Ataru Nishimura

Nox4 Is a Major Source of Superoxide Production in Human Brain Pericytes

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.

Involvement of platelet-derived growth factor receptor β in fibrosis through extracellular matrix protein production after ischemic stroke.

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.

Detrimental role of pericyte Nox4 in the acute phase of brain ischemia

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.

Possible involvement of basic FGF in the upregulation of PDGFRβ in pericytes after ischemic stroke

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.

Extracellular Acidification Activates cAMP Responsive Element Binding Protein via Na+/H+ Exchanger Isoform 1–Mediated Ca 2+ Oscillation in Central Nervous System Pericytes

Objective—We have previously shown that Na+/H+ exchanger isoform 1 (NHE1) plays an important role in Ca2+ signaling and cell proliferation in human central nervous system (CNS) pericytes. The aims of the present study were to elucidate how NHE1–induced Ca2+ 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 Ca2+ oscillation and subsequently phosphorylated Ca2+/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. Conclusion—Our results indicate that extracellular acidosis induces Ca2+ oscillation via NHE1, leading to Ca2+/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.

Prehospital antiplatelet use and functional status on admission of patients with non-haemorrhagic moyamoya disease: a nationwide retrospective cohort study (J-ASPECT study).

Objectives To elucidate the association between antiplatelet use in patients with non-haemorrhagic moyamoya disease before hospital admission and good functional status on admission in Japan. Design Retrospective, multicentre, non-randomised, observational study. Setting Nationwide registry data in Japan. Participants A total of 1925 patients with non-haemorrhagic moyamoya disease admitted between 1 April 2012 and 31 March 2014 in Japan. Main outcome measure We performed propensity score-matched analysis to examine the association between prehospital antiplatelet use and no significant disability on hospital admission, as defined by a modified Rankin Scale score of 0 or 1. Results Propensity-matched patients who received prehospital antiplatelet drugs were associated with a good outcome on hospital admission (OR adjusted for all covariates, 3.82; 95% CI 1.22 to 11.99) compared with those who did not receive antiplatelet drugs prior to hospital admission. Conclusions Prehospital antiplatelet use was significantly associated with good functional status on hospital admission among patients with non-haemorrhagic moyamoya disease in Japan. Our results suggest that prehospital antiplatelet use should be considered when evaluating outcomes of patients with non-haemorrhagic moyamoya disease.

Comparing intracerebral hemorrhages associated with direct oral anticoagulants or warfarin

Objectives This cross-sectional survey explored the characteristics and outcomes of direct oral anticoagulant (DOAC)–associated nontraumatic intracerebral hemorrhages (ICHs) by analyzing a large nationwide Japanese discharge database. Methods We analyzed data from 2,245 patients who experienced ICHs while taking anticoagulants (DOAC: 227; warfarin: 2,018) and were urgently hospitalized at 621 institutions in Japan between April 2010 and March 2015. We compared the DOAC- and warfarin-treated patients based on their backgrounds, ICH severities, antiplatelet therapies at admission, hematoma removal surgeries, reversal agents, mortality rates, and modified Rankin Scale scores at discharge. Results DOAC-associated ICHs were less likely to cause moderately or severely impaired consciousness (DOAC-associated ICHs: 31.3%; warfarin-associated ICHs: 39.4%; p = 0.002) or require surgical removal (DOAC-associated ICHs: 5.3%; warfarin-associated ICHs: 9.9%; p = 0.024) in the univariate analysis. Propensity score analysis revealed that patients with DOAC-associated ICHs also exhibited lower mortality rates within 1 day (odds ratio [OR] 4.96, p = 0.005), within 7 days (OR 2.29, p = 0.037), and during hospitalization (OR 1.96, p = 0.039). Conclusions This nationwide study revealed that DOAC-treated patients had less severe ICHs and lower mortality rates than did warfarin-treated patients, probably due to milder hemorrhages at admission and lower hematoma expansion frequencies.

Status and Future Perspectives of Utilizing Big Data in Neurosurgical and Stroke Research

The management, analysis, and integration of Big Data have received increasing attention in healthcare research as well as in medical bioinformatics. The J-ASPECT study is the first nationwide survey in Japan on the real-world setting of stroke care using data obtained from the diagnosis procedure combination-based payment system. The J-ASPECT study demonstrated a significant association between comprehensive stroke care (CSC) capacity and the hospital volume of stroke interventions in Japan; further, it showed that CSC capabilities were associated with reduced in-hospital mortality rates. Our study aims to create new evidence and insight from ‘real world’ neurosurgical practice and stroke care in Japan using Big Data. The final aim of this study is to develop effective methods to bridge the evidence-practice gap in acute stroke healthcare. In this study, the authors describe the status and future perspectives of the development of a new method of stroke registry as a powerful tool for acute stroke care research.

Ruptured Aneurysm of an Aberrant Internal Carotid Artery Successfully Treated with Simultaneous Intervention and Surgery in a Hybrid Operating Room

BACKGROUND Aberrant internal carotid artery (aICA) is an anatomic anomaly whereby the internal carotid artery courses through the tympanic cavity without separation by bone. Because aICA is rare, there are no definite treatment strategies for aICA and its complications. CASE DESCRIPTION We report a case of aICA accompanied by pseudoaneurysm formation and massive bleeding. The patient was a 31-year-old woman with a 2-year history of hearing loss, ear fullness, and pulsatile tinnitus in her left ear. After a diagnosis of otitis media with effusion, she underwent a myringotomy and massive arterial bleeding occurred. After the bleeding was temporarily stopped, aICA and pseudoaneurysm formation on the aICA were shown. To prevent rebleeding, we performed endovascular internal trapping around the pseudoaneurysm after performing common carotid artery to radial artery to middle cerebral artery bypass grafting. After surgery, the aneurysm disappeared. In addition, no new neurologic complications were observed, and the patients hearing improved and the tinnitus diminished. CONCLUSIONS This is the first case report of an aICA complicated by pseudoaneurysm formation successfully treated with simultaneous endovascular trapping and high-flow bypass in a hybrid operating room.

Early Reperfusion After Brain Ischemia Has Beneficial Effects Beyond Rescuing Neurons

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 &bgr;-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 &bgr; 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.