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.ope

Novel Lactam Type Pyridine Derivatives Improves Myocardium Dysfunction Derived from Ischemic Injury

The extended acute myocardial ischemia (AMI) results in cardiac myocytes death. In the present study, we show that lactam pyridine derivative, SK-D80375, have the effects on cell survival in hypoxic cardiomyocytes and might be used as an anti-ischemic drug. The lactam pyridine derivatives are inhibitors of the late sodium current, which decreases sodium-dependent intracellular calcium overload in ischemia/reperfusion-injured hearts. We found that pretreatment with SK-D80375 significantly decreased the level of intracellular Ca2+ and the expression level of the Na+-Ca2+ exchanger by 39±2.5% and 19±0.5%, respectively in hypoxic cardiomyocytes compared to untreated controls. In addition, the expression level of sarcoplasmic reticulum Ca2+ ATPase 2a was significantly increased by 37±1.5% in SK-D80375-treated hypoxic cardiomyocytes compared to untreated controls. The induction of Hsp70 was observed in SK-D80375-treated hypoxic cardiomyocytes with dose-dependent manner and the highest level of Hsp70 was induced at the concentration of 2.5 μM SK-D80375. The echocardiographic analysis showed that heart function was significantly improved in SK-D80375-injected ischemic hearts. These results demonstrate that lactam pyridine derivative, SK-D80375, have beneficial effects on hypoxia-induced cell death, therefore, might be used as a novel anti-ischemia drugope

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

중간엽 줄기세포의 부착성에 미치는 활성 산소 종의 역할

Dept. of Medical Science/석사[한글] 심근 손상의 복원을 위한 줄기 세포 치료는 세포 -이식 후 세포의 적은 부착성에 의해 치료에 있어 제한을 받는다. 심근 경색 후 허혈은 활성 산소 종의 발생을 증가 시켜 중간엽 줄기 세포와 그 주변의 상호작용과 관련을 갖는다. 세포내의 활성 산소 종은 세포 부착, 유입과 증식에 있어서 중요한 역할을 한다. 중간엽 줄기 세포와 그 주변의 상호 작용에서의 활성 산소 종의 역할을 확인하고자 하였다. 과산화수소의 처리 후, 중간엽 줄기세포는 과산화수소의 농도가 증가함에 따라 분리가 되나, 유리기 scavenger인 NAC(N-acetyl-L-cysteine, 2 mM)을 처리하였을 경우 세포의 분리가 저해 되는 것을 확인할 수 있다. 섬유상 단백질이나 3차원 세포기질을 처리 한 배양 접시에 과산화수소를 50 μM 처리한 경우와 처리하지 않은 경우의 비교군 실험에서, 과산화수소의 처리가 이루어지지 않은 3차원 세포 기질의 배양접시에서 섬유상 단백질을 처리한 배양접시와 비교하였을 경우 두 배 가량 높은 양의 중간엽 줄기 세포의 부착 성이 보였다. 또, 과산화수소에 의해 세포 내 활성 산소 종의 생성이 70% 증가 하였다. FAK(Focal Adhesion Kinase) 인산화와 MAPK와 Src의 활성과 paxillin의 발현을 포함한 다른 하위 신호는 산화적 신호에 의해 의미 있게 감소하였다. 과산화수소를 처리한 중간엽 줄기세포의 발현은 FAK에서 77.1±2.8%, Src에서 62.2±4.3%, paxillin에서 36.2±4.8% 그리고 vinculin에서 29.6±2.6%, 각각 감소하였다. 또한, 세포 부착에서 주요한 물질인 integrin의 발현에서 대조 군과 비교 하여 과산화수소를 처리한 경우, Integrin αV와 fibronectin의 발현은 60%, 32.5% 그리고 integrin β1은 79.4% 감소하였다. H2O2 처리 이후, 중간엽 줄기세포의 부착에서 integrin-유도 활성 산소 종 생성의 조절과 관련이 있는 Small GTPase Rac-1의 발현은 과산화수소 처리 군에서 대조 군과 비교 하였을 경우 75.4% 감소했다. 결과적으로 활성 산소 종은 세포부착의 조절과 주변 상호 조절에서 세포부착과 하위 신호물질을 저해하는 것을 나타내었다. 이러한 결과로 경색된 심장의 중간엽 줄기세포 이식에 있어 더 나은 결과를 가져 오게 될 방법을 발견하게 만들 수 있을 것을 기대된다. [영문]Stem cell therapy for repair of myocardial injury has inherent limitations due to the poor attachment of cells after cell transplantation. In ischemic surroundings after myocardial infarction (MI) and interaction of mesenchymal stem cells (MSCs) with niche is associated with increased production of reactive oxygen species (ROS). The intracellular ROS plays 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. After treatment of H2O2 (50 μM) on MSCs, detachment of MSCs was dose-dependently increased, but cell detachment was inhibited by pretreatment with the free radical scavenger, N-acetyl-L-cysteine (NAC, 2 mM). The H2O2 treated MSCs were detached on 3D-matrix gel about 2 - fold, compared with fibronectin coated plate. Intracellular ROS production was enhanced about 70.0% in H2O2-treated cells compared with non-treated cells and phosphorylation of FAK (77.1 ± 2.8%) and Src (62.2 ± 4.3%) were decreased. Expression level of paxillin and vinculin was reduced to 36.2 ± 4.8% and 29.6 ± 2.6%, respectively in H2O2-treated MSCs. Expression of integrin-related adhesion molecules was also depressed; integrin αV (60.0%), integrin β1 (79.4%), and fibronectin (32.5%) in MSCs treated with H2O2. The transcript level of Rac-1 was decreased about 75.4% in H2O2-treated cells compared with non-treated cells. These results indicate that ROS suppress the cell adhesion and downstream signaling molecules in the regulation of cell adhesion. These results also contribute to a better understanding of the transplantation of MSCs on infarcted heart.ope

Protective effects of protein transduction domain-metallothionein fusion proteins against hypoxia- and oxidative stress-induced apoptosis in an ischemia/reperfusion rat model

Ischemic heart diseases caused by insufficient oxygen supply to the cardiac muscle require pharmaceutical agents for the prevention of the progress and recurrence. Metallothionein (MT) has a potential as a protein therapeutic for the treatment of this disease due to its anti-oxidative effects under stressful conditions. In spite of its therapeutic potential, efficient delivery systems need to be developed to overcome limitations such as low transduction efficiency, instability and short half-life in the body. To enhance intra-cellular transduction efficiency, Tat sequence as a protein transduction domain (PTD) was fused with MT in a recombinant method. Anti-apoptotic and anti-oxidative effects of Tat-MT fusion protein were evaluated under hyperglycemia and hypoxia stress conditions in cultured H9c2 cells. Recovery of cardiac functions by anti-apoptotic and anti-fibrotic effects of Tat-MT was confirmed in an ischemia/reperfusion (I/R) rat myocardial infarction model. Tat-MT fusion protein effectively protected H9c2 cells under stressful conditions by reducing intracellular ROS production and inhibiting caspase-3 activation. Tat-MT fusion protein inhibited apoptosis, reduced fibrosis area and enhanced cardiac functions in I/R. Tat-MT fusion protein could be a promising therapeutic for the treatment of ischemic heart diseases.ope

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.ope

Modification of mesenchymal stem cells for cardiac regeneration.

IMPORTANCE OF THE FIELD: Mesenchymal stem cells (MSCs) have the greatest potential for use in cell-based therapy of human heart diseases, especially in myocardial infarcts. The therapeutic potential of MSCs in myocardial repair is based on the ability of MSCs to directly differentiate into cardiac tissue and on the paracrine actions of factors released from MSCs. However, the major obstacle in the clinical application of MSC-based therapy is the poor viability of the transplanted cells due to harsh microenvironments like ischemia, inflammation and/or anoikis in the infarcted myocardium. Recently, various approaches have been implemented in an effort to improve the survival of implanted MSCs through ex vivo manipulation of MSCs. AREAS COVERED IN THIS REVIEW: Major obstacles in MSC-based therapy are discussed, along with recent advances for enhancing therapeutic potential of engrafted MSCs from the past decade. WHAT THE READER WILL GAIN: This review focuses primarily on ex vivo manipulation of MSCs before transplantation, which includes pretreatment, preconditioning and genetic modification of MSCs, and future directions. TAKE HOME MESSAGE: Modification of MSCs before transplantation has developed into a promising option for enhancing the beneficial effects of MSC-based therapy for cardiac repair after myocardial infarction.ope

Prolongation and enhancement of the anti-apoptotic effects of PTD-Hsp27 fusion proteins using an injectable thermo-reversible gel in a rat myocardial infarction model

Ischemic heart disease has emerged as a leading cause of death worldwide. Conventional surgery-based therapy for this disease, especially myocardial infarction, requires additional pharmaceutical agents using heart's endogenous protective mechanism to suppress the progress and recurrence of the disease. Heat shock protein 27 (Hsp27) has been considered to be a potentially therapeutic protein for the treatment of ischemic heart disease due to its anti-apoptotic and protective effects on cardiomyocytes under stressful conditions. Despite the potency of Hsp27, low transduction efficiency, protein instability, and a short half-life in the body have limited its in vivo applications. Protein transduction domains (PTD) were recombinantly fused with Hsp27 to enhance transduction efficiency. Although the intracellular delivery of the PTD-Hsp27 fusion proteins was significantly enhanced compared with Hsp27, the instability and short half-life of the PTD-Hsp27 fusion proteins still need to be improved for in vivo applications. Injectable thermo-reversible gel system was developed and found to be effective in stabilizing and retarding the release of the PTD-Hsp27 fusion proteins both in vitro and in vivo. PTD-Hsp27-loaded thermo-reversible gels were locally administered to the heart muscle in a ligation/reperfused rat myocardial infarction model and the long-term therapeutic efficacy was observed by measuring the inhibition of apoptosis and the area of fibrosis.ope