Byeong-Wook Song

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

Mesenchymal Stem Cells Pretreated with Delivered Hph‐1‐Hsp70 Protein Are Protected from Hypoxia‐Mediated Cell Death and Rescue Heart Functions from Myocardial Injury

Mesenchymal stem cell (MSC) therapy for myocardial injury has inherent limitations due to the poor viability of MSCs after cell transplantation. In this study, we directly delivered Hsp70, a protein with protective functions against stress, into MSCs, using the Hph‐1 protein transduction domain ex vivo for high transfection efficiency and low cytotoxicity. Compared to control MSCs in in vitro hypoxic conditions, MSCs delivered with Hph‐1‐Hsp70 (Hph‐1‐Hsp70‐MSCs) displayed higher viability and anti‐apoptotic properties, including Bcl2 increase, reduction of Bax, JNK phosphorylation and caspase‐3 activity. Hsp70 delivery also attenuated cellular ATP‐depleting stress. Eight animals per group were used for in vivo experiments after occlusion of the left coronary artery. Transplantation of Hph‐1‐Hsp70‐MSCs led to a decrease in the fibrotic heart area, and significantly reduced the apoptotic positive index by 19.5 ± 2%, compared to no‐treatment controls. Hph‐1‐Hsp70‐MSCs were well‐integrated into the infarcted host myocardium. The mean microvessel count per field in the infarcted myocardium of the Hph‐1‐Hsp70‐MSC‐treated group (122.1 ± 13.5) increased relative to the MSC‐treated group (75.9 ± 10.4). By echocardiography, transplantation of Hph‐1‐Hsp70‐MSCs resulted in additional increases in heart function, compared to the MSCs‐transplanted group. Our results may help formulate better clinical strategies for in vivo MSC cell therapy for myocardial damage. STEM CELLS 2009;27:2283–2292

Integrin-Linked Kinase Is Required in Hypoxic Mesenchymal Stem Cells for Strengthening Cell Adhesion to Ischemic Myocardium†‡

Mesenchymal stem cells (MSCs) therapy has limitations due to the poor viability of MSCs after cell transplantation. Integrin‐mediated adhesion is a prerequisite for cell survival. As a novel anti‐death strategy to improve cell survival in the infarcted heart, MSCs were genetically modified to overexpress integrin‐linked kinase (ILK). The survival rate of ILK‐transfected MSCs (ILK‐MSCs) was augmented by about 1.5‐fold and the phosphorylation of ERK1/2 and Akt in ILK‐MSCs were increased by about three and twofold, respectively. ILK‐MSCs demonstrated an increase of twofold in the ratio of Bcl‐2/Bax and inhibited caspase‐3 activation, compared with hypoxic MSCs. The adhesion rate of ILK‐MSCs also had a 32.2% increase on the cardiac fibroblast‐derived three‐dimensional matrix and ILK‐MSCs showed higher retention by about fourfold compared to unmodified MSCs. Six animals per group were used for the in vivo experiments analyzed at 1 week after occlusion of the left coronary artery. ILK‐MSC transplanted rats had a 12.0% ± 3.1% smaller infarct size than MSC‐treated rats after ligation of left anterior descending coronary artery. Transplantation of ILK‐MSCs not only led to a 16.0% ± 0.4% decrease in the fibrotic heart area, but also significantly reduced the apoptotic positive index by two‐thirds when compared with ligation only. The mean microvessel count per field in the infarcted myocardium of ILK‐MSCs group was increased relative to the sham group and MSCs group. In conclusion, the ILK gene transduction of MSCs further assisted cell survival and adhesion, and improved myocardial damage when compared with MSC only after transplantation. STEM CELLS 2009;27:1358–1365

Cordycepin inhibits vascular smooth muscle cell proliferation

Percutaneous transluminal coronary angioplasty (PTCA) is a common procedure for treating atherosclerosis, but its efficacy is limited because of the occurrence of restenosis within 3-6 months after angioplasty. Restenosis is induced by the remodeling of the vessel wall and/or the accumulation of cells and extracellular matrix (ECM) in the intimal layer. Therefore, the matrix metalloproteinase (MMP) system may be a potential therapeutic target for the treatment of restenosis or atherosclerosis. Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, antioxidant, and anti-inflammatory activities. The effect of cordycepin on restenosis has not yet been clearly elucidated. Therefore, in the present study, we tested the role of cordycepin on the MMP system in vascular smooth muscle cells. In the carotid artery of a balloon-injured Sprague-Dawley (SD) rat, neointimal formation was reduced by treatment with cordycepin (20 microM/day, i.p), which inhibited the proliferation of rat aortic smooth muscle cells (RaoSMCs). To investigate the mechanism by which cordycepin inhibits the remodeling of the vessel wall and/or the accumulation of cells and ECM, we examined the activation of MMP systems in collagen type I-activated RaoSMCs. Cordycepin markedly inhibited the activation of MMP-2 and -9 as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in a dose-dependent manner in collagen type I-activated RaoSMCs. Moreover, cordycepin suppressed cycloxygenase-2 (COX-2) expression related to hyperplasia of RAoSMCs. Taken together, these data suggest that cordycepin may induce antiproliferation in RAoSMCs via the modulation of vessel wall remodeling. Therefore, cordycepin may be a potential therapeutic approach to treat restenosis.

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.

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.

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.

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.