Onju Ham

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 H2O2 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, αV and β1, in H2O2 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 MSCs. STEM CELLS 2010;28:555–563

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is critical for successful cartilage regeneration. Several methods have been developed to attempt to chondrogenic differentiation, because chondrogenic differentiated cells can form stable cartilage and induce expression of a cartilage-specific phenotype. In this study, we found that both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling.

Up-regulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3β protein expression

Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response.

Antiarrhythmic Potential of Mesenchymal Stem Cell Is Modulated by Hypoxic Environment

OBJECTIVES The purpose of this study was to evaluate the antiarrhythmic potential of mesenchymal stem cells (MSC) under a different environment. BACKGROUND Little is known about how environmental status affects antiarrhythmic potential of MSCs. METHODS To investigate the effect of paracrine factors secreted from MSCs under different circumstances on arrhythmogenicity in rats with myocardial infarction, we injected paracrine media (PM) secreted under hypoxic, normoxic conditions (hypoxic PM and normoxic PM), and MSC into the border zone of infarcted myocardium in rats. RESULTS We found that the injection of hypoxic PM, but not normoxic PM, markedly restored conduction velocities, suppressed focal activity, and prevented sudden arrhythmic deaths in rats. Underlying this electrophysiological alteration was a decrease in fibrosis, restoration of connexin 43, alleviation of Ca(2+) overload, and recovery of Ca(2+)-regulatory ion channels and proteins, all of which is supported by proteomic data showing that several paracrine factors including basic fibroblast growth factor, insulinlike growth factor 1, hepatocyte growth factor, and EF-hand domain-containing 2 are potential mediators. When compared with PM, MSC injection did not reduce or prevent arrhythmogenicity, suggesting that the antiarrhythmic or proarrhythmic potential of MSC is mainly dependent on paracrine factors. CONCLUSIONS A hypoxic or normoxic environment surrounding MSC affects the type and properties of the growth factors or cytokines, and these secreted molecules determine the characteristics of the electro-anatomical substrate of the surrounding myocardium.

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ.

A change in intracellular free calcium (Ca(2+)) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca(2+) signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca(2+)-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca(2+) overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca(2+) overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca(2+)-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca(2+) overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

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 Ca2+ 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.

Therapeutic Potential of Differentiated Mesenchymal Stem Cells for Treatment of Osteoarthritis

Osteoarthritis (OA) is a chronic, progressive, and irreversible degenerative joint disease. Conventional OA treatments often result in complications such as pain and limited activity. However, transplantation of mesenchymal stem cells (MSCs) has several beneficial effects such as paracrine effects, anti-inflammatory activity, and immunomodulatory capacity. In addition, MSCs can be differentiated into several cell types, including chondrocytes, osteocytes, endothelia, and adipocytes. Thus, transplantation of MSCs is a suggested therapeutic tool for treatment of OA. However, transplanted naïve MSCs can cause problems such as heterogeneous populations including differentiated MSCs and undifferentiated cells. To overcome this problem, new strategies for inducing differentiation of MSCs are needed. One possibility is the application of microRNA (miRNA) and small molecules, which regulate multiple molecular pathways and cellular processes such as differentiation. Here, we provide insight into possible strategies for cartilage regeneration by transplantation of differentiated MSCs to treat OA patients.

Enhancement of MSC adhesion and therapeutic efficiency in ischemic heart using lentivirus delivery with periostin.

Many approaches have shown beneficial effects of modified mesenchymal stem cells (MSCs) for treatment of infarcted myocardium, but have primarily focused on enhancing the survival of transplanted MSCs. Here, we show the dual benefits of periostin-overexpressing MSCs (p-MSCs) for infarcted myocardium. P-MSCs led to the marked histological and functional recovery of infarcted myocardium by enhancing survival of MSCs and directly preventing apoptosis of cardiomyocytes. Survival of p-MSCs themselves and cardiomyocytes co-cultured with p-MSCs or treated with the conditioned media from p-MSCs was significantly increased under hypoxic conditions. Decreases in adhesion-related integrins were reversed in cardiomyocytes co-cultured with p-MSCs, followed by increases in p-PI3K and Akt, indicating that periostin activates the PI3K pathway through adhesion-related integrins. When p-MSCs were injected into myocardial infarcted rats, histological pathology and cardiac function were significantly improved compared to MSC-injected controls. Thus, periostin might be a new target of therapeutic treatments using MSCs as carriers for infarcted myocardium.

MicroRNA‐365 Inhibits the Proliferation of Vascular Smooth Muscle Cells by Targeting Cyclin D1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle‐specific cyclin D1 was found to be a potential target of miR‐365 by direct binding. Through an in vitro experiment, we showed that exogenous miR‐365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR‐365 was observed to block G1/S transition in platelet‐derived growth factor‐bb (PDGF‐bb)‐induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF‐bb, angiotensin II (Ang II), and serum, led to the downregulation of miR‐365 expression levels. The expression of miR‐365 was confirmed in balloon‐injured carotid arteries. Taken together, our results suggest an anti‐proliferative role for miR‐365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR‐365 may influence neointimal formation in atherosclerosis patients. J. Cell. Biochem. 115: 1752–1761, 2014.

Overexpression of phosphoinositide-3-kinase class II alpha enhances mesenchymal stem cell survival in infarcted myocardium

The efficacy of mesenchymal stem cell (MSC) therapy for myocardial regeneration is limited by the poor survival of stem cells after transplantation into the infarcted heart. To improve the cell survival of MSCs in the infarcted heart, MSCs were genetically engineered to overexpress phosphoinositide-3-kinase class II alpha (PI3K-C2α). PI3K-C2α overexpression increased PI3K expression and the cell viability of MSCs. Furthermore, levels of survival-related phosphorylation were elevated in PI3K-C2α-MSCs. But, the level of apoptotic proteins downregulated and the number of PI-positive cells decreased in PI3K-C2α-MSCs compared to hypoxic MSCs. Nine rats per group had 1×10(6) cells (20 μl PBS) transplanted after myocardial infarction. One week after transplantation, infarct size and area of fibrosis were reduced in the PI3K-C2α-MSC-transplanted group. The number of TUNEL positive cells declined, while the mean microvessel count per field was higher in the PI3K-C2α-MSC group than the MSC-injected group. Heart function was improved in the PI3K-C2α-MSCs group as assessed using a Millar catheter at 3weeks after transplantation. These findings suggest that overexpression of PI3K-C2α in MSCs can assist cell survival and enhance myocardial regeneration.