Role of cardiac progenitor cell‐derived exosome‐mediated microRNA‐210 in cardiovascular disease

Abstract Cardiac progenitor cells are considered to be one of the most promising stem cells for heart regeneration and repair. The cardiac protective effect of CPCs is mainly achieved by reducing tissue damage and/or promoting tissue repair through a paracrine mechanism. Exosome is a factor that plays a major role in the paracrine effect of CPCs. By delivering microRNAs to target cells and regulating their functions, exosomes have shown significant beneficial effects in slowing down cardiac injury and promoting cardiac repair. Among them, miRNA‐210 is an important anoxic‐related miRNA derived from CPCs exosomes, which has great cardiac protective effect of inhibiting myocardial cell apoptosis, promoting angiogenesis and improving cardiac function. In addition, circulating miR‐210 may be a useful biomarker for the prediction or diagnosis of related cardiovascular diseases. In this review, we briefly reviewed the mechanism of miR‐210 derived from CPCs exosomes in cardiac protection in recent years.


| INTRODUC TI ON
Cardiovascular disease (CVD) is still an important cause of human death, and its incidence is increasing year by year. Current treatments for these diseases slow the progression of the disease, but ultimately fail to prevent irreversible damage to the heart muscle, leading to pump failure. Therefore, how to repair and regenerate damaged cardiomyocytes is a key and difficult problem in the current treatment. Cardiac progenitor cells (CPCs) are a group of stem cells found in adult heart muscle and are one of the best stem cell types currently available to repair the heart. At first, it was believed that direct differentiation and replacement of damaged heart tissues after transplantation were the main mechanism of CPCs to promote cardiac recovery. However, with the progress of research, it was found that CPCs mainly reduced tissue damage and/or promoted tissue repair through paracrine. Exosome, an extracellular vesicle, is an important factor for heart repair and protection in the paracrine effect of CPCs. They are rich in important biological information such as proteins, lipids, mRNAs and MicroRNAs (miRNAs) and are important messengers and carriers for information transmission between cells and regulate the biological functions of other cells.
Among them, miRNAs regulate cardiac functions by acting on themselves or distant target cells through autocrine or paracrine, so as to play a role in the treatment of CVDs. CPCs exosomes contain a variety of miRNAs, which have the effects of anti-apoptosis, antifibrosis, promoting angiogenesis, reducing infarction area and scar formation. Among them, miR-210 is an important hypoxia-related miRNA, which has certain advantages in inhibiting cell apoptosis, promoting angiogenesis and improving cardiac function. In this paper, the role and research prospect of CPC-derived exosomes and miR-210 in CVDs were reviewed in order to provide new ideas for the treatment of CVDs.

| E XOSOME FROM C ARD IAC PROG ENITOR CELL S
Cardiac progenitor cells, also known as cardiac stem cells, 1 are a small group of stem-like cells in the heart, which are immature cardiac cells that can be differentiated into progenitor cells. Studies have reported that CPCs can promote myocardial cell proliferation, inhibit apoptosis, promote angiogenesis, reduce myocardial fibrosis and other beneficial effects and can increase myocardial blood flow, reduce inflammation, reduce the scar after myocardial infarction size, thereby increasing myocardial blood supply, reducing inflammation, reducing scar size after myocardial infarction, and even preventing myocardial injury after ischaemia/reperfusion, improving cardiac function. [2][3][4] However, there has been considerable controversy about the main mechanism of CPC-mediated cardiac repair, including the hypothesis of direct transdifferentiation and paracrine effect regulating endogenous repair. 5 Early studies have shown that transplanted CPCs can be directly transformed into cardiomyocytes to repair or regenerate damaged hearts. However, in the process of a large number of experimental studies, it has been gradually found that CPCs mainly protects endogenous tissues through some indirect mechanism of action, thus promoting cardiac recovery. CADUCEUS clinical trial showed that cardiac function continued to increase after CPCs transplantation, 4,6 and the number of cardiac cells, blood vessels and endothelial cells increased after transplantation, all of which benefited from the paracrine effect of CPCs, but the indirect mechanism of the transplanted cells still could not be detected.
Therefore, many studies have focused on the exploration of a paracrine mechanism of CPCs, and extracellular vesicles (EVs), as the basic medium of intercellular interaction, have attracted much attention. 7 Researchers observed using transmission electron microscopy CPCs multivesicular body contains the secrete body sample EVs in mice and human heart, 8 then studies further describe the ultrastructure of the outer body sample EVs secretion. 9 The results showed that the diameter of EVs secreted by CPCs was about 30-90nm and had a typical lipid double-layer structure, 10 which was similar in size and structure to exosomes. Therefore, they are exosomes and bioactive components that play a major role in the paracrine effect of CPCs.
The formation of exosomes begins with the process of invagination of cells, which form early nucleus (EEs). Under the control of related proteins, the early endosomes are formed by multiple intraluminal vesicles through the internal bud process. Polycystic bodies (MVBs) fuse with cell membranes under the regulation of Rab enzyme, secreting intraluminal vesicles, that is exosomes. [11][12][13] Different types of cells secrete exosomes in normal or pathological conditions, carrying biologically active molecules such as proteins, lipids and nucleic acids, including mRNA, miRNA, long-chain noncoding RNA (lncRNA) and DNA. It has its specificity due to different sources. In vivo, secreted vesicles can be internalized by adjacent cells or circulated in the blood, eventually interacting with the cells within a certain distance, affecting the physiological pathways in the recipient cells. 14 19,20 Experimental studies have shown that CPC exosome of mice has the effect of pro-angiogenic and anti-fibrotic. 18,21 Among them, the pro-angiogenic activity of CPC exosome in vitro and in vivo was mainly caused by the stimulation of endothelial cell migration by its matrix metalloproteinase (MMP) content. 22 In different animal models of myocardial infarction (MI) (mice and pigs), injection of CPC-derived exosomes at the edge of MI reduced infarct size, increased vascular density and recovered left ventricular ejection fraction compared with that of earlier studies. 10,18,23 Exosomes derived from CPCs have the same cardioprotective functions as CPCs, but, which are more stable and easier to save than cells. Moreover, exosomes carry a large number of bioactive molecules, such as mRNA and miRNA, which are conducive to signal transmission between cells, and are the important material basis for their function. Many studies have suggested that EVs secreted by CPCs have cardioprotective effects, and its potential mechanisms involve miRNAs. Analysis of miRNA expression in exosomes derived from CPCs revealed that several overexpressed major miRNAs were enriched in CPC exosomes, including miR-132, 10,29 miR-210, 10,21 miR-21, [30][31][32] miR-17, 21 miR-103, 21 miR-146a, 18,33 miR-133a, 34-36 miR-451, 20 miR-20a, 21 miR-15b, 21 miR-181a and miR-323-5p, 10,18 whose biological effects are very broad, such as regulating cell differentiation and proliferation, angiogenesis, inhibition of apoptosis and fibrosis, playing an important role in cardiac protection (Table 1). a specific site of the miR-210 promoter to activate its expression. In anoxic myocardial cells, the expression of miR-210 can also be upregulated by Akt, p53 and the upstream regulatory factor NF-kappa B transcription factor p50 (NFkB1) of miR-210, thereby reducing the production of mitochondrial ROS and exerting the protective effect on the myocardium. 37,38 Insulin can induce miR-210 expression through the PI3K/Akt pathway. 39 The study has also found that overexpression of miR-210 is also regulated by oxidized low-density lipoprotein (ox-LDL). 40 MiR-210 exerts its corresponding biological effects by regulating the expression of target genes. In normal hypoxic environment, it can induce the up-regulation of miR-210 and regulate the occurrence and development of cardiovascular disease in many aspects. 41 Its role is mainly in inhibiting cardiomyocyte apoptosis, promoting angiogenesis and improving cardiac function.

| Inhibition of cardiomyocyte apoptosis
Cardiomyocyte apoptosis, also called programmed cell death, is re-

| Promoting angiogenesis
Acute myocardial infarction is prone to complications such as cardiogenic shock and chronic heart failure (CHF). The main reason is the decrease of myocardial blood flow brought about by coronary artery occlusion, which leads to myocardial cell death and myocardial remodelling. Therefore, improving myocardial function after AMI may play an active role in reducing the incidence of re-  [60][61][62] but some studies also believe that miR-210 plays the above role by down-regulating the level of EFNA3 mRNA. 42 Meanwhile, Ctgf, PTP1b, GPD1-L and PHD2 exert similar angiogenic effect under the regulation of miR-210. 42,63 These studies proved that miR-210 has a higher potential of angiogenesis, providing evidence for cardiac protective effect of miR-210 ( Figure 1).

| Improve heart function
The continuous progress of various cardiovascular diseases will eventually lead to a decline in heart function, and eventually de-

| MIR-210 A S A B I OMARK ER OF C ARDIOVA SCUL AR DIS E A S E S
Because the membrane structure of exosomes enhances the ability to protect miRNAs, circulating miRNAs can stably exist in peripheral blood and resist degradation of endogenous RNases. And it can be detected in human body fluids. Therefore, some peripheral circulation miRNAs are considered as potential biomarkers of related cardiovascular disease. 64,65 In patients with aortic stenosis (AS), circulating miR-210 significantly increased compared with healthy patients, and its expression level could predict the prognosis of patients. 66 At the same time, miR-210 is also auxiliary biomarkers and the prognosis of chronic heart failure. MiR-210 under anoxic conditions present expression, and heart failure in pathophysiology, is caused by cardiac ejection in the body peripheral tissue relative lack of oxygen, so with the degree of heart failure, peripheral hypoxia also becomes more serious, miR-210 expression levels are higher. The expression level of miR-210 was significantly increased in patients with severe heart failure (NYHA III and IV), suggesting that miR-210 could be used as a potential biomarker to predict the prognosis of patients with heart failure. 67 However, research have shown that up-regulation of miR-210 can participate in the development of atherosclerosis by promoting endothelial cell apoptosis, and may become an effective therapeutic target for atherosclerosis, 68 which is consistent with the miR-210 over-expressed in the serum of patients with atherosclerotic occlusion disorder, therefore, miR-210 can be used as the biomarkers in the diagnosis of atherosclerosis. 69 Therefore, we speculate that circulating miR-210 may be a useful marker of predicting cardiovascular disease or diagnosis of related cardiovascular diseases and guide clinical treatment and prognosis.

| FUTURE PER S PEC TIVE S
In recent years, stem cell therapy has become a research hot spot in the cardiovascular field, bringing hope to heart regeneration and repair, as well as questions and challenges. The potential of cardiac regeneration and repair of CPCs has been gradually valued. More and more studies have shown that the paracrine effect is the main mechanism of CPCs, which can activate the endogenous cardiac repair mechanism, thereby promoting endothelial angiogenesis, inhibiting myocardial apoptosis, anti-fibrosis, reducing the scar after MI, and playing a role in cardiovascular protection, repair and regeneration. The exosomes derived from CPCs have the same cardioprotective function as CPCs and carry a large number of microRNAs that are closely related to their structure and function, which contribute to intercellular signal transmission.
They participate in a variety of cardiovascular pathophysiological processes and are an important material basis for its functioning.
MiR-210 is an important hypoxia-associated miRNA derived from exosomes of CPCs. Experimental researches have certificated that hypoxia conditions induce miR-210 overexpression, which significantly inhibits cardiomyocyte apoptosis, promotes angiogenesis, improves cardiac function and embodies the heart of CPC protect and repair function. In addition, miR-210 can enhance the role of mesenchymal stem cells (MSC) in the treatment of CVDs under hypoxia conditions, providing a useful therapeutic strategy for stem cell therapy in cardiac repair, 50,70 and miRNAs can be detected in many body fluids. Therefore, miR-210 not only has unique advantages in the treatment of cardiovascular diseases, but also may be an excellent biomarker, which is expected to be an alternative to cell therapy. However, current research on miRNA still faces many problems. A miRNA can regulate multiple target genes, and multiple miRNAs can also jointly regulate the same gene. In addition, because the miRNAs and their potential targets are not fully complementary, it becomes very difficult to identify miRNA targets through informatic approaches. Therefore, it is impossible to accurately determine the mode of action of miRNAs and the mechanism by which it affects mRNA translation and gene expression.
This complex regulatory network increases the limitations of clinical applications of miRNAs. At the same time, it is still not obvious whether circulating miRNAs is useful as meaningful biomarkers, and whether their detection methods are operable, and lack of large-scale prospective studies to confirm. Therefore, the role of miR-210 in cardiovascular disease has to be continuously explored and discovered in order to provide effective biological targets for clinical diagnosis and treatment.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (No. 81473634).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.