Specific differentiation of bone marrow-derived mesenchymal stem cells by regulation of protein kinase system

Dept. of Medical Science/박사[한글]현재까지 알려진 많은 신호 기전을 조절하는 단백질들 중 하나인 단백질 키나아제는 줄기세포의 분화 조절에서 중요한 역할을 할 수 있으며 이는 세포의 운명과 같은 중요한 생물학적 과정이 다양한 신호기전의 섬세한 조합에 의해 결정되어지기 때문이다. 게다가, 단백질 키나아제의 활성을 조절하는 세포 투과성 저분자화합물은 특정 단백질의 기능을 시간적, 양적 그리고 모듈단위로 조절할 수 있는 장점을 가지고 있다. 따라서 본 연구에서는 간엽줄기세포의 분화를 체계적으로 조절하는 화학물질과 기전에 대해 알아보고자 하였으며 다양한 신호기전의 조합을 조절할 수 있는 주요한 단백질 키나아제 군의 억제제 스크리닝을 통해 몇몇 단백질 키나아제가 간엽줄기세포의 분화에 상당한 영향을 미침을 발견하였다. 우선, PKA 억제제인 H-89를 줄기세포에 처리한 결과, 생체외에서 연골세포로의 분화과정이 증진되었다. H-89를 처리한 뒤 연골세포로의 분화 정도를 측정하는 alcian blue 염색 정도가 증가하였다. 다양한 농도 (0.1-1 μM)의 H-89를 간엽줄기세포에 처리할 경우, 연골세포의 세포외 기질에 존재하는 단백질들 중 하나인 aggrecan의 발현이 유도되었다. 간엽줄기세포가 연골세포로 분화되는 과정에서 ERK의 활성은 증가하였으나 H-89과 U0126을 동시에 처리할 경우 ERK의 활성은 증가하지 않았다. 연골세포로의 분화과정에서 세포 생착과 관련된 분자의 발현에서 상당한 변화가 유발됨을 RT-PCR을 통해 확인하였다. PKC 활성제인 phorbol myristate acetate (PMA)의 처리는 간엽줄기세포에서 심근세포의 특이적 표현인자 (cardiac troponin T, myosin light chain, myosin heavy chain, NK2 transcription factor-related, locus 5, 그리고 Myocyte-specific enhancer factor 2)의 발현을 유발시켰다. 분화 유도된 심근세포는 기능인자인 adrenergic과 muscarinic 수용체가 발현되고 norepinephrine을 처리하면 발현된 α1-adrenergic 수용체를 통해 ERK의 인산화가 유발되었다. 칼슘 관련 인자(sarcoplasmic reticulum Ca2+-ATPase 그리고 L-type Ca2+ channel)의 mRNA 발현 수준은 분화 유도된 간엽줄기세포와 심근세포에서 유사성을 보였다. GSK-3β inhibitor 처리에 의해 내피유사세포로의 분화가 유도되었으며 분화 유도된 세포는 내피세포의 특이적 표현인자(CD31, CD34, eNOS, VE-cadherin, VCAM-1, 그리고 VEGF-R2) 발현의 상당히 증가하였으며, 또 모세혈관과 유사한 구조의 형성과 같은 기능적 특성을 보였다. 간엽줄기세포에 GSK-3β inhibitor를 처리하면 세포주기 중 S기가 감소하고 전반적으로 생육이 저하되었다. 신호 물질의 분석을 통해 GSK-3β inhibitor 처리에 의한 내피세포로의 분화에는 GSK-3β/β-catenin 하위신호기전이 관련되어 있음을 알 수 있었다. 본 연구는 단백질 키나아제의 조절이 간엽줄기세포의 체계적인 분화 조절 방법이 될 수 있으며 이 체계는 간엽줄기세포에 대한 학문적 이해와 새로운 재생의학의 발달에 기여할 것으로 기대된다. [영문]Protein kinases, the members of a large family of proteins involved in modulating many known signaling pathways, are likely to play important roles in regulating stem cell differentiation programs because important bio-processes such as cell fate are likely to be determined by an elaborate orchestration of multiple signaling pathways. In addition, cell permeable small molecules to modulate activation of protein kinase have several merits that include the ability for temporal, tunable and modular control of specific protein function. Therefore, research efforts focused on search for chemical reagent and mechanism that regulates systematic differentiation of MSCs. It was found that some of protein kinase cause recognizable changes in differentiation rates of MSCs based on screening inhibitors of major protein kinase subfamilies to alter the orchestration of multiple signaling pathways. At first, chondrogenesis of MSCs in vitro was significantly improved in cells treated with H-89, a PKA inhibitor. After treatment of H-89, the data of alcian blue staining assay for the quantitation of chondrogenesis was increased. Aggrecan, one of the extracellular matrix genes in chondrocytes, was induced in MSCs treated with various concentrations of H-89 (0.1?1 μM). During chondrogenesis of MSC, activation of ERKs was increased in MSCs treated with H-89 but cotreatment with H-89 and U0126 did not lead to a change in ERK activation. Semiquantitative RT-PCR showed a significantly changed level of cell adhesion molecule during chondrogenesis. MSCs gave rise to cardiomyocytes expressing of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain, NK2 transcription factor-related, locus 5, and Myocyte-specific enhancer factor 2) by addition of phorbol myristate acetate (PMA), a PKC activator. Differentiated cardiomyocytes expressed functional adrenergic and muscarinic receptors and norepinephrine induced phosphorylation of ERK via α1-adrenoceptors. The mRNA level of Ca2+-related factor (sarcoplasmic reticulum Ca2+-ATPase, L-type Ca2+ channel) in differentiated MSCs was similar to that in cardiomyocytes. Differentiation into endothelial-like cells was induced by cultivation of cells in the presence of GSK-3β inhibitor. The differentiated cells showed a strong increase of expression of endothelial-specific markers (CD31, CD34, eNOS, VE-cadherin, VCAM-1, and VEGF-R2) and functional behavior of endothelial cells such as a formation of capillary-like structure. Treatment of proliferating MSCs with GSK-3β inhibitor leads to depletion of S-phase cells and over all decreased proliferation. The differentiation of MSCs into endothelial-like cells by the treatment of GSK-3β inhibitor was associated with GSK-3β/β-catenin signaling. The current study suggests that the regulation of protein kinase may emerge as a remarkable tool for systematic differentiation of MSCs. This system would greatly contribute to a novel understanding of MSC biology and the development of novel regenerative medicine.ope

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.ope

Tissue transglutaminase is essential for integrin-mediated survival of bone marrow-derived mesenchymal stem cells

Autologous mesenchymal stem cell (MSC) transplantation therapy for repair of myocardial injury has inherent limitations due to the poor viability of the stem cells after cell transplantation. Adhesion is a prerequisite for cell survival and also a key factor for the differentiation of MSCs. As a novel prosurvival modification strategy, we genetically engineered MSCs to overexpress tissue transglutaminase (tTG), with intention to enhance adhesion and ultimately cell survival after implantation. tTG-transfected MSCs (tTG-MSCs) showed a 2.7-fold and greater than a twofold increase of tTG expression and surface tTG activity, respectively, leading to a 20% increased adhesion of MSCs on fibronectin (Fn). Spreading and migration of tTG-MSCs were increased 4.75% and 2.52%, respectively. Adhesion of tTG-MSCs on cardiogel, a cardiac fibroblast-derived three-dimensional matrix, showed a 33.1% increase. Downregulation of tTG by transfection of small interfering RNA specific to the tTG resulted in markedly decreased adhesion and spread of MSCs on Fn or cardiogel. tTG-MSCs on Fn significantly increased phosphorylation of focal adhesion related kinases FAK, Src, and PI3K. tTG-MSCs showed significant retention in infarcted myocardium by forming a focal adhesion complex and developed into cardiac myocyte-like cells by the expression of cardiac-specific proteins. Transplantation of 1 × 106 MSCs transduced with tTG into the ischemic rat myocardium restored normalized systolic and diastolic cardiac function. tTG-MSCs further restored cardiac function of infarcted myocardium as compared with MSC transplantation alone. These findings suggested that tTG may play an important role in integrin-mediated adhesion of MSCs in implanted tissues.ope

Enhanced calreticulin expression promotes calcium-dependent apoptosis in postnatal cardiomyocytes

Calreticulin (CRT) is one of the major Ca2+ binding chaperone proteins of the endoplasmic reticulum (ER) and an unusual luminal ER protein. Postnatally elevated expression of CRT leads to impaired development of the cardiac conductive system and may be responsible for the pathology of complete heart block. In this study, the molecular mechanisms that affect Ca2+-dependent signal cascades were investigated using CRT-overexpressing cardiomyocytes. In particular, we asked whether calreticulin plays a critical role in the activation of Ca2+-dependent apoptosis. In the cells overexpressing CRT, the intracellular calcium concentration was significantly increased and the activity of PKC and level of SECAR2a mRNA were reduced. Phosphorylation of Akt and ERKs decreased compared to control. In addition the activity of the anti-apoptotic factor, Bcl-2, was decreased and the activities of pro-apoptotic factor, Bax, p53 and caspase 8 were increased, leading to a dramatic augmentation of caspase 3 activity. Our results suggest that enhanced CRT expression in mature cardiomyocytes disrupts intracellular calcium regulation, leading to calcium-dependent apoptosis.ope

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

Effect of propofol on calcium homeostasis in hypoxia-reoxygenated neonatal rat cardiomyocytes

Intracellular Ca2+ overload induced by hypoxia-reoxygenation alters Ca2+ homeostasis, which plays an important role in myocardial cell injury. Even though propofol is known as a radical scavenger with Ca2+ channel blocking properties, little is known about cardioprotective effect associated with Ca2+ homeostasis in cardiomyocytes. In the present study, we showed that propofol protects cardiomyocytes against hypoxia-reoxygenation injury. In propofol-treated cardiomyocytes, we observed a decrease in the expression of pro-apoptotic protein Bax, cytochrome c, caspase-3 activation and intracellular Ca2+ content. We also found that propofol treatment enhanced expression of anti-apoptotic protein Bcl-2 and activation of ERK concerned with survival. Propofol attenuated alterations of genes involving Ca2+-regulatory mechanism and significantly modulated abnormal changes of SERCA2a genes in hypoxia-reoxygenated neonatal cardiomyocytes. These results suggest that propofol modulates the expression of genes involved in Ca2+ homeostasis, thereby producing cardioprotective effects through a reduction in apoptotic cell death.ope

Allopurinol modulates reactive oxygen species generation and Ca2+ overload in ischemia-reperfused heart and hypoxia-reoxygenated cardiomyocytes

Myocardial oxidative stress and Ca2+ overload induced by ischemia-reperfusion may be involved in the development and progression of myocardial dysfunction in heart failure. Xanthine oxidase, which is capable of producing reactive oxygen species, is considered as a culprit regarding ischemia-reperfusion injury of cardiomyocytes. Even though inhibition of xanthine oxidase by allopurinol in failing hearts improves cardiac performance, the regulatory mechanisms are not known in detail. We therefore hypothesized that allopurinol may prevent the xanthine oxidase-induced reactive oxygen species production and Ca2+ overload, leading to decreased calcium-responsive signaling in myocardial dysfunction. Allopurinol reversed the increased xanthine oxidase activity in ischemia-reperfusion injury of neonatal rat hearts. Hypoxia-reoxygenation injury, which simulates ischemia-reperfusion injury, of neonatal rat cardiomyocytes resulted in activation of xanthine oxidase relative to that of the control, indicating that intracellular xanthine oxidase exists in neonatal rat cardiomyocytes and that hypoxia-reoxygenation induces xanthine oxidase activity. Allopurinol (10 μM) treatment suppressed xanthine oxidase activity induced by hypoxia-reoxygenation injury and the production of reactive oxygen species. Allopurinol also decreased the concentration of intracellular Ca2+ increased by enhanced xanthine oxidase activity. Enhanced xanthine oxidase activity resulted in decreased expression of protein kinase C and sarcoendoplasmic reticulum calcium ATPase and increased the phosphorylation of extracellular signal-regulated protein kinase and p38 kinase. Xanthine oxidase activity was increased in both ischemia-reperfusion-injured rat hearts and hypoxia-reoxygenation-injured cardiomyocytes, leading to reactive oxygen species production and intracellular Ca2+ overload through mechanisms involving p38 kinase and extracellular signal-regulated protein kinase (ERK) via sarcoendoplasmic reticulum calcium ATPase (SERCA) and protein kinase C (PKC). Xanthine oxidase inhibition with allopurinol modulates reactive oxygen species production and intracellular Ca2+ overload in hypoxia-reoxygenation-injured neonatal rat cardiomyocytes.ope

Losartan inhibits proliferation and inflammation of vascular smooth muscle cells by modulation of uric acid transporter

Hyperuricemia has known to be a risk factor for coronary artery disease, hypertension, and heart failure. Among angiotensin II receptor blockers(ARBs), only losartan lowers serum uric acid(UA) level compared with other ARBs. Recent studies have reported that UAcauses vascular smooth muscle cells(VSMCs) proliferation and inflammation by entering cells via a functional uric acid transporter(UAT). We investigated whether losartan could inhibit the UA-stimulated proliferative and inflammatory signaling activation by modulation of UAT in cultured rat aortic VSMCs. Following 72 hours incubation of UA, proliferation of VSMCs was increased dose-dependently. Losartan inhibited UA-stimulatedproliferation in dose-dependent manner with an IC50 value of about 1 M. Losartan attenuatedp38, ERKs activation, COX-2 expression, and MCP-1 production. Losartan significantly decreased expression of UAT in UA-stimulated VSMCs(33% reduction, p<0.05), which suggests inhibitory effect of losartan on the uptake of UA via UAT. These data provide that losartan may involve anti-inflammatory and antiproliferative activity by modulating UAT. Therefore, losartan may provide beneficial effect on vascular changes in hyperuricemic conditionope

Association of soluble receptor for advanced glycation end-product with increasing central aortic stiffness in hypertensive patients.

OBJECTIVES: In humans, a secreted isoform of (soluble) receptor for advanced glycation end-products (sRAGE) may act as a decoy receptor of advanced glycation end-product. The level of sRAGE may reflect the activity of cell surface receptor for advanced glycation end-product. But there has been no study that has demonstrated the association of sRAGE with central aortic stiffness. Here, we studied the relation of plasma sRAGE level and arterial pulse wave velocity in hypertensive patients. MATERIALS AND METHODS: A total of 415 patients were enrolled (men; 57.6%, mean age; 53.2±10.8 years), with 25.8% (n=107) of these being diabetic. All patients underwent pulse wave velocity (PWV) and blood sampling of sRAGE, high-sensitive C-reactive protein, and other serologic markers. RESULTS: The log-transformed sRAGE was significantly correlated with the marker of central aortic stiffness heart to femoral PWV (hfPWV; r=0.165, P=0.001). It also showed a significant correlation with hfPWV in patients with diabetes (r=0.301, P=0.002), but not in patients without diabetes (r=0.115, P=0.055). By multiple linear regression analysis, the log-transformed sRAGE was independently correlated with hfPWV (β=0.13, P=0.004) when controlled for other variables. CONCLUSIONS: This study has demonstrated, for the first time, that serum sRAGE level was independently correlated with a marker of central aortic stiffness. This result suggests the potential role of RAGE in the pathogenesis of aortic stiffness.ope