Songhwa Kang

Selective Interference Targeting of Lnk in Umbilical Cord‐Derived Late Endothelial Progenitor Cells Improves Vascular Repair, Following Hind Limb Ischemic Injury, via Regulation of JAK2/STAT3 Signaling

The Lnk adaptor protein is a strong negative regulator that affects self‐renewal of hematopoietic stem cells and vascular repair in injured tissues. However, the signaling mechanisms through which these proteins influence the vascular regeneration function of endothelial progenitor cells (EPCs) remain unknown. In this study, we investigated the effect of Lnk‐targeted small interfering RNA (si‐lnk) on the clonogenic proliferative potential and vascular regenerative function of EPCs and the activation of the JAK/STAT3 signaling pathway. Treatment with stem cell factor (SCF) increased the clonogenic proliferation of si‐lnk EPCs. Importantly, activation of the JAK2/STAT3 pathway was enhanced in SCF‐sensitized si‐lnk EPCs. In a hind limb model of ischemia, transplantation of si‐lnk EPCs increased the blood flow ratio, capillary density, proliferation, and survival of transplanted cells, and the secretion of pivotal angiogenic cytokines at ischemic sites. These results provide strong evidence that si‐lnk regulates the clonogenic proliferative potential of EPCs through the activation of the JAK2/STAT3 signaling pathway, thereby accelerating angiogenesis and promoting repair in injured hind limb ischemia. Stem Cells 2014;33:1490–1500

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear. Methods In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34+ and CD34− cells, and determined the optimal concentrations of CD34+ cells and CD34− cells for spindle-shaped EPC differentiation. Results Functionally, the coculture of CD34+ and CD34− cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34+ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34+ and CD34− cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3+) markedly accelerated the early EPC status of CD34+ cells, while macrophages (CD11b+) or megakaryocytes (CD41+) specifically promoted large EPC colonies. Conclusion Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34+ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.

Regular Exercise Training Increases the Number of Endothelial Progenitor Cells and Decreases Homocysteine Levels in Healthy Peripheral Blood

Endothelial progenitor cells (EPCs) are known to play an important role in the repair of damaged blood vessels. We used an endothelial progenitor cell colony-forming assay (EPC-CFA) to determine whether EPC numbers could be increased in healthy individuals through regular exercise training. The number of functional EPCs obtained from human peripheral blood-derived AC133 stem cells was measured after a 28-day regular exercise training program. The number of total endothelial progenitor cell colony-forming units (EPC-CFU) was significantly increased compared to that in the control group (p=0.02, n=5). In addition, we observed a significant decrease in homocysteine levels followed by an increase in the number of EPC-CFUs (p=0.04, n=5), indicating that the 28-day regular exercise training could increase the number of EPC colonies and decrease homocysteine levels. Moreover, an inverse correlation was observed between small-endothelial progenitor cell colony-forming units (small-EPC-CFUs) and plasma homocysteine levels in healthy men (r=-0.8125, p=0.047). We found that regular exercise training could increase the number of EPC-CFUs and decrease homocysteine levels, thus decreasing the cardiovascular disease risk in men.

Regulation of ROS-independent ERK signaling rescues replicative cellular senescence in ex vivo expanded human c-kit-positive cardiac progenitor cells

BACKGROUNDS Although the rescue of cellular senescence during ex vivo expansion of human-derived cardiac progenitor cells (hCPC) is critical for the application of autologous stem cell therapy in cardiovascular disease, the underlying molecular pathways during replicative senescence in hCPC have not been fully defined. Thus, we examined whether the regulation of mitogen-activated protein kinases activation could facilitate the recovery of human c-kit-positive hCPCs (hCPC(c-kit+)) and whether senescence is reactive oxygen species (ROS)-dependent or -independent. METHODS AND RESULTS To investigate the molecular pathways of replicative cellular senescence, we first evaluated cellular senescence in ex vivo-expanded hCPC(c-kit+) by using senescence-associated β-galactosidase (SA-β-gal) activity with enlarged cytoplasm and observed increased expression of cell senescence-related pivotal molecules, including TP53, cleavage Mdm2 (cMdm2), and Mdm2. Unexpectedly, we found that the extracellular signal-regulated kinase (ERK) was markedly activated in aged hCPC(c-kit+), with reduced proliferative activity. SA-β-gal activity and cytoplasm size in senescent hCPC(c-kit+) were significantly reduced, with reduced TP53 and cMdm2 expression after treatment with a specific ERK inhibitor (U0126). We examined whether the signaling in ERK inhibitory rescue of hCPC(c-kit+) senescence is ROS-dependent. Interestingly, the increased ROS level was not changed after treatment with a specific ERK inhibitor. Similarly, the increased expression levels of endogenous antioxidant enzymes, e.g., peroxiredoxin (Prdx)-1 and 2, in senescent hCPC(c-kit+) were not changed after treatment with a specific ERK inhibitor. CONCLUSIONS From the above results, we conclude that the specific inhibition of ERK during cellular senescence might rescue bioactivities of senescent hCPC(c-kit+) in a ROS-independent manner.

High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker

Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.

Oleuropein attenuates hydrogen peroxide-induced autophagic cell death in human adipose-derived stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with self-renewing properties; thus, transplanting functionally enhanced MSCs might be a promising strategy for cell therapy against ischemic diseases. However, extensive oxidative damage in ischemic tissue affects the cell fate of transplanted MSCs, eventually resulting in cell damage and autophagic cell death. Oleuropein (OLP) is a bioactive compound isolated from olives and olive oil that harbors antioxidant properties. This study aimed to investigate the potential cytoprotective effects of OLP against oxidative stress and autophagic cell death in MSCs. We found that short-term priming with OLP attenuated H2O2-induced apoptosis by regulating the pro-apoptotic marker Bax and the anti-apoptotic markers Bcl-2 and Mcl-1. Notably, OLP inhibits H2O2 -induced autophagic cell death by modulating autophagy-related death signals, including mTOR (mammalian target of rapamycin), ULK1 (unc-51 like autophagy activating kinase 1), Beclin-1, AMPK (AMP-activated protein kinase), and LC3 (microtubule-associated protein 1a/1b-light chain 3). Our data suggest that OLP might reduce H2O2-induced autophagy and cell apoptosis in MSCs by regulating both the AMPK-ULK axis and the Bcl-2-Mcl-1 axis. Consequently, short-term cell priming with OLP might enhance the therapeutic effect of MSCs against ischemic vascular diseases, which provides an important potential improvement for emerging therapeutic strategies.

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice.

AIM Dysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D). METHODS The colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKAy mice, using EPC colony-forming assay (EPC-CFA). RESULTS T2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types. CONCLUSION T2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.

Cytoprotective Roles of a Novel Compound, MHY-1684, against Hyperglycemia-Induced Oxidative Stress and Mitochondrial Dysfunction in Human Cardiac Progenitor Cells

Diabetic cardiomyopathy (DCM) is tightly linked to heart disorders and dysfunction or death of the cardiomyocytes including resident cardiac progenitor cells (CPCs) in diabetic patients. In order to restore loss of function of resident or transplanted CPCs, much research has focused on novel therapeutic strategies including the discovery of novel function-modulating factors such as reactive oxygen species (ROS) scavengers. Here, we developed and defined a novel antioxidant, MHY-1684, for enhancing the angiogenic potential of CPCs against ROS-related DCM. Short-term treatment with MHY-1684 restored ROS-induced CPC cell death. Importantly, MHY-1684 decreased hyperglycemia-induced mitochondrial ROS generation and attenuated hyperglycemia-induced mitochondrial fragmentation. We observed that the activation process of both Drp1 (phosphorylation at the site of Ser616) and Fis-1 is drastically attenuated when exposed to high concentrations of D-glucose with MHY-1684. Interestingly, phosphorylation of Drp1 at the site of Ser637, which is an inhibitory signal for mitochondrial fusion, is restored by MHY-1684 treatment, suggesting that this antioxidant may affect the activation and inhibition of mitochondrial dynamics-related signaling and mitochondrial function in response to ROS stress. In conclusion, our finding of the novel compound, MHY-1684, as an ROS scavenger, might provide an effective therapeutic strategy for CPC-based therapy against diabetic cardiomyopathy.