Il-Kwon Kim

Small-diameter blood vessels engineered with bone marrow-derived cells

Objective:The objective of this study is to investigate if bone marrow–derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. Summary Background Data:BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle–like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. Methods:Canine BMCs were differentiated in vitro into smooth muscle α-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. Results:The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. Conclusions:Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.

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

Circulating miRNAs as Potential Marker for Pulmonary Hypertension

MircoRNAs (miRNAs) are small non-coding RNAs that govern the gene expression and, play significant role in the pathogenesis of heart failure. The detection of miRNAs in circulation of pulmonary hypertensive (PH) human subjects remains elusive. In the current study, we determined the pattern of miRNAs of mild-to-severe human PH subjects and, compared them with the control subjects by miRNA array. Blood was obtained using fluoroscopic and waveform guided catheterization from the distal (pulmonary artery) port of the catheter. A total 40 human subjects were included in the study and, the degree of PH was determined by mean pulmonary arterial pressure. Among several miRNAs in the array, we validated 14 miRNAs and, the data were consistent with the array profile. We identified several novel downregulated miRNAs (miR-451, miR-1246) and upregulated miRNAs (miR-23b, miR-130a and miR-191) in the circulation of PH subjects. Our study showed novel set of miRNAs which are dysregulated in PH and, are directly proportional to the degree of PH. These miRNAs may be considered as potential biomarker for early detection of PH.

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.

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.

Evidence for In Vivo Growth Potential and Vascular Remodeling of Tissue-Engineered Artery

Nondegradable synthetic polymer vascular grafts currently used in cardiovascular surgery have no growth potential. Tissue-engineered vascular grafts (TEVGs) may solve this problem. In this study, we developed a TEVG using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices, and tested whether the TEVGs exhibit growth potential and vascular remodeling in vivo. Vascular smooth muscle-like cells and endothelial-like cells were differentiated from bone marrow mononuclear cells in vitro. TEVGs were fabricated by seeding these cells onto decellularized porcine abdominal aortas and implanted into the abdominal aortas of 4-month-old, bone marrow donor pigs (n = 4). Eighteen weeks after implantation, the dimensions of TEVGs were measured and compared with those of native abdominal aortas. Expression of molecules associated with vascular remodeling was examined with reverse transcription-polymerase chain reaction assay and immunohistochemistry. Eighteen weeks after implantation, all TEVGs were patent with no sign of thrombus formation, dilatation, or stenosis. Histological and immunohistochemical analyses of the retrieved TEVGs revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. The outer diameter of three of the four TEVGs increased in proportion to increases in body weight and outer native aorta diameter. Considerable extents of expression of molecules associated with extracellular matrix (ECM) degradation (i.e., matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase) and ECM precursors (i.e., procollagen I, procollagen III, and tropoelastin) occurred in the TEVGs, indicating vascular remodeling associated with degradation of exogenous ECMs (implanted decellularized matrices) and synthesis of autologous ECMs. This study demonstrates that the TEVGs with autologous BMCs and decellularized tissue matrices exhibit growth potential and vascular remodeling in vivo of tissue-engineered artery.

Inhibition of nuclear factor-κB in the lungs prevents monocrotaline-induced pulmonary hypertension in mice.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with significant morbidity and mortality in patients with various lung and heart diseases. PAH is characterized by vascular obstruction which leads to a sustained increased pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure. Limited PAH therapies indicate that novel approaches are urgently needed for the treatment of PAH. Nuclear factor-&kgr;B (NF-&kgr;B) has been shown to play an important role in different cardiac pathologies; however, the role of NF-&kgr;B remains limited in the setting of PAH. Here, we investigated whether NF-&kgr;B inhibition in the lungs using Club (Clara) cell-10 promoter driving I&kgr;B&agr; mutant had any effect in monocrotaline (MCT)-induced PAH mouse model. Our data revealed that MCT-induced PAH and right ventricular hypertrophy were associated with NF-&kgr;B activation, inflammatory response, and altered expression of bone morphogenetic protein receptor 2, inhibitor of differentiation, and Notch-3 signaling molecules in wild-type mice; and all these alterations were prevented in I&kgr;B&agr; mutant mice treated with MCT. Moreover, endothelial cell apoptosis and endothelial-to-mesenchymal transition occurred in the lungs of MCT-treated wild-type mice and were restored in I&kgr;B&agr; mutant+MCT mice, indicating an association with NF-&kgr;B signaling. In lung microvascular endothelial cells, I&kgr;B&agr; (AA) mutant plasmid restored the decreased bone morphogenetic protein receptor 2 protein level and reversed the endothelial-to-mesenchymal transition process induced by transforming growth factor-&bgr;1. We conclude that NF-&kgr;B regulates bone morphogenetic protein receptor 2–inhibitor of differentiation–Notch-3 axis genes and the subsequent endothelial cell apoptosis and endothelial-to-mesenchymal transition events in the lungs, providing new mechanistic information about MCT-induced PAH and right ventricular hypertrophy.

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.

Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction.

Transplanting cord blood‐derived cells has been shown to augment neovascularization in ischaemic tissue.