Novel Lactam Type Pyridine Derivatives Improves Myocardium Dysfunction Derived from Ischemic Injury

The extended acute myocardial ischemia (AMI) results in cardiac myocytes death. In the present study, we show that lactam pyridine derivative, SK-D80375, have the effects on cell survival in hypoxic cardiomyocytes and might be used as an anti-ischemic drug. The lactam pyridine derivatives are inhibitors of the late sodium current, which decreases sodium-dependent intracellular calcium overload in ischemia/reperfusion-injured hearts. We found that pretreatment with SK-D80375 significantly decreased the level of intracellular Ca2+ and the expression level of the Na+-Ca2+ exchanger by 39±2.5% and 19±0.5%, respectively in hypoxic cardiomyocytes compared to untreated controls. In addition, the expression level of sarcoplasmic reticulum Ca2+ ATPase 2a was significantly increased by 37±1.5% in SK-D80375-treated hypoxic cardiomyocytes compared to untreated controls. The induction of Hsp70 was observed in SK-D80375-treated hypoxic cardiomyocytes with dose-dependent manner and the highest level of Hsp70 was induced at the concentration of 2.5 μM SK-D80375. The echocardiographic analysis showed that heart function was significantly improved in SK-D80375-injected ischemic hearts. These results demonstrate that lactam pyridine derivative, SK-D80375, have beneficial effects on hypoxia-induced cell death, therefore, might be used as a novel anti-ischemia drugope

Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex.

The integrity of transplanted mesenchymal stem cells (MSCs) for cardiac regeneration is dependent on cell-cell or cell-matrix adhesion, which is inhibited by reactive oxygen species (ROS) generated in ischemic surroundings after myocardial infarction. Intracellular ROS play a key role in the regulation of cell adhesion, migration, and proliferation. This study was designed to investigate the role of ROS on MSC adhesion. In H(2)O(2) treated MSCs, adhesion and spreading were inhibited and detachment was increased in a dose-dependent manner, and these effects were significantly rescued by co-treatment with the free radical scavenger, N-acetyl-L-cysteine (NAC, 1 mM). A similar pattern was observed on plates coated with different matrices such as fibronectin and cardiogel. Hydrogen peroxide treatment resulted in a marked decrease in the level of focal adhesion-related molecules, such as phospho-FAK and p-Src in MSCs. We also observed a significant decrease in the integrin-related adhesion molecules, alpha V and beta1, in H(2)O(2) treated MSCs. When injected into infarcted hearts, the adhesion of MSCs co-injected with NAC to the border region was significantly improved. Consequently, we observed that fibrosis and infarct size were reduced in MSC and NAC-injected rat hearts compared to in MSC-only injected hearts. These results indicate that ROS inhibit cellular adhesion of engrafted MSCs and provide evidence that the elimination of ROS might be a novel strategy for improving the survival of engrafted MSCsope

Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca(2+) homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium.ope

PDGFRβ 타겟 신규 microRNA-9에 의한 신생내막 형성 제어

Dept. of Medical Science/박사Vascular smooth muscle cell (VSMC) proliferation and migration are important phenomena in atherosclerosis and in other vasculopathies. These diseases are promoted by the release of various cytokines and growth factors into the intima. These alterations down-regulate the expression of VSMC-specific contractile phenotype genes and promote VSMC proliferation and migration. The PDGF/PDGF receptor (PDGFR) interaction, which is a vascular disease cue, induces VSMC proliferation and migration, and eventually, vascular disease occurs. Additionally, the PDGF/PDGFR signaling pathway regulates phenotypic switching from a contractile to a proliferative phenotype. In the present study, microRNA-9 (miR-9) was identified as a novel regulator of VSMC proliferation and migration that suppresses PDGFRβ signaling. VSMC-specific phenotypic switching to a contractile phenotype was also observed under miR-9 overexpression conditions. Furthermore, several small molecules that regulate the induction of endogenous miR-9 expression were screened to overcome the limitation of directly transplanting miRs into damaged tissue in vivo. Among these molecules, SQ22536 (Drug 8) induced endogenous miR-9 expression. In addition, Drug 8 regulated endogenous miR-9 through two different mechanisms: 1) epigenetic modification by regulating histone deacetylase (HDAC) expression and 2) transcriptional regulation by phosphorylating and activating the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) gene. Moreover, Drug 8 regulated VSMC proliferation and migration by suppressing PDGFRβ expression and its downstream signaling molecules, such as miR-9. Furthermore, when Drug 8 was injected in a rat balloon injury model, neointimal formation decreased, and the expression of proliferation-related proteins was down-regulated. Taken together, these findings suggest that miR-9 induction through small molecule treatment is an effective therapeutic strategy to repress neointimal hyperplasia.prohibitio

심근 재생을 위한 허혈성 중간엽 줄기세포의 Fas 억제

Dept. of Medical Science/석사The use of mesenchymal stem cells (MSCs) as a therapy for myocardial disease is limited by poor viability of the transplanted cells. Reactive oxygen species (ROS) are generated in ischemic surroundings after myocardial infarction. Ischemic heart generated Fas ligand (FasL). FasL belongs to the TNF family that induces apoptosis in target cells binding Fas. This study aimed to determine if Fas-FasL complex mediated the death of MSCs in the ischemic heart. Expression levels of FasL were detected in a myocardial ischemia-reperfusion injury model. Expression of Fas was significantly increased in H2O2-treated MSCs in vitro. Survival of FasL-treated MSCs was decreased in H2O2-treatment compared to non-treated cells. When Fas was blocked using a Fas recombinant Fc chimera (Fas/Fc), caspase-8 and caspase-3 levels were lower than in FasL-treated MSCs. Increased survival was observed in Fas/Fc-treated MSCs compared to FasL-treated cells. These results indicate that Fas-Fas L complex interferes with the survival of MSCs and confirm a major role for the Fas-Fas L cytotoxic pathway in cardiac infarction.ope

Anti-death strategies against oxidative stress in grafted mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess the potential for use in cell-based therapy for repair of myocardial injury. The therapeutic potential of MSCs is based on the capacity of MSCs to differentiate into cardiac tissue and release paracrine factors. However, a major problem in the clinical application of MSC-based therapy is the poor viability of transplanted MSCs at the site of graft due to harsh microenvironment conditions, such as ischemia and/or anoikis. Ischemia after myocardial infarction (MI) and interaction of MSCs with their niche is associated with increased production of reactive oxygen species (ROS). ROS hinder cell adhesion and induce detachment of cells, which induces anoikis signals in implanted MSCs. Therefore, strategies to regulate oxidative stress following the implantation of MSCs are therapeutically attractive. In this review, we first describe ROS as a major obstacle in MSC-based therapy and focus on manipulation of implanted MSCs to reduce ROS-mediated anoikis.ope

Development of human parabiosis models from stem cell-derived lung organoids

줄기세포 폐오가노이드 기반 인간 파라바이오시스 모델 개발KGM113201

ROS-mediated bidirectional regulation of miRNA results in distinct pathologic heart conditions

Under distinct pathological heart conditions, the expression of a single miRNA can display completely opposite patterns. However, the mechanism underlying the bidirectional regulation of a single miRNA and the clinical implications of this regulation remain largely unknown. To address this issue, we examined the regulation of miR-1, one of the most abundant miRNAs in the heart, during cardiac hypertrophy and ischemia/reperfusion (I/R). Our data indicated that different magnitudes and chronicities of ROS levels in cardiomyocytes resulted in differential expression of miR-1, subsequently altering the expression of myocardin. In animal models, the administration of a miR-1 mimic attenuated cardiac hypertrophy by suppressing the transverse aortic constriction-induced increase in myocardin expression, whereas the administration of anti-miR-1 ameliorated I/R-induced cardiac apoptosis and deterioration of heart function. Our findings indicated that a pathologic stimulus such as ROS can bidirectionally alter the expression of miRNA to contribute to the development of pathological conditions exhibiting distinct phenotypes and that the meticulous adjustment of the pathological miRNA levels is required to improve clinical outcomes.ope

PLCδ1 Protein Rescues Ischemia-reperfused Heart by the Regulation of Calcium Homeostasis

Myocardial Ca2+ overload induced by ischemia/reperfusion (I/R) is a major element of myocardial dysfunction in heart failure. Phospholipase C (PLC) plays important roles in the regulation of the phosphoinositol pathway and Ca2+ homeostasis in various types of cells. Here, we investigated the protective role of PLCδ1 against myocardial I/R injury through the regulation of Ca2+ homeostasis. To investigate its role, PLCδ1 was fused to Hph1, a cell-permeable protein transduction domain (PTD), and treated into rat neonatal cardiomyocytes and rat hearts under respective hypoxia-reoxygenation (H/R) and ischemia-reperfusion conditions. Treatment with Hph1-PLCδ1 significantly inhibited intracellular Ca2+ overload, reactive oxygen species generation, mitochondrial permeability transition pore opening, and mitochondrial membrane potential elevation in H/R neonatal cardiomyocytes, resulting in the inhibition of apoptosis. Intravenous injections of Hph1-PLCδ1 in rats with I/R-injured myocardium caused significant reductions in infarct size and apoptosis and also improved systolic and diastolic cardiac functioning. Furthermore, a small ions profile obtained using time-of-flight secondary ion mass spectrometry showed that treatment with Hph1-PLCδ1 leads to significant recovery of calcium-related ions toward normal levels in I/R-injured myocardium. These results suggest that Hph1-PLCδ1 may manifest as a promising cardioprotective drug due to its inhibition of the mitochondrial apoptotic pathway in cells suffering from I/R injury.ope

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1μM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the β-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.ope