Koichi Arimura

PDGF Receptor β Signaling in Pericytes Following Ischemic Brain Injury

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.

Neurotrophin production in brain pericytes during hypoxia: a role of pericytes for neuroprotection.

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.

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.

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.

Surgical Management of Intracranial Artery Dissection

Intracranial artery dissection (IAD) is a relatively rare cause of stroke, but it has been recognized increasingly with recent advances of the neuroimaging technique. Since rebleeding occurs frequently in the acute stage in the ruptured IAD, urgent surgical treatment should be performed to prevent rebleeding. On the other hand, surgical treatment for unruptured IAD is controversial because it has little risk for bleeding. However, surgical treatment for unruptured IAD may be considered if the formation or enlargement of the aneurysmal dilatation has been confirmed. Since there are several proposed surgical strategies for IAD, it is important to select an appropriate strategy on a case-by-case basis. If the risk of infarction due to vessel occlusion is high, combined bypass surgery should be considered.

Acceleration-selective Arterial Spin-labeling MR Angiography Used to Visualize Distal Cerebral Arteries and Collateral Vessels in Moyamoya Disease

Purpose To evaluate and compare the performance of acceleration-selective arterial spin labeling (AccASL) magnetic resonance (MR) angiography in the visualization of cerebral arteries and collateral vessels in patients with Moyamoya disease with that of time-of-flight (TOF) MR angiography, with digital subtraction angiography (DSA) as the reference standard. Materials and Methods Thirty-six cerebral hemispheres from 22 patients with Moyamoya disease underwent TOF and AccASL MR angiography and DSA. Qualitative evaluations included imaging of the terminal internal carotid artery (ICA), distal middle cerebral arteries (MCAs), Moyamoya vessels, and leptomeningeal anastomosis (LMA) collaterals with reference to DSA. Quantitative evaluations included assessment of contrast-to-noise ratio (CNR) and number of vessels in MCA branches. The linear mixed-effect model was used to compare the two methods. Results Mean scores for qualitative evaluation were significantly higher with AccASL angiography than with TOF angiography for imaging distal MCAs (3.9 ± 0.3 [standard deviation] vs 2.9 ± 1.1; P < .001), Moyamoya vessels (3.6 ± 0.6 vs 2.7 ± 0.9, P < .001), and LMA collaterals (3.8 ± 0.6 vs 1.8 ± 0.7, P < .001). Scores for steno-occlusive degree around the terminal ICAs were better with TOF angiography than with AccASL angiography (2.6 ± 0.5 vs 2.4 ± 0.6, P = .023). CNRs in the M4 segment were significantly higher with AccASL angiography (11.9 ± 12.9, P < .001) than with TOF angiography (4.1 ± 7.9). The number of vessels was significantly higher with AccASL angiography (18.3 ± 5.0, P < .001) than with TOF angiography (8.9 ± 4.9). The increase in the number of vessels from TOF angiography to AccASL angiography was greater in patients with severe ICA steno-occlusion (late ICA stage group, 11.4 ± 4.5; early ICA stage group, 6.8 ± 4.0; P = .007) and well-developed leptomeningeal anastomosis (mildly developed LMA group, 7.1 ± 4.3; well-developed LMA group, 11.3 ± 4.5; P = .011). Conclusion AccASL MR angiography enables better visualization of distal cerebral arteries and collateral vessels in patients with Moyamoya disease than does TOF MR angiography, while TOF MR angiography enables better visualization of stenosis of proximal arteries. Both methods work in a mutually beneficial manner in the assessment of cerebral arteries.

Endovascular parent-artery occlusion of large or giant unruptured internal carotid artery aneurysms. A long-term single-center experience ☆

The development of stent-like devices has increased treatment options for complex internal carotid artery (ICA) aneurysms, but the optimal treatment remains unclear. The purpose of this study was to evaluate the safety and efficacy of endovascular parent-artery occlusion (PAO) for ICA aneurysms. We retrospectively reviewed 28 patients with unruptured ICA aneurysms ⩾10mm treated with PAO between April 2002 and March 2015 at our institution. Patients who developed neurologic symptoms or with venous-phase delay >2s during balloon test occlusion were not treated by PAO. Patients with venous-phase delays of 1-2s underwent superficial temporal artery to middle cerebral artery (STA-MCA) bypass prior to PAO. The median patient age was 65 (range, 26-84)years. Nineteen aneurysms (68%) were located in the cavernous segment. The median aneurysm size was 25 (range 11-40)mm. Venous-phase delay of 1-2s was observed in five patients. Perioperative ischemic complications (N=9, 32%), which occurred within 30days after treatment, were significantly associated with venous-phase delays of 1-2s (p<0.01) and history of hypertension (p<0.01). Six-month morbidity was observed in one (3.6%) patient. Complete occlusion at final follow-up and delayed (i.e. ⩾31days after treatment) ischemic events were observed in 100% and 0% of patients, respectively, over a median period of 63 (range, 6-147) months. Despite the high frequency of perioperative ischemic episodes, endovascular PAO with selective use of STA-MCA bypass showed excellent long-term outcomes in patients with unruptured ICA aneurysms ⩾10mm.