a Patient With PROC p.Lys193del., J Stroke Cerebrovasc Dis., 10.1016/j.jstrokecerebrovasdis.2019.104597., 29, 5, 104597-104597, 2020.05, We report a 58-year-old woman who suddenly developed brain infarction with weakness of the left lower extremity and left perioral dysesthesia during postoperative tamoxifen therapy for breast cancer and prednisolone therapy for rheumatoid arthritis. Diffusion-weighted images detected multiple areas of hyperintensity in the posterior circulation system of the brain. Despite extensive examinations, we could not identify any embolic sources except hypoplasia of the right vertebral artery. We found decreased activity of protein C against its antigen level (activity: 59% versus antigen: 122%) with enhanced activity of coagulation factor VIII (178%) and von Willebrand factor (285%). DNA sequencing identified trinucleotide deletion of the PROC gene leading to 1 amino acid deletion at Lys-193 (p.Lys193del). We speculate that the PROC gene polymorphism may have participated in tamoxifen- and prednisolone- associated hypercoagulable state, leading to development of an embolic stroke in this patient..
20. Kana Ueki, Yoshinobu Wakisaka, Kuniyuki Nakamura, Yuji Shono, Shinichi Wada, Yoji Yoshikawa, Yuta Matsukuma, Takeshi Uchiumi, Dongchong Kang, Takanari Kitazono, Tetsuro Ago, Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes due to m.3243A > G mutation in a 76-year-old woman., J Neurol Sci., DOI: 10.1016/j.jns.2020.116791, 2020.05.
21. Matsuo R, Ago T, Kiyuna F, Sato N, Nakamura K, Kuroda J, Wakisaka Y, Kitazono T; Fukuoka Stroke Registry Investigators., Smoking Status and Functional Outcomes After Acute Ischemic Stroke., Stroke, 10.1161/STROKEAHA.119.027230., 51, 3, 846-852, 2020.03, 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: Unique identifier: 000000800..
22. Shibahara T, Ago T, Nakamura K, Tachibana M, Yoshikawa Y, Komori M, Yamanaka K, Wakisaka Y, Kitazono T., Pericyte-Mediated Tissue Repair through PDGFRβ Promotes Peri-Infarct Astrogliosis, Oligodendrogenesis, and Functional Recovery after Acute Ischemic Stroke., eNeuro, 10.1523/ENEURO.0474-19.2020., 7, 2, 2020.03.
23. Nakamura K, Ikeuchi T, Nara K, Rhodes CS, Zhang P, Chiba Y, Kazuno S, Miura Y, Ago T, Arikawa-Hirasawa E, Mukouyama YS, Yamada Y., Perlecan regulates pericyte dynamics in the maintenance and repair of the blood-brain barrier., J Cell Biol., 10.1083/jcb.201807178., 218, 10, 3506-3525, 2019.10, Ischemic stroke causes blood-brain barrier (BBB) breakdown due to significant damage to the integrity of BBB components. Recent studies have highlighted the importance of pericytes in the repair process of BBB functions triggered by PDGFRβ up-regulation. Here, we show that perlecan, a major heparan sulfate proteoglycan of basement membranes, aids in BBB maintenance and repair through pericyte interactions. Using a transient middle cerebral artery occlusion model, we found larger infarct volumes and more BBB leakage in conditional perlecan (Hspg2)-deficient (Hspg2 - / - -TG) mice than in control mice. Control mice showed increased numbers of pericytes in the ischemic lesion, whereas Hspg2 - / - -TG mice did not. At the mechanistic level, pericytes attached to recombinant perlecan C-terminal domain V (perlecan DV, endorepellin). Perlecan DV enhanced the PDGF-BB-induced phosphorylation of PDGFRβ, SHP-2, and FAK partially through integrin α5β1 and promoted pericyte migration. Perlecan therefore appears to regulate pericyte recruitment through the cooperative functioning of PDGFRβ and integrin α5β1 to support BBB maintenance and repair following ischemic stroke..
24. Komori M, Ago T, Wakisaka Y, Nakamura K, Tachibana M, Yoshikawa Y, Shibahara T, Yamanaka K, Kuroda J, Kitazono T., Early initiation of a factor Xa inhibitor can attenuate tissue repair and neurorestoration after middle cerebral artery occlusion., Brain Res., 10.1016/j.brainres.2019.05.020, 1718, 201-211, 2019.05, The timing of anti-coagulation therapy initiation after acute cardioembolic stroke remains controversial. We investigated the effects of post-stroke administration of a factor Xa inhibitor in mice, focusing on tissue repair and functional restoration outcomes. We initiated administration of rivaroxaban, a Xa inhibitor, immediately after permanent distal middle cerebral artery occlusion (pMCAO) in CB-17 mice harboring few leptomeningeal anastomoses at baseline. Rivaroxaban initiated immediately after pMCAO hindered the recovery of blood flow in ischemic areas by inhibiting leptomeningeal anastomosis development, and led to impaired restoration of neurologic functions with less extensive peri-infarct astrogliosis. Within infarct areas, angiogenesis and fibrotic responses were attenuated in rivaroxaban-fed mice. Furthermore, inflammatory responses, including the accumulation of neutrophils and monocytes/macrophages, local secretion of pro-inflammatory cytokines, and breakdown of the blood-brain barrier, were enhanced in infarct areas in mice treated immediately with rivaroxaban following pMCAO. The detrimental effects were not found when rivaroxaban was initiated after transient MCAO or on day 7 after pMCAO. Collectively, early post-stroke initiation of a factor Xa inhibitor may suppress leptomeningeal anastomosis development and blood flow recovery in ischemic areas, thereby resulting in attenuated tissue repair and functional restoration unless occluded large arteries are successfully recanalized..
25. Zhang P, Ishikawa M, Rhodes C, Doyle A, Ikeuchi T, Nakamura K, Chiba Y, He B, Yamada Y, Pannexin-3 Deficiency Delays Skin Wound Healing in Mice due to Defects in Channel Functionality., J Invest Dermatol., 10.1016/j.jid.2018.08.033., 139, 4, 909-918, 2019.04, Pannexin-3 (Panx3) is a gap junction protein that is required for regulating cell cycle exit and the differentiation of osteoblasts and chondrocytes during skeletal development. However, the role of Panx3 in skin tissue regeneration remains unclear. After dorsal skin punch biopsies, Panx3-knockout mice exhibited a significant delay in wound healing with insufficient re-epithelialization, decreased inflammatory reaction, and reduced collagen remodeling. Panx3 expression coincided with inflammatory reactions both in vivo and in vitro. By applying exogenous tumor necrosis factor-α to mimic inflammation in vitro, Panx3 expression was induced in HaCaT cells. In addition, Panx3 depletion reduced epithelial-mesenchymal transition during skin wound healing. A protein essential for signaling in epithelial-mesenchymal transition, transforming growth factor-β interacted with Panx3 by modulating intracellular adenosine triphosphate levels and thereby enhanced HaCaT cell migration ability with Panx3 overexpression. In conclusion, Panx3 plays a key role in the skin wound healing process by controlling keratinocytes and keratinocyte-mesenchyme cross-talk via hemichannel and endoplasmic reticulum Ca channel functions, which differs from another gap junction, connexin 43 (Cx43), during skin wound healing. 2+.
26. Tachibana M, Ago T, Wakisaka Y, Kuroda J, Shijo M, Yoshikawa Y, Komori M, Nishimura A, Makihara N, Nakamura K, Kitazono T., Early Reperfusion After Brain Ischemia Has Beneficial Effects Beyond Rescuing Neurons., Stroke., 10.1161/STROKEAHA.117.016689, 48, 8, 2222-2230, 2017.08, 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..
27. Nishimura A, Ago T, Kuroda J, Arimura K, Tachibana M, Nakamura K, Wakisaka Y, Sadoshima J, Iihara K, Kitazono T., Detrimental role of pericyte Nox4 in the acute phase of brain ischemia., J Cereb Blood Flow Metab., 10.1177/0271678X15606456, 36, 6, 1143-54, 2016.06, Pericytes are mural cells abundantly present in cerebral microvessels and play important roles, including the formation and maintenance of the blood-brain barrier. Nox4 is a major source of reactive oxygen species in cardiovascular cells and modulate cellular functions, particularly under pathological conditions. In the present study, we found that the expression of Nox4 was markedly induced in microvascular cells, including pericytes, in peri-infarct areas after middle cerebral artery occlusion stroke models in mice. The upregulation of Nox4 was greater in a permanent middle cerebral artery occlusion model compared with an ischemia/reperfusion transient middle cerebral artery occlusion model. We performed permanent middle cerebral artery occlusion on mice with Nox4 overexpression in pericytes (Tg-Nox4). Infarct volume was significantly greater with enhanced reactive oxygen species production and blood-brain barrier breakdown in peri-infarct areas in Tg-Nox4, compared with littermate controls. In cultured brain pericytes, Nox4 was significantly upregulated by hypoxia and was promptly downregulated by reoxygenation. Phosphorylation of NFκB and production of matrix metalloproteinase 9 were significantly increased in both cultured pericytes overexpressing Nox4 and in peri-infarct areas in Tg-Nox4. Collectively, Nox4 is upregulated in pericytes in peri-infarct areas after acute brain ischemia and may enhance blood-brain barrier breakdown through activation of NFκB and matrix metalloproteinase 9, thereby causing enlargement of infarct volume..
28. Nakamura K*, Arimura K*, Nishimura A, Tachibana M, Yoshikawa Y, Makihara N, Wakisaka Y, Kuroda J, Kamouchi M, Ooboshi H, Kitazono T, Ago T. * equally contributed, Possible involvement of basic FGF in the upregulation of PDGFRβ in pericytes after ischemic stroke., Brain Res., 10.1016/j.brainres.2015.11.003, 1630, 98-108, 2016.01, Central nervous system (CNS) pericytes have been recognized as an indispensable component of the neurovascular unit. The expression of platelet-derived growth factor receptor β (PDGFRβ) is markedly increased in CNS pericytes after brain ischemia. It has been elucidated that PDGFRβ, expressed in pericytes and pericyte-derived fibroblast-like cells, plays important roles in the maintenance of the blood-brain barrier (BBB) and in the repair process in infarct areas. The aim of this study was to uncover how the PDGFRβ expression is regulated in pericytes after brain ischemia. We found that basic fibroblast growth factor (bFGF), but neither hypoxia at 1% O2 nor acidification at pH 6.5, significantly upregulated the PDGFRβ expression in human cultured CNS pericytes. SU5402, an inhibitor of FGF receptor (FGFR), and inhibitors of its downstream effectors Akt and Erk abolished the bFGF-induced upregulation of PDGFRβ. On the other hand, acidification significantly upregulated the expression of bFGF, while hypoxia upregulated the expression of FGFR1 in the pericytes. The expression of bFGF and FGFR1 was markedly induced in the ischemic hemisphere after ischemic insult in a middle cerebral artery occlusion stroke model. Immunofluorescent double labeling demonstrated that the expression of bFGF and FGFR1 was co-localized with PDGFRβ-positive cells in peri-infarct areas. Moreover, treatment with bFGF enhanced cell growth and the PDGF-BB-induced migratory activity of cultured pericytes, which were significantly suppressed by SU5402 or Sunitinib, an inhibitor of PDGFR. These data suggested that increased bFGF upregulates the expression of PDGFRβ and may enhance PDGFRβ-mediated pericyte functions after brain ischemia..
29. Kuniyuki Nakamura, Tetsuro Ago, Akihiro Tsuchimoto, Nozomi Noda, Asako Nakamura, Toshiharu Ninomiya, Takeshi Uchiumi, Kazuhiko Tsuruya, Masahiro Kamouchi, Hiroaki Ooboshi, Takanari Kitazono, A CADASIL-Like Case with a Novel Noncysteine Mutation of the NOTCH3 Gene and Granular Deposits in the Renal Arterioles., Case reports in neurological medicine, 10.1155/2015/431461, 2015, 431461-431461, 2015.03, We herein report the finding of a 62-year-old male, who developed dysarthria and dysphagia, with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy- (CADASIL-) like cerebral lesions. He also suffered from slowly progressive renal failure with the findings of granular deposits similar to electron-dense granular osmiophilic material in the renal arterioles. We found a novel heterozygous missense mutation of the NOTCH3 gene, c.4039G>C in exon 24, resulting in a p.Gly1347Arg substitution in its extracellular domain. The noncysteine substitution may underlie the pathogenesis of white matter lesions in the brain and of the chronic renal failure in the present case..
30. Makihara N, Arimura K, Ago T, Tachibana M, Nishimura A, Nakamura K, Matsuo R, Wakisaka Y, Kuroda J, Sugimori H, Kamouchi M, Kitazono T., Involvement of platelet-derived growth factor receptor β in fibrosis through extracellular matrix protein production after ischemic stroke., Exp Neurol., 10.1016/j.expneurol.2014.12.007, 264, 127-34, 2015.02, Fibrosis is concomitant with repair processes following injuries in the central nervous system (CNS). Pericytes are considered as an origin of fibrosis-forming cells in the CNS. Here, we examined whether platelet-derived growth factor receptor β (PDGFRβ), a well-known indispensable molecule for migration, proliferation, and survival of pericytes, was involved in the production of extracellular matrix proteins, fibronectin and collagen type I, which is crucial for fibrosis after ischemic stroke. Immunohistochemistry demonstrated induction of PDGFRβ expression in vascular cells of peri-infarct areas at 3-7days in a mouse stroke model. The PDGFRβ-expressing cells extended from peri-infarct areas toward the ischemic core after day 7 while expressing fibronectin and collagen type I in the infarct areas. In contrast, desmin and α-smooth muscle actin, markers of pericytes, were only expressed in vascular cells. In PDGFRβ heterozygous knockout mice, the expression of fibronectin and collagen type I was attenuated at both mRNA and protein levels with an enlargement of the infarct volume after ischemic stroke compared with that in wild-type littermates. In cultured brain pericytes, the expression of PDGF-B, PDGFRβ, fibronectin, and collagen type I, but not desmin, was significantly increased by serum depletion (SD). The SD-induced upregulation of fibronectin and collagen type I was suppressed by SU11652, an inhibitor of PDGFRβ, while PDGF-B further increased the SD-induced upregulation. In conclusion, the expression level of PDGFRβ may be a crucial determinant of fibrosis after ischemic stroke. Moreover, PDGFRβ signaling participates in the production of fibronectin and collagen type I after ischemic stroke..
31. Kuroda J, Ago T, Nishimura A, Nakamura K, Matsuo R, Wakisaka Y, Kamouchi M, Kitazono T., Nox4 is a major source of superoxide production in human brain pericytes., J Vasc Res., 10.1159/000369930, 51, 6, 429-38, 2015.01, BACKGROUND: Pericytes are multifunctional cells surrounding capillaries and postcapillary venules. In brain microvasculature, pericytes play a pivotal role under physiological and pathological conditions by producing reactive oxygen species (ROS). The aims of this study were to elucidate the source of ROS and its regulation in human brain pericytes. METHODS: The expression of Nox enzymes in the cells was evaluated using RT-PCR and western blot. Superoxide production was determined by superoxide dismutase-inhibitable chemiluminescence. Silencing of Nox4 was performed using RNAi, and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: Nox4 was predominant among the Nox family in human brain pericytes. Membrane fractions of cells produced superoxide in the presence of NAD(P)H. Superoxide production was almost abolished with diphenileneiodonium, a Nox inhibitor; however, inhibitors of other possible superoxide-producing enzymes had no effect on NAD(P)H-dependent superoxide production. Pericytes expressed angiotensin II (Ang II) receptors, and Ang II upregulated Nox4 expression. Hypoxic conditions also increased the Nox4 expression. Silencing of Nox4 significantly reduced ROS production and attenuated cell proliferation. CONCLUSION: Our study showed that Nox4 is a major superoxide-producing enzyme and that its expression is regulated by Ang II and hypoxic stress in human brain pericytes. In addition, Nox4 may promote cell growth..
32. Nakamura K, Kamouchi M, Arimura K, Nishimura A, Kuroda J, Ishitsuka K, Tokami H, Sugimori H, Ago T, Kitazono T., Extracellular acidification activates cAMP responsive element binding protein via Na+/H+ exchanger isoform 1-mediated Ca2+ oscillation in central nervous system pericytes., Arterioscler Thromb Vasc Biol., 10.1161/ATVBAHA.112.254946, 32, 11, 2670-7, 2012.11, 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..
33. Ishitsuka K, Ago T, Arimura K, Nakamura K, Tokami H, Makihara N, Kuroda J, Kamouchi M, Kitazono T., Neurotrophin production in brain pericytes during hypoxia: a role of pericytes for neuroprotection., Microvasc Res., 10.1016/j.mvr.2012.02.009, 83, 3, 352-9, 2012.05, 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..
34. Arimura K, Ago T, Kamouchi M, Nakamura K, Ishitsuka K, Kuroda J, Sugimori H, Ooboshi H, Sasaki T, Kitazono T., PDGF receptor β signaling in pericytes following ischemic brain injury., Curr Neurovasc Res., 9, 1, 1-9, 2012.02, 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..
35. Shono Y, Kamouchi M, Kitazono T, Kuroda J, Nakamura K, Hagiwara N, Ooboshi H, Ibayashi S, Iida M., Change in Intracellular pH causes the toxic Ca2+ entry via NCX1 in neuron- and glia-derived cells., Cell Mol Neurobiol., 10.1007/s10571-009-9470-7, 30, 3, 453-60, 2010.04, Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca(2+) in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca(2+) during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca(2+) and pH were measured using the fluorescence of fura-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca(2+) transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca(2+) increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca(2+), diminished by a reduction of external Na(+), and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca(2+). Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca(2+) due to reversal of Ca(2+) transport via Na(+)/Ca(2+) exchanger coactivated with Na(+)/H(+) exchanger, which can cause cell death..
36. Ishikawa E, Ooboshi H, Kumai Y, Takada J, Nakamura K, Ago T, Sugimori H, Kamouchi M, Kitazono T, Ibayashi S, Iida M., Midkine gene transfer protects against focal brain ischemia and augments neurogenesis., J Neurol Sci., 10.1016/j.jns.2009.05.026, 285, 1-2, 78-84, 2009.10, 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..
37. Jiro Toyonaga, Kazuhiko Tsuruya, Kohsuke Masutani, Hiroto Maeda, Kuniyuki Nakamura, Masatomo Taniguchi, Hideki Hirakata, Mitsuo Iida, Hemorrhagic shock and obstructive uropathy due to a large rectus sheath hematoma in a patient on anticoagulant therapy., Internal medicine (Tokyo, Japan), 48, 24, 2119-22, 2009.09, A 54-year-old woman was transferred to our hospital with disseminated intravascular coagulation, and was treated with heparin. On hospitalization day 13, she developed lower abdominal pain and mass followed by circulatory shock. She became oliguric and laboratory tests showed serum creatinine of 3.5 mg/dL and hemoglobin of 7.4 g/dL. Computed tomography showed hematoma in the left rectus sheath, compressing the urinary bladder exteriorly, which resulted in worsening of bilateral hydronephrosis. Conservative treatment resulted in resolution of the rectus sheath hematoma and improvement of renal function. Rectus sheath hematoma can be treated conservatively without surgical intervention even in complicated cases..
38. Nakamura K, Kamouchi M, Kitazono T, Kuroda J, Shono Y, Hagiwara N, Ago T, Ooboshi H, Ibayashi S, Iida M., Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes., Microvasc Res., 10.1016/j.mvr.2008.12.001, 77, 3, 327-34, 2009.05, 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..
39. Nakamura K, Kamouchi M, Kitazono T, Kuroda J, Matsuo R, Hagiwara N, Ishikawa E, Ooboshi H, Ibayashi S, Iida M., Role of NHE1 in calcium signaling and cell proliferation in human CNS pericytes., Am J Physiol Heart Circ Physiol., 10.1152/ajpheart.01203.2007, 294, 4, H1700-7, 2008.04, The central nervous system (CNS) pericytes play an important role in brain microcirculation. Na(+)/H(+) exchanger isoform 1 (NHE1) has been suggested to regulate the proliferation of nonvascular cells through the regulation of intracellular pH, Na(+), and cell volume; however, the relationship between NHE1 and intracellular Ca(2+), an essential signal of cell growth, is still not known. The aim of the present study was to elucidate the role of NHE1 in Ca(2+) signaling and the proliferation of human CNS pericytes. The intracellular Ca(2+) concentration was measured by fura 2 in cultured human CNS pericytes. The cells showed spontaneous Ca(2+) oscillation under quasi-physiological ionic conditions. A decrease in extracellular pH or Na(+) evoked a transient Ca(2+) rise followed by Ca(2+) oscillation, whereas an increase in pH or Na(+) did not induce the Ca(2+) responses. The Ca(2+) oscillation was inhibited by an inhibitor of NHE in a dose-dependent manner and by knockdown of NHE1 by using RNA interference. The Ca(2+) oscillation was completely abolished by thapsigargin. The proliferation of pericytes was attenuated by inhibition of NHE1. These results demonstrate that NHE1 regulates Ca(2+) signaling via the modulation of Ca(2+) release from the endoplasmic reticulum, thus contributing to the regulation of proliferation in CNS pericytes..
40. Kazunori Toyoda, Ken Uda, Akiko Shirakawa, Kotaro Yasumori, Kuniyuki Nakamura, Tooru Inoue, Yasushi Okada, Combined and staged endovascular recanalization of cervical and intracranial arteries in hyperacute ischemic stroke., Internal medicine (Tokyo, Japan), 46, 23, 1935-6, 2007.12.
41. Kamouchi M, Kitazono T, Ago T, Wakisaka M, Kuroda J, Nakamura K, Hagiwara N, Ooboshi H, Ibayashi S, Iida M., Hydrogen peroxide-induced Ca2+ responses in CNS pericytes., Neurosci Lett., 416, 1, 12-6, 416(1):12-6, 2007.04, 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..