Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines

Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.     

自体骨髓单核细胞移植对急性心肌梗死的作用

目的探讨未经诱导的自体骨髓单核细胞可否在梗死心肌环境中存活并分化为心肌细胞及血管内皮细胞。方法40只日本大耳雄兔随机分为两组：移植组及对照组,每组各20只。采用结扎冠状动脉左前降支的方法建立急性心梗模型,以心电图证实模型成功,由超声心动图评价心功能。模型建立后7天,将BrdU标记的自体骨髓单核细胞注射到移植组动物心肌梗死区及周边区,而对照组动物相同部位仅注射等量生理盐水。移植后6周,收集动物心脏进行组织学及免疫组化分析。结果抗BrdU免疫组化发现移植组动物心肌梗死区及周边区内均存在染色阳性的移植细胞,且周边区内的移植细胞呈心肌细胞及血管内皮细胞的形态特点,同时这些细胞抗心肌特异性肌动蛋白抗体染色阳性,证实其肌源性分化。另外,移植组动物梗死周边区血管密度显著高于对照组（P〈0.05）,但两组动物在心肌梗死区内的血管密度没有统计学差异（P〉0.05）。移植后6周,两组动物心功能均有改善,移植组明显优于对照组（P〈0.05）。结论自体骨髓单核细胞移植于梗死心肌后,可在梗死区及周边区存活,并在周边区分化为血管内皮细胞及具有心肌细胞形态特点的细胞、增加梗死周边区的血管密度,改善心功能。     

Carvedilol Enhances Mesenchymal Stem Cell Therapy for Myocardial Infarction via Inhibition of Caspase-3 Expression

Adult stem cells have shown great promise toward repairing infarcted heart and restoring cardiac function. Mesenchymal stem cells (MSCs), because of their inherent multipotent nature and their ability to secrete a multitude of growth factors and cytokines, have been used for cardiac repair with encouraging results. Preclinical studies showed that MSCs injected into infarcted hearts improve cardiac function and attenuate fibrosis. Although stem cell transplantation is a promising therapeutic option to repair the infarcted heart, it is faced with a number of challenges, including the survival of the transplanted cells in the ischemic region, due to excessive oxidative stress present in the ischemic region. The objective of this study was to determine the effect of Carvedilol (Carv), a nonselective β-blocker with antioxidant properties, on the survival and engraftment of MSCs in the infarcted heart. MSCs were subjected to a simulated host-tissue environment, similar to the one present in the infarcted myocardium, by culturing them in the presence of hydrogen peroxide (H(2)O(2)) to induce oxidative stress. MSCs were treated with 2.5 μM Carv for 1 h in serum-free medium, followed by treatment with H(2)O(2) for 2 h. The treated cells exhibited significant protection against H(2)O(2)-induced cell death versus untreated controls as determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. Likewise, transplantation of MSCs after permanent left coronary artery ligation and treatment of animals after myocardial infarction (MI) with Carv (5 mg/kg b.wt.) led to significant improvement in cardiac function, decreased fibrosis, and caspase-3 expression compared with the MI or MSC-alone groups.     

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1⁺ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1^– cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1⁺ cells did not. We therefore believe that the SSEA-1⁺ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.     

静脉移植骨髓间质干细胞改善大鼠梗死心脏功能

目的:探讨经外周静脉途径移植骨髓间质干细胞治疗急性心肌梗死的有效性和可能的机制。方法:采用左前降支冠状动脉结扎术制备大鼠心肌梗死模型,24h后经尾静脉注入同种异体骨髓间质干细胞或磷酸缓冲液;分别于移植后3d和1个月取材,应用ELISA和免疫组化技术分析细胞移植对血管内皮生长因子(VEGF)表达的影响,通过免疫组化染色观察移植细胞的分化情况并计数血管;超声心动图连续检测大鼠心功能在术后各时间点的变化。结果:细胞移植术后3d,移植组VEGF蛋白的表达量明显高于对照组(P<0.01),免疫组化显示移植组心脏的梗死区及其周边区域的细胞内VEGF阳性着色明显强于对照组,1个月时明显减弱。移植术后1个月,在部分移植细胞中检测到心肌细胞或血管内皮细胞特异性蛋白的表达,血管计数显示移植组血管数量较同期对照组增加1.4倍(P<0.01);同时移植组心功能较对照组明显改善(P<0.01)。结论:通过促进梗死周边缺血心肌的血管新生和移植细胞分化,骨髓间质干细胞经外周静脉移植可作为急性心肌梗死治疗的另一条有效路径。     

Cellular mechanisms underlying cardiac engraftment of stem cells

Introduction: Over the past decade, it has become clear that long-term engraftment of any ex vivo expanded cell product transplanted into injured myocardium is modest and all therapeutic regeneration is mediated by stimulation of endogenous repair rather than differentiation of transplanted cells into working myocardium. Given that increasing the retention of transplanted cells boosts myocardial function, focus on the fundamental mechanisms limiting retention and survival of transplanted cells may enable strategies to help to restore normal cardiac function.

Areas covered: This review outlines the challenges confronting cardiac engraftment of ex vivo expanded cells and explores means of enhancing cell-mediated repair of injured myocardium.

Expert opinion: Stem cell therapy has already come a long way in terms of regenerating damaged hearts though the poor retention of transplanted cells limits the full potential of truly cardiotrophic cell products. Multifaceted strategies directed towards fundamental mechanisms limiting the long-term survival of transplanted cells will be needed to enhance transplanted cell retention and cell-mediated repair of damaged myocardium for cardiac cell therapy to reach its full potential.     

Cellular Antioxidant Levels Influence Muscle Stem Cell Therapy

Although cellular transplantation has been shown to promote improvements in cardiac function following injury, poor cell survival following transplantation continues to limit the efficacy of this therapy. We have previously observed that transplantation of muscle-derived stem cells (MDSCs) improves cardiac function in an acute murine model of myocardial infarction to a greater extent than myoblasts. This improved regenerative capacity of MDSCs is linked to their increased level of antioxidants such as glutathione (GSH) and superoxide dismutase. In the current study, we demonstrated the pivotal role of antioxidant levels on MDSCs survival and cardiac functional recovery by either reducing the antioxidant levels with diethyl maleate or increasing antioxidant levels with N-acetylcysteine (NAC). Both the anti- and pro-oxidant treatments dramatically influenced the survival of the MDSCs in vitro. When NAC-treated MDSCs were transplanted into infarcted myocardium, we observed significantly improved cardiac function, decreased scar tissue formation, and increased numbers of CD31⁺ endothelial cell structures, compared to the injection of untreated and diethyl maleate–treated cells. These results indicate that elevating the levels of antioxidants in MDSCs with NAC can significantly influence their tissue regeneration capacity.     

超声心动图评价干细胞移植对心肌梗死大鼠左心室功能的影响

目的采用二维超声心动图和组织多普勒超声心动图评价骨髓干细胞(bonemarrow-drivedmesenchymalstemcells,MSCs)移植前后大鼠的心脏形态和左心功能,为干细胞移植治疗心肌梗死的临床应用提供参考数据。方法40只Wistar大鼠随机分为对照组和移植组。应用液氮冷冻法制作心肌梗死模型后,移植组大鼠心肌内注射经5-氮胞苷诱导后的MSCs。手术前、术后1周、术后1个月分别应用二维超声心动图和组织多普勒显像测定两组大鼠的心脏功能。结果术后1个月移植组和对照组大鼠分别存活13只和15只。数据分析表明:移植组术后1个月,大鼠心脏功能与术后1周时相比明显改善(P<0.05),而对照组无改善。结论诱导后的MSCs移植到心肌梗死区及其周围可防止心功能恶化,改善心脏功能。MSCs移植可能成为治疗心肌梗死的一种切实有效的方法。     

同种异体骨髓间质干细胞移植联合血管内皮细胞生长因子基因转染治疗心肌梗死

背景:骨髓间质干细胞和血管内皮细胞生长因子移植具有促进血管再生、改善心功能的作用,但是二者联合应用是否优于单独应用尚不清楚。目的:观察同种异体骨髓间质干细胞移植联合血管内皮生长因子基因转染对大鼠急性心肌梗死血管再生和心功能的影响。设计:大鼠骨髓间质干细胞培养采用单一样本观察;细胞移植和基因转染采用随机对照动物实验。单位:华中科技大学协和医院心内科,同济医学院心血管病研究所。材料:健康雄性Wistar大鼠94只。表达载体PAdTrack/VEGF165。方法:实验于2004-06/2005-06在同济医学院心血管病研究所实验室完成。①体外分离、纯化、培养大鼠骨髓间质干细胞,以5-溴-2’-脱氧尿苷标记细胞。②制备、抽提、纯化、鉴定质粒PAdTrack/VEGF165。③结扎冠状动脉建立急性心肌梗死模型2周后,随机将其分为4组,每组均为12只,干细胞 质粒组、干细胞组、质粒组、对照组,分别进行心肌内大鼠骨髓间质干细胞移植和/或VEGF165转染、DMEM注射。④4周后行免疫组织化学和超声心动图检查。主要观察指标:①各组大鼠梗死及缺血区免疫组织化学和苏木精-伊红染色检查。②血管计数。③超声心动图检查。结果:48只大鼠进入结果分析。①干细胞 质粒组和干细胞组梗死及缺血心肌处可见大量5-溴-2’-脱氧尿苷标记的移植细胞,其中缺血心肌处部分移植细胞分化为血管内皮细胞并形成新生毛细血管。②Ⅷ因子染色阳性的新生血管密度分布为干细胞 质粒组>质粒组>干细胞组>对照组(P均<0.01)。③细胞移植和基因转染治疗后室壁厚度和室壁运动幅度改善,射血分数值增加幅度为干细胞 质粒组>干细胞组>质粒组>对照组(P均<0.01)。结论:同种异体骨髓间质干细胞移植联合血管内皮生长因子基因转染能进一步增强大鼠梗死缺血区血管再生、改善室壁厚度和心功能。     

Modulation of human mesenchymal stem cell function in a three‐dimensional matrix promotes attenuation of adverse remodelling after myocardial infarction

The application of tissue engineering (TE) practices for cell delivery offers a unique approach to cellular cardiomyoplasty. We hypothesized that human mesenchymal stem cells (hMSCs) applied to the heart in a collagen matrix would outperform the same cells grown in a monolayer and directly injected for cardiac cell replacement after myocardial infarction in a rat model. When hMSC patches were transplanted to infarcted hearts, several measures for left ventricle (LV) remodelling and function were improved, including fractional area change, wall thickness, –dP/dt and LV end‐diastolic pressure. Neovessel formation throughout the LV infarct wall after hMSC patch treatment increased by 37% when compared to direct injection of hMSCs. This observation was correlated with increased secretion of angiogenic factors, with accompanying evidence that these factors enhanced vessel formation (30% increase) and endothelial cell growth (48% increase) in vitro. These observations may explain the in vivo observations of increased vessel formation and improved cardiac function with patch‐mediated cell delivery. Although culture of hMSC in collagen patches enhanced angiogenic responses, there was no effect on cell potency or viability. Therefore, hMSCs delivered as a cardiac patch showed benefits above those derived from monolayers and directly injected. hMSCs cultured and delivered within TE constructs may represent a good option to maximize the effects of cellular cardiomyoplasty. Copyright © 2011 John Wiley & Sons, Ltd.     

Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (MSC-Akt) efficiently repaired infarcted rat myocardium and improved cardiac function. Controversy still exists over the mechanisms by which MSC contribute to tissue repair. Herein, we tested if cellular fusion of MSC plays a determinant role in cardiac repair. We injected MSC expressing Cre recombinase, with or without Akt, into Cre reporter mice. In these mice, LacZ is expressed only after Cre-mediated excision of a loxP-flanked stop signal and is indicative of fusion. MSC engraftment within infarcted myocardium was transient but significantly enhanced by Akt. MSC fusion with cardiomyocytes was observed as early as 3 days, but was infrequent, and we found a low rate of differentiation of MSC into cardiomyocytes. MSC-Akt decreased infarct size at 3 days and restored early cardiac function. In conclusion, MSC-Akt improved early repair despite transient engraftment, low levels of cellular fusion, and differentiation. These new observations further confirm our recently reported data that early paracrine mechanisms mediated by MSC are responsible for enhancing the survival of existing myocytes and that Akt could alter the secretion of various cytokines and growth factors.     

Engineering and Visualization of Bacteria for Targeting Infarcted Myocardium

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (ΔppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P_BAD), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of -arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ΔppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.     

体外构建工程化心肌组织的研究与应用

背景：＂工程化心肌组织＂是应用组织工程的方法,构建出具有天然心肌组织特征的心肌,将它移植到体内,最终修复或完全替代病损组织。但以往研究获得的工程化心肌组织存在较多的缺陷,仍不能满足实际需要。目的：总结体外构建工程化心肌组织方法的研究进展。方法：由第一作者用计算机检索中国期刊全文数据库（CNKI：2000/2010）和Medline（2000/2010）数据库,检索词分别为＂心肌,组织工程,心肌构建物＂和＂Myocardial Constructionmaterials,Tissue Engineering,Myocardial＂。从种子细胞、支架材料、体外培养环境和生物反应体系、工程化心肌组织的再血管化、移植实验5方面进行总结,对不同的种子细胞及生物支架材料和体外培养的环境、移植工程化组织的再血管化等方面进行了介绍。共检索到150篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章。结果与结论：适合心肌细胞生存和心肌细胞形成的新型生物活性支架,理想的种子细胞、体外培养环境与生物反应体系、移植工程化组织的再血管化,动物移植实验等方面都是组织工程化心肌组织再造的关键所在。构建的组织工程化心肌组织应同时具备良好的收缩功能、稳定的电生理特性、力学强度和柔韧性、无免疫原性及自身能血管化或在移植后能迅速血管化的条件,但未来还需进一步研究。     

SDF-1α as a therapeutic stem cell homing factor in myocardial infarction

Myocardial infarction is associated with persistent muscle damage, scar formation and depressed cardiac performance. Recent studies have demonstrated the clinical significance of stem cell-based therapies after myocardial infarction with the aim to improve cardiac remodeling and function by inducing the reconstitution of functional myocardium and formation of new blood vessels. Stem cell homing signals play an important role in stem cell mobilization from the bone marrow to the ischemic cardiac environment and are therefore crucial for myocardial repair. To date, the most prominent stem cell homing factor is the chemokine SDF-1α/CXCL12. This protein was shown to be significantly upregulated in many experimental models of myocardial infarction and in patients suffering from ischemic cardiac diseases, suggesting the involvement in the pathophysiology of these disorders. A number of studies focused on manipulating SDF-1α and its receptor CXCR4 as central regulators of the stem cell mobilization process. Targeted expression of SDF-1α after myocardial infarction was shown to result in increased engraftment of bone marrow-derived stem cells into infarcted myocardium. This was accompanied by beneficial effects on cardiomyocyte survival, neovascularization and cardiac function. Thus, the SDF-1/CXCR4 axis seems to be a promising novel therapeutic approach to improve post-infarction therapy by attracting circulating stem cells to remain, survive and possibly differentiate in the infarct area. This review will summarize clinical trials of stem cell therapy in patients with myocardial infarction. We further discuss the basic findings about SDF-1α in stem cell recruitment and its therapeutic implications in experimental myocardial infarction.     

同种异体骨髓间质干细胞移植联合VEGF基因转染治疗心肌梗死的实验研究

目的：探讨同种异体骨髓间质千细胞（BMSCs）移植联合血管内皮生长因子（VEGF）基因转染对大鼠急性心肌梗死血管再生和心功能的影响：方法：体外分离、纯化、培养大鼠BMSCs，以BrdU标记细胞；制备、抽提、纯化质粒PAdTrack／VEGF165。结扎冠状动脉建立急性心肌梗死模型2周后，随机将其分为4组（n=12），进行心肌内BMSCs移植和／或VEGF165转染，组Ⅰ：BMSCs移植＋VEGF165转染；组Ⅱ：BMSCs移值；组Ⅲ：VEGF165转染；组Ⅳ：DMEM注射作为对照组。4周后行免疫组化和超声心动图检查：结果：组Ⅰ和组Ⅱ梗死及缺血心肌处可见大量BrdU标记的移植细胞，其中缺血心肌处部分移植细胞分化为血管内皮细胞并形成新生毛细血管。Ⅷ因子染色阳性的新生血管密度分布为组Ⅰ〉组Ⅲ〉组Ⅱ〉组Ⅳ（P均〈0．01）。细胞移植和基因转染治疗后室壁厚度和室壁运动幅度改善，EF值增加幅度（△EF）组Ⅰ〉组Ⅱ〉组Ⅲ〉组Ⅳ（P均〈0．01）：结论：同种异体骨髓间质干细胞移植联合VEGF基因转染能进一步增强大鼠梗死缺血区血管再生、改善室壁厚度和心功能，有利于心肌梗死后的康复。     

Role of host tissues for sustained humoral effects after endothelial progenitor cell transplantation into the ischemic heart

Noncellular differentiation effects have emerged as important mechanisms mediating therapeutic effects of stem or progenitor cell transplantation. Here, we investigated the expression patterns and sources of humoral factors and their regional and systemic biological effects after bone marrow (BM)-derived endothelial progenitor cell (EPC) transplantation into ischemic myocardium. Although most of the transplanted EPCs disappeared within a week, up-regulation of multiple humoral factors was sustained for longer than two weeks, which correlated well with the recovery of cardiac function. To determine the source of the humoral factors, we injected human EPCs into immunodeficient mice. Whereas the expression of human EPC (donor)-derived cytokines rapidly decreased to a nondetectable level within a week, up-regulation of mouse (recipient)-derived cytokines, including factors that could mobilize BM cells, was sustained. Histologically, we observed higher capillary density, a higher proliferation of myocardial cells, a lower cardiomyocyte apoptosis, and reduced infarct size. Furthermore, after EPC transplantation, BM-derived stem or progenitor cells were increased in the peripheral circulation and incorporated into the site of neovascularization and myocardial repair. These data indicate that myocardial EPC transplantation induces humoral effects, which are sustained by host tissues and play a crucial role in repairing myocardial injury.