Esm1 and Stc1 as Angiogenic Factors Responsible for Protective Actions of Adipose-Derived Stem Cell Sheets on Chronic Heart Failure After Rat Myocardial Infarction.

BACKGROUND: Although adipose-derived stem cell (ADSC) sheets improve the cardiac function after myocardial infarction (MI), underlying mechanisms remain to be elucidated. The aim of this study was to determine the fate of transplanted ADSC sheets and candidate angiogenic factors released from ADSCs for their cardiac protective actions.Methods and Results:MI was induced by ligation of the left anterior descending coronary artery. Sheets of transgenic (Tg)-ADSCs expressing green fluorescence protein (GFP) and luciferase or wild-type (WT)-ADSCs were transplanted 1 week after MI. Both WT- and Tg-ADSC sheets improved cardiac functions evaluated by echocardiography at 3 and 5 weeks after MI. Histological examination at 5 weeks after MI demonstrated that either sheet suppressed fibrosis and increased vasculogenesis. Luciferase signals from Tg-ADSC sheets were detected at 1 and 2 weeks, but not at 4 weeks, after transplantation. RNA sequencing of PKH (yellow-orange fluorescent dye with long aliphatic tails)-labeled Tg-ADSCs identified mRNAs of 4 molecules related to angiogenesis, including those of Esm1 and Stc1 that increased under hypoxia. Administration of Esm1 or Stc1 promoted tube formation by human umbilical vein endothelial cells. CONCLUSIONS: ADSC sheets improved cardiac contractile functions after MI by suppressing cardiac fibrosis and enhancing neovascularization. Transplanted ADSCs existed for >2 weeks on MI hearts and produced the angiogenic factors Esm1 and Stc1, which may improve cardiac functions after MI.

ß-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

BACKGROUND: Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a ß-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. CONCLUSIONS: ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation.

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.

Pretreatment with an angiotensin II receptor blocker abolished ameliorating actions of adipose-derived stem cell sheets on cardiac dysfunction and remodeling after myocardial infarction.

INTRODUCTION: Cell sheets using myoblasts have been developed for the treatment of heart failure after myocardial infarction (MI) bridging to heart transplantation. Stem cells are supposed to be better than myoblasts as a source of cells, since they possess a potential to proliferate and differentiate into cardiomyocytes, and also have capacity to secrete angiogenic factors. Adipose-derived stem cells (ASCs) obtained from fat tissues are expected to be a new cell source for ASC sheet therapies. Administration of angiotensin II receptor blockers (ARBs) is a standard therapy for heart failure after MI. However, it is not known whether ARBs affect the cell sheet therapy. This study aimed to examine ameliorating effects of ASC sheets on heart failure and remodeling after MI, and how pretreatment with ARBs prior to the creation of MI and ASC sheet transplantation modifies the effects of ASC sheets. METHODS: ASCs were isolated from fat tissues of wild-type rats, and ASC sheets were engineered on temperature-responsive dishes. In in vitro studies using cultured cells, mRNA levels of vascular endothelial growth factor (VEGF) in ASCs were determined by RT-PCR in the presence of angiotensin II and/or an ARB, irbesartan, under normoxia and hypoxia; mRNA and protein levels of angiotensin II receptor type 1a (AT1aR), type 1b (AT1bR) and type 2 (AT2R) were also determined by RT-PCR and western blotting. In in vivo studies using a rat MI model, effects of transplanted ASC sheets and/or irbesartan on cardiac functions and remodeling after MI were evaluated by echocardiography, histological analysis and molecular biological techniques. RESULTS: In the in vitro studies, ASCs expressed higher levels of VEGF mRNA under hypoxia. They also expressed mRNA and protein of AT1aR but not AT1bR or AT2R. Under normoxia, angiotensin II increased the level of VEGF mRNA in ASCs, which was abolished by irbesartan. Under hypoxia, irbesartan reduced the level of VEGF mRNA in ASCs regardless of whether angiotensin II was present or not. In the in vivo studies, ASC sheets improved cardiac functions after MI, leading to decreased interstitial fibrosis and increased capillary density in border zones. These effects of ASC sheets were abolished by oral administration of irbesartan before MI and their transplantation. CONCLUSIONS: ASC sheets ameliorated cardiac dysfunctions and remodeling after MI via increasing VEGF expression, which was abolished by pretreatment with irbesartan before the creation of MI and transplantation.

Protective Effects of Topiroxostat on an Ischemia-Reperfusion Model of Rat Hearts.

BACKGROUND: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. CONCLUSIONS: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.