Zhen-Ao Zhao

A brief review: adipose-derived stem cells and their therapeutic potential in cardiovascular diseases

Adipose-derived stem cells (ADSCs) are easily obtained and expanded, and have emerged as a novel source of adult stem cells for the treatment of cardiovascular diseases. These cells have been shown to have the capability of differentiating into cardiomyocytes, vascular smooth muscle cells, and endothelial cells. Furthermore, ADSCs secrete a series of paracrine factors to promote neovascularization, reduce apoptosis, and inhibit fibrosis, which contributes to cardiac regeneration. As a novel therapy in the regenerative field, ADSCs still face various limitations, such as low survival and engraftment. Thus, engineering and pharmacological studies have been conducted to solve these problems. Investigations have moved into phase I and II clinical trials examining the safety and efficacy of ADSCs in the setting of myocardial infarction. In this review, we discuss the differentiation and paracrine functions of ADSCs, the strategies promoting their therapeutic efficacy, and their clinical usage.

microRNA-206 is involved in survival of hypoxia preconditioned mesenchymal stem cells through targeting Pim-1 kinase

BackgroundOverexpression of Pim-1 in stem/progenitor cells stimulated cell cycling and enhanced cardiac regeneration in vivo. We proposed that hypoxic preconditioning could increase survival of bone marrow mesenchymal stem cells (MSCs) via upregulation of Pim-1 and aimed to determine the microRNAs that modulate the expression of Pim-1.Methods and resultsMSCs were subjected to hypoxia exposure. The expression of Pim-1 in MSCs was enhanced in a time-dependent manner, detected by quantitative PCR and western blot. miR-206 is predicted as one of the potential microRNAs that target Pim-1. The expression of miR-206 was decreased in hypoxic MSCs and reversely correlated with Pim-1 expression. Luciferase activity assay further confirmed Pim-1 as a putative target of miR-206. In addition, gain and loss-of-function studies with miR-206 mimics and inhibitors showed that inhibition of miR-206 in hypoxic MSCs promoted the migration ability of the cells, prevented cell apoptosis, and protected membrane potential of mitochondria, while the benefits were all blocked by Pim-1 inhibitor. In an acute model of myocardial infarction, transplanted hypoxic MSCs showed a significantly improved survival as compared with hypoxic MSCs overexpressing miR-206.ConclusionsHypoxic preconditioning could increase short-term survival of bone marrow MSCs via upregulation of Pim-1, and miR-206 was one of the critical regulators in this process.

Direct reprogramming of fibroblasts into cardiomyocytes

Cardiovascular diseases are the leading causes of death in the world. The limited regenerative capacity of adult cardiomyocytes is the major barrier for heart regeneration. After myocardial infarction, myofibroblasts are the dominant cell type in the infarct zone. Therefore, it is a good idea to reprogram terminally differentiated myofibroblasts into cardiomyocyte-like cells directly, providing a good strategy to simultaneously reduce scar tissue and increase functional cardiomyocytes. Transcription factors were first identified to reprogram myofibroblasts into cardiomyocytes. Thereafter, microRNAs and/or small molecules showed great potential to optimize the reprogramming process. Here, we systemically summarize and compare the major progress in directed cardiac reprogramming including transcription factors and miRNAs, especially the small molecules. Furthermore, we discuss the challenges needed to be overcome to apply this strategy clinically.

MicroRNA-133 overexpression promotes the therapeutic efficacy of mesenchymal stem cells on acute myocardial infarction

BackgroundOur study aim was to evaluate the therapeutic efficacy and mechanisms of miR-133-overexpressing mesenchymal stem cells (MSCs) on acute myocardial infarction.MethodsRat MSCs were isolated and purified by whole bone marrow adherent culturing. After transfection with the agomir or antagomir of miR-133, MSCs were collected for assay of cell vitality, apoptosis, and cell cycle progression. At the same time, exosomes were isolated from the supernatant to analyze the paracrine miR-133. For in-vivo studies, constitutive activation of miR-133 in MSCs was achieved by lentivirus-mediated miR-133 overexpression. A rat myocardial infarction model was created by ligating the left anterior descending coronary artery, while control MSCs (vector-MSCs) or miR-133-overexpressed MSCs (miR-133-MSCs) were injected into the zone around the myocardial infarction. Subsequently, myocardial function was evaluated by echocardiography on days 7 and 28 post infarction. Finally the infarcted hearts were collected on days 7 and 28 for myocardial infarct size measurement and detection of snail 1 expression.ResultsHypoxia-induced apoptosis of MSCs obviously reduced, along with enhanced expression of total poly ADP-ribose polymerase protein, after miR-133 agomir transfection, while the apoptosis rate increased in MSCs transfected with miR-133 antagomir. However, no change in cell viability and cell-cycle distribution was observed in control, miR-133-overexpressed, and miR-133-interfered MSCs. Importantly, rats transplanted with miR-133-MSCs displayed more improved cardiac function after acute myocardial infarction, compared with those that received vector-MSC injection. Further studies indicated that cardiac expression of snail 1 was significantly repressed by adjacent miR-133-overexpressing MSCs, and both the inflammatory level and the infarct size decreased in miR-133-MSC-injected rat hearts.ConclusionsmiR-133-MSCs obviously improved cardiac function in a rat model of myocardial infarction. Transplantation of miR-133-overexpressing MSCs provides an effective strategy for cardiac repair and modulation of cardiac-related diseases.

Regulatory roles of interferon-inducible protein 204 on differentiation and vasculogenic activity of endothelial progenitor cells

BackgroundEndothelial progenitor cells (EPCs) have shown great potential in angiogenesis either by their differentiation into endothelial cells or by secretion of angiogenic factors. Interferon-inducible protein 204 (Ifi204) has been reported to participate in the regulation of cell growth and differentiation. However, its role in differentiation of EPCs remains unknown. We proposed that Ifi204 could modulate the differentiation and regenerative abilities of EPCs.MethodsIfi204-expressing lentivirus and Ifi204 siRNA were introduced into EPCs to overexpress and suppress the expression of Ifi204. Using fluorescence-activated cell sorting, immunocytochemistry, and quantitative PCR, endothelial markers including CD31, VE-cadherin, and vWF were detected in the modified EPCs. An in-vitro incorporation assay and a colony-forming assay were also performed.ResultsEvidence showed that Ifi204 inhibition decreased the endothelial differentiation and vasculogenic activities of EPCs in vitro. In mice with hindlimb ischemia, downregulation of Ifi204 in EPCs, which was tracked by our newly synthesized nanofluorogen, impaired neovascularization, with a corresponding reduction in hindlimb blood reperfusion by postoperative day 14.ConclusionsIfi204 is required for EPC differentiation and neovascularization in vitro and in vivo. The regulatory roles of Ifi204 in EPC differentiation may benefit the clinical therapy of ischemic vascular diseases.

Functional mutant GATA4 identification and potential application in preimplantation diagnosis of congenital heart diseases

Congenital heart diseases (CHDs) affect nearly 1% of all neonates and show an increasing tendency. The complex inheritance patterns and multifactorial etiologies make these defects difficult to be identified before complete manifestation. Genetic screening has identified hundreds of specific mutant sites for CHDs based on cardiac transcriptional factors. GATA4 is a master regulator required for ventral morphogenesis and heart tube formation. Its mutation is most widely studied in CHDs. In the past decades, over 100 GATA4 mutant sites have been reported, but only a few functional sites have been identified. Thus, it is important to distinguish deleterious sites from neutral sites. In silico prediction of functional sites using bioinformatics tools can provide the valuable information, but it is not solid enough. Here, the roles of GATA4 in heart development is discussed in detail and its mutation sites in protein coding region are summarized systematically, providing an integrated resource for GATA4 mutations. Furthermore, we discussed the advantage and disadvantage of different methods for functional mutation identification. Especially, the disease model of induced pluripotent stem cell is emerging as a powerful tool to assess GATA4 mutations in human. In the recent years, single-cell based high-throughput sequencing is being applied in preimplantation diagnosis and assisted reproduction progressively, providing a new strategy for the prevention of congenital diseases as we discussed. Based on functional mutant sites identification, preimplantation diagnosis will contribute to CHDs prevention eventually.

The Application of Induced Pluripotent Stem Cells in Cardiac Disease Modeling and Drug Testing

In recent decades, cardiovascular diseases have become the greatest health threat to human beings, and thus it is particularly important to explore the subtle underlying pathogenesis of cardiovascular diseases. Although many molecular pathways have been explored to be essential in the development of cardiovascular diseases, their clinical significances are still uncertain. With the emergence of induced pluripotent stem cells (iPSCs), a unique platform for cardiovascular diseases has been established to model cardiovascular diseases on specific genetic background in vitro. This review summarizes current progresses of iPSCs in cardiovascular disease modeling and drug testing. This review highlighted iPSC-based cardiovascular disease modeling and drug testing. The technical advances in iPSC-based researches and various clinically relevant applications are discussed. With further intensive research, iPSC technology will shape the future of clinical translational research in cardiovascular diseases.

Long noncoding RNA Meg3 regulates cardiomyocyte apoptosis in myocardial infarction

Myocardial infarction (MI), with a major process of cardiomyocyte death, remains a leading cause of morbidity and mortality worldwide. To date, it has been shown that lncRNAs play important roles in cardiovascular pathology. However, the detailed studies on lncRNAs regulating cardiomyocyte death in myocardial infarction are still limited. In this study, we found a progressively upregulated expression of Meg3 in mouse injured heart after MI. Gain-of-function and loss-of-function approaches further revealed pro-apoptotic functions of Meg3 in rodent cardiomyocytes. Moreover, Meg3 was directly upregulated by p53 in hypoxic condition, and involved in apoptotic regulation via its direct binding with RNA-binding protein FUS (fused in sarcoma). Afterwards, adult MI mice that underwent intramyocardial injection with adeno-associated virus serotype 9 (AAV9) system carrying Meg3 shRNA showed a significant improvement of cardiac function. Moreover, we also found that MEG3 was increased in clinical heart failure samples, and had conservatively pro-apoptotic function in human cardiomyocytes that were differentiated from the human embryonic stem cells. Together, these results indicate that p53-induced Meg3–FUS complex plays an important role in cardiomyocyte apoptosis post-MI, and its specific knockdown in cardiomyocytes with AAV9 system represents a promising method to treat MI for preclinical investigation.