||Tomoya Shibahara, Kuniyuki Nakamura, Yoshinobu Wakisaka, Masahiro Shijo, Kei Yamanaka, Masamitsu Takashima, Hayato Takaki, Masaoki Hidaka, Takanari Kitazono, Tetsuro Ago, PDGFR β-positive cell-mediated post-stroke remodeling of fibronectin and laminin α 2 for tissue repair and functional recovery, J Cereb Blood Flow Metab
., 10.1177/0271678X221145092, 43, 4, 518-530, 2023.04.
||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..
||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..
||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.
||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..
||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..
||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..
||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..
||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..
||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..