血管内皮前駆細胞の血管新生作用に対する水溶性スタチン(プラバスタチン)の影響

脂溶性HMG-CoAリダクターゼ・インヒビター(脂溶性スタチン)による血管内皮前駆細胞(EPC)動員,治療的血管形成作用改善効果は多数の報告がされている.しかしながら水溶性スタチン(プラバスタチン)のEPCに対する作用は充分に解明されていない.本研究により,水溶性スタチンがEPC内に取り込まれ,細胞内シグナルを刺激することでEPC動態に作用することを明らかとする.^C標識プラバスタチンにより,HepG2細胞を対照群としてEPC内へのプラバスタチン取り込みを測定した結果,EPC群は有意にプラバスタチンを細胞内に取り込んだ(*29.15±2.80vs7.82±0.47count/min/mg protein,^*p<0.000).EPCの遊走能測定実験において,プラバスタチン群は対照群と比較して,有意に遊走能を増加させた.また,抗アポトーシス作用測定実験において,プラバスタチン群は対照群と比較して有意にアポトーシスを抑制していた.更に,ウェスタンブロットを行ったところ,EPC内においてプラバスタチンはPI-3-kinase/Akt経路を刺激することにより,eNOSのリン酸化を行うことが判明した.この反応はPI-3/Akt阻害剤により消失することにより,PI-3-kinase/Akt経路依存性であることが確かめられた.In vivoにおいて,プラバスタチンをマウスに投与したところ,プラバスタチンは対照群と比較して投与時間依存性に末梢血液中のEPC数を増加させることが判明した.これらの結果により,水溶性スタチンであるプラバスタチンは,EPC内のPI-3-kinase/Akt/eNOS経路により,EPC遊走能・抗アポトーシス作用を活性化することが明らかとなった.Previous studies have demonstrated that hydrophobic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) mobilize circulating endothelial progenitor cells (EPCs), and contribute to therapeutic vasculogenesis. However, the effects of hydrophilic statin (pravastatin) on EPC kinetics remain to be investigated. Here, we investigated whether pravastatin stimulates EPC kinetics via intracellular signal transduction following its uptake by EPCs. Pravastatin uptake by cultured EPCs was measured by 14C-radio labeled molecules, and was compared with that by HepG2 cells as a negative control (^*29.15 ± 2.80 vs 7.82 ± 0.47 count/min/mg protein, ^*p<0.0001). EPC migratory activity toward pravastatin was significantly stimulated when compared with the vehicle group. EPC antiapoptotic assay by DAPI staining and cell death detection ELISA demonstrated abrogated apoptosis in the pravastatin group. In addition, pravastatin activated the PI-3-kinase/Akt pathway leading to endothelial nitricoxide synthase eNOS) activation. Co-treatment with PI-3/Akt inhibitors blocked pravastatin-induced Akt activation, indicating Akt activation through PI-3-kinase phosphorylation. When pravastatin was orally administered to nude mice, the number of circulating EPCs increased in a time-dependent fashion. These findings suggest that hydrophilic pravastatin exerts provasculogenic effects via the upregualtion of EPC migration and survival through PI-3-kinase/Akt/eNOS pathway activation following intracellular uptake

血管内皮前駆細胞の血管新生作用に対する水溶性スタチン(プラバスタチン)の影響

脂溶性HMG-CoAリダクターゼ・インヒビター(脂溶性スタチン)による血管内皮前駆細胞(EPC)動員,治療的血管形成作用改善効果は多数の報告がされている.しかしながら水溶性スタチン(プラバスタチン)のEPCに対する作用は充分に解明されていない.本研究により,水溶性スタチンがEPC内に取り込まれ,細胞内シグナルを刺激することでEPC動態に作用することを明らかとする.^<14>C標識プラバスタチンにより,HepG2細胞を対照群としてEPC内へのプラバスタチン取り込みを測定した結果,EPC群は有意にプラバスタチンを細胞内に取り込んだ(*29.15±2.80vs7.82±0.47count/min/mg protein,^*p<0.000).EPCの遊走能測定実験において,プラバスタチン群は対照群と比較して,有意に遊走能を増加させた.また,抗アポトーシス作用測定実験において,プラバスタチン群は対照群と比較して有意にアポトーシスを抑制していた.更に,ウェスタンブロットを行ったところ,EPC内においてプラバスタチンはPI-3-kinase/Akt経路を刺激することにより,eNOSのリン酸化を行うことが判明した.この反応はPI-3/Akt阻害剤により消失することにより,PI-3-kinase/Akt経路依存性であることが確かめられた.In vivoにおいて,プラバスタチンをマウスに投与したところ,プラバスタチンは対照群と比較して投与時間依存性に末梢血液中のEPC数を増加させることが判明した.これらの結果により,水溶性スタチンであるプラバスタチンは,EPC内のPI-3-kinase/Akt/eNOS経路により,EPC遊走能・抗アポトーシス作用を活性化することが明らかとなった.Previous studies have demonstrated that hydrophobic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) mobilize circulating endothelial progenitor cells (EPCs), and contribute to therapeutic vasculogenesis. However, the effects of hydrophilic statin (pravastatin) on EPC kinetics remain to be investigated. Here, we investigated whether pravastatin stimulates EPC kinetics via intracellular signal transduction following its uptake by EPCs. Pravastatin uptake by cultured EPCs was measured by 14C-radio labeled molecules, and was compared with that by HepG2 cells as a negative control (^*29.15 ± 2.80 vs 7.82 ± 0.47 count/min/mg protein, ^*p<0.0001). EPC migratory activity toward pravastatin was significantly stimulated when compared with the vehicle group. EPC antiapoptotic assay by DAPI staining and cell death detection ELISA demonstrated abrogated apoptosis in the pravastatin group. In addition, pravastatin activated the PI-3-kinase/Akt pathway leading to endothelial nitricoxide synthase eNOS) activation. Co-treatment with PI-3/Akt inhibitors blocked pravastatin-induced Akt activation, indicating Akt activation through PI-3-kinase phosphorylation. When pravastatin was orally administered to nude mice, the number of circulating EPCs increased in a time-dependent fashion. These findings suggest that hydrophilic pravastatin exerts provasculogenic effects via the upregualtion of EPC migration and survival through PI-3-kinase/Akt/eNOS pathway activation following intracellular uptake

Regeneration-associated cell transplantation contributes to tissue recovery in mice with acute ischemic stroke.

Various cell-based therapeutic strategies have been investigated for vascular and tissue regeneration after ischemic stroke. We have developed a novel cell population, called regeneration-associated cells (RACs), by quality- and quantity-controlled culture of unfractionated mononuclear cells. RACs were trans-arterially injected into 10-week-old syngeneic male mice at 1, 3, 5 or 7 days after permanent middle cerebral artery occlusion (MCAO) to determine the optimal timing for administration in terms of outcome at day 21. Next, we examined the effects of RACs injection at day 1 after MCAO on neurological deficits, infarct volume, and mediators of vascular regeneration and anti-inflammation at days 7 and 21. Infarct volume at day 21 was significantly reduced by transplantation of RACs at day 1 or 3. RACs injected at day 1 reduced the infarct volume at day 7 and 21. Angiogenesis and anti-inflammatory mediators, VEGF and IL-10, were increased at day 7, and VEGF was still upregulated at day 21. We also observed significantly enhanced ink perfusion in vivo, tube formation in vitro, and definitive endothelial progenitor cell colonies in colony assay. These results suggest that RAC transplantation in MCAO models promoted significant recovery of neural tissues through intensified anti-inflammatory and angiogenic effects

Batroxobin accelerated tissue repair via neutrophil extracellular trap regulation and defibrinogenation in a murine ischemic hindlimb model.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin restores ischemic tissue injury in terms of anti-inflammatory effects. In an in vitro flow cytometry assay for human neutrophil extracellular traps (NETs), batroxobin (DF-521; Defibrase) inhibited human NETs induced by tumor necrosis factor-α (TNF-α) in the presence of human fibrinogen. Next, the effect of batroxobin was investigated by immunohistochemistry of the anterior tibial muscle (ATM) in an ischemic hindlimb model using C57BL/6J mice intraperitoneally injected with DF-521 versus the saline control. NETs and fibrinogen deposition in the ischemic ATM decreased in DF-521-treated mice on day 2 after ischemia. Meanwhile, reverse transcription-quantitative PCR assay of the ischemic ATM unveiled continuous downregulation in the expression of the genes; Tnf-α and nitric oxide synthase2 (Nos2) with hypoxia-inducible factor-1α (Hif-1α) and vascular endothelial growth factor-a (Vegf-a) from day 3 to day 7, but the upregulation of arginase-1 (Arg-1) and placental growth factor (Plgf) with myogenin (Myog) on day 7. Daily intraperitoneal DF-521 injection for the initial 7 days into mice with ischemic hindlimbs promoted angiogenesis and arteriogenesis on day 14. Moreover, DF-521 injection accelerated myofiber maturation after day 14. Laser doppler imaging analysis revealed that blood perfusion in DF-521-injected mice significantly improved on day 14 versus the saline control. Thus, DF-521 improves microcirculation by protecting NETs with tissue defibrinogenation, thereby protecting against severe ischemic tissue injury and accelerating vascular and skeletal muscular regeneration. To our knowledge, batroxobin might be the first clinically applicable NET inhibitor against ischemic diseases

Regeneration-associated cells improve recovery from myocardial infarction through enhanced vasculogenesis, anti-inflammation, and cardiomyogenesis.

BackgroundConsidering the impaired function of regenerative cells in myocardial infarction (MI) patients with comorbidities and associated risk factors, cell therapy to enhance the regenerative microenvironment was designed using regeneration-associated cells (RACs), including endothelial progenitor cells (EPCs) and anti-inflammatory cells.MethodsRACs were prepared by quality and quantity control culture of blood mononuclear cells (QQMNCs). Peripheral blood mononuclear cells (PBMNCs) were isolated from Lewis rats and conditioned for 5 days using a medium containing stem cell factors, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6 to generate QQMNCs.ResultsA 5.3-fold increase in the definitive colony-forming EPCs and vasculogenic EPCs was observed, in comparison to naïve PBMNCs. QQMNCs were enriched with EPCs (28.9-fold, PConclusionQQMNCs conferred potent angiogenic and anti-inflammatory properties to the regenerative microenvironment, enhancing myocardiogenesis and functional recovery of rat MI hearts

Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model

Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure–volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM