Dongchao Lv

Cardiac Telocytes and Fibroblasts in Primary Culture: Different Morphologies and Immunophenotypes

Telocytes (TCs) are a peculiar type of interstitial cells with very long prolongations termed telopodes. TCs have previously been identified in different anatomic structures of the heart, and have also been isolated and cultured from heart tissues in vitro. TCs and fibroblasts, both located in the interstitial spaces of the heart, have different morphologies and functionality. However, other than microscopic observation, a reliable means to make differential diagnosis of cardiac TCs from fibroblasts remains unclear. In the present study, we isolated and cultured cardiac TCs and fibroblasts from heart tissues, and observed their different morphological features and immunophenotypes in primary culture. Morphologically, TCs had extremely long and thin telopodes with moniliform aspect, stretched away from cell bodies, while cell processes of fibroblasts were short, thick and cone shaped. Furthermore, cardiac TCs were positive for CD34/c-kit, CD34/vimentin, and CD34/PDGFR-β, while fibroblasts were only vimentin and PDGFR-β positive. In addition, TCs were also different from pericytes as TCs were CD34 positive and α-SMA weak positive while pericytes were CD34 negative but α-SMA positive. Besides that, we also showed cardiac TCs were homogenously positive for mesenchymal marker CD29 but negative for hematopoietic marker CD45, indicating that TCs could be a source of cardiac mesenchymal cells. The differences in morphological features and immunophenotypes between TCs and fibroblasts will provide more compelling evidence to differentiate cardiac TCs from fibroblasts.

MicroRNAs in diabetic cardiomyopathy and clinical perspectives

Diabetes is a progressive metabolic disorder that can ultimately lead to serious chronic vascular complications including renal failure, vision loss, and cardiac dysfunction (Ruiz and Chakrabarti, 2013). Diabetic cardiomyopathy is responsible for higher incidence of sudden cardiac death and represents the leading cause of morbidity and mortality among the diabetic patients (Aksnes et al., 2007; Chavali et al., 2013). Previous studies have indicated that oxidative stress and mitochondrial dysfunction were critically involved in the etiology of diabetes-induced cardiac dysfunction (Sugamura and Keaney, 2011; Styskal et al., 2012), that could subsequently induce a cascade of complex pathophysiological events characterized by early impairments of diastolic function, development of cardiomyocyte hypertrophy, myocardial fibrosis and cardiomyocyte apoptosis, eventually leading to heart failure (Huynh et al., 2014). However, the underlying mechanisms of diabetic cardiomyopathy are far from understood and current therapeutic strategies do not specifically aim at diabetic cardiomyopathy and diabetes-induced heart failure. MicroRNAs (miRNAs, miRs), a novel class of non-coding RNAs of 22~24 nucleotides in length, act as post-transcriptional regulators of gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNA that induces mRNA degradation and/or translational repression (Lim et al., 2005; Van Rooij, 2011). Given that miRNAs are crucially involved in many critical biological processes including cell proliferation, apoptosis, necrosis, migration and differentiation (Bartel, 2004), desregulated miRNAs contribute to many human diseases including diabetes (Tyagi et al., 2011; Shantikumar et al., 2012; McClelland and Kantharidis, 2014) and cardiovascular diseases (Xiao et al., 2012; Fu et al., 2013; Vickers et al., 2014). Recent studies demonstrate that aberrant expression of miRNAs also participates in the pathogenetic processes mediating diabetic cardiomyopathy, where miR-1, -133, -141, -206, -223 have been reported upregulated, whereas miR-133a, -373, and -499 downregulated (Shen et al., 2011; Shantikumar et al., 2012; Asrih and Steffens, 2013). Thus, it is of crucial importance to gain insight into the role of miRNAs in the development of diabetic cardiomyopathy which will help clarify the molecular mechanisms as well as identify novel therapeutic strategies for diabetic cardiomyopathy. Cardiomyocyte hypertrophy, myocardial fibrosis, and cardiomyocyte apoptosis are important features of diabetic cardiomyopathy (Ruiz and Chakrabarti, 2013). Downregulation of miR-133a induces cardiomyocyte hypertrophy via upregulating the expression of MEF2A and MEF2C, two transcription factors involved in myocardial hypertrophy (Feng et al., 2010). While upregulation of miR-1 and -206 contributes to increased cardiomyocyte apoptosis, via repressing the expression of heat shock protein (Hsp) 60, PIM 1, and IGF-1 receptor (Yu et al., 2008; Shan et al., 2010; Katare et al., 2011). In addition, miR-373 is downregulated in diabetic heart, which is supposed to induce cardiac fibrosis via regulating the expression of p300 (Feng et al., 2008; Chen et al., 2010; Shen et al., 2011; Chavali et al., 2014). Thus, these reports indicate the critical contribution of miRNAs in cardiomyocyte hypertrophy, myocardial fibrosis, and cardiomyocyte apoptosis during the development of diabetic cardiomyopathy. Hyperglycemia, oxidative stress and mitochondrial damage are involved in the etiology of diabetes-induced cardiac dysfunction and diabetic cardiomyopathy (Shantikumar et al., 2012; Asrih and Steffens, 2013; McClelland and Kantharidis, 2014). Previous study has shown that miR-499 and -133a were markedly downregulated in the diabetic cardiomyocytes, while normalization of oxidant/antioxidant level by the treatment of N-acetylcysteine (NAC) restored the impaired expression of these miRNAs, indicating that hyperglycemia-induced downregulation of miR-499 and -133a was oxidative stress dependent (Yildirim et al., 2013). Similarly, miR-373 was downregulated by hyperglycemia-induced oxidative stress in diabetic cardiomyopathy via p38 MAPK pathway (Shen et al., 2011). MiR-141, a critical regulator of the inner mitochondrial phosphate carrier (Slc25a3), has been shown upregulated in diabetic heart, thus leading to the impaired mitochondrial ATP production in the pathogenesis of diabetic cardiomyopathy (Baseler et al., 2012). In terms of cardiomyocyte glucose metabolism, miR-223 was shown to be upregulated in the left ventricular biopsies of diabetic patients, which induced Glut4 protein level in cardiomyocytes and contributed to cardiomyocyte glucose uptake in vitro, indicating that overexpression of miR-223 might be a compensatory response to restore glucose metabolism in diabetic heart (Lu et al., 2010). Accumulating evidence has indicated that circulating miRNAs can be used as sensitive biomarkers for certain diseases such as cardiovascular diseases and cancers (Fabbri, 2010; Tijsen et al., 2012; Xu et al., 2012). Despite that diabetes is among the major risk factors for cardiovascular complications, researches investigating circulating miRNAs in diabetic patients are quite limited. Zampetaki et al. reported deregulation of 12 plasma miRNAs (miR-24, -21, -20b, -15a, -126, -191, -197, -223, -320, -486, -150, and -28-3p) in diabetic subjects, among which miR-126 emerged as a predictor of diabetes mellitus (Zampetaki et al., 2010). A separate study identified 7 upregulated serum miRNAs (miR-9, -29a, -30d, -34, -124, -146a, and -375) in newly diagnosed type 2 diabetic patients as compared to susceptible controls (Kong et al., 2011). Another study identified elevation of miR-144, 192, and 29a in the whole blood of diabetic patients (Karolina et al., 2011), whereas no change was found in miR-126 level which was inconsistent with the report published by Zampetaki et al. (2010). This may be explained by different biosamples detected (plasma vs. whole blood) in these two studies (Zampetaki et al., 2010; Karolina et al., 2011). In addition, miR-503 was found to be enriched in the plasma of diabetic patients with critical limb ischemia (Caporali et al., 2011). However, to date, no specific circulating miRNA has been identified in diabetic cardiomyopathy. The diagnostic and predicted value of circulating miRNAs as biomarkers for diabetic cardiomyopathy remains to be further explored. Taken together, desregulated miRNAs are potentially involved in the etiology and pathogenetic processes of diabetic cardiomyopathy. An in-depth understanding of their functional roles and molecular mechanisms in the development of diabetic cardiomyopathy will provide better prospects to identify sensitive clinical biomarkers and novel therapeutic targets for diabetic cardiomyopathy.

miR-19b attenuates H2O2-induced apoptosis in rat H9C2 cardiomyocytes via targeting PTEN

Myocardial ischemia-reperfusion (I-R) injury lacks effective treatments. The miR-17-92 cluster plays important roles in regulating proliferation, apoptosis, cell cycle and other pivotal processes. However, their roles in myocardial I-R injury are largely unknown. In this study, we found that miR-19b was the only member of the miR-17-92 cluster that was downregulated in infarct area of heart samples from a murine model of I-R injury. Meanwhile, downregulation of miR-19b was also detected in H2O2-treated H9C2 cells in vitro mimicking oxidative stress occurring during myocardial I-R injury. Using flow cytometry and Western blot analysis, we found that overexpression of miR-19b decreased H2O2-induced apoptosis and improved cell survival, while downregulation of that had inverse effects. Furthermore, PTEN was negatively regulated by miR-19b at the protein level while silencing PTEN could completely block the aggravated impact of miR-19b inhibitor on H2O2-induced apoptosis in H9C2 cardiomyocytes, indicating PTEN as a downstream target of miR-19b controlling H2O2-induced apoptosis. These data indicate that miR-19b overexpression might be a novel therapy for myocardial I-R injury.

Exercise-induced circulating extracellular vesicles protect against cardiac ischemia–reperfusion injury

Extracellular vesicles (EVs) serve an important function as mediators of intercellular communication. Exercise is protective for the heart, although the signaling mechanisms that mediate this cardioprotection have not been fully elucidated. Here using nano-flow cytometry, we found a rapid increase in plasma EVs in human subjects undergoing exercise stress testing. We subsequently identified that serum EVs were increased by ~1.85-fold in mice after 3-week swimming. Intramyocardial injection of equivalent quantities of EVs from exercised mice and non-exercised controls provided similar protective effects against acute ischemia/reperfusion (I/R) injury in mice. However, injection of exercise-induced EVs in a quantity equivalent to the increase seen with exercise (1.85 swim group) significantly enhanced the protective effect. Similarly, treatment with exercise-induced increased EVs provided additional anti-apoptotic effect in H2O2-treated H9C2 cardiomyocytes mediated by the activation of ERK1/2 and HSP27 signaling. Finally, by treating H9C2 cells with insulin-like growth factor-1 to mimic exercise stimulus in vitro, we found an increased release of EVs from cardiomyocytes associated with ALIX and RAB35 activation. Collectively, our results show that exercise-induced increase in circulating EVs enhances the protective effects of endogenous EVs against cardiac I/R injury. Exercise-derived EVs might serve as a potent therapy for myocardial injury in the future.

Targeting microRNAs in Pathological Hypertrophy and Cardiac Failure.

MicroRNAs (miRNAs) are a novel class of endogenous, short, non-coding, posttranscriptional RNAs, which play important roles in regulating lots of important biological functions. Evidences show that altered expression of miRNAs are involved in pathological hypertrophy and cardiac failure, making it possible to target miRNAs as a novel therapy. In this review, we focus on very recent progresses in the regulation of miRNAs in pathological hypertrophy and cardiac failure. In addition, we also discuss the potential of using miRNAs as a new therapy for pathological hypertrophy and cardiac failure.

miR-19b controls cardiac fibroblast proliferation and migration

Cardiac fibrosis is a fundamental constituent of a variety of cardiac dysfunction, making it a leading cause of death worldwide. However, no effective treatment for cardiac fibrosis is available. Therefore, novel therapeutics for cardiac fibrosis are highly needed. Recently, miR‐19b has been found to be able to protect hydrogen peroxide (H2O2)‐induced apoptosis and improve cell survival in H9C2 cardiomyocytes, while down‐regulation of miR‐19b had opposite effects, indicating that increasing miR‐19b may be a new therapeutic strategy for attenuating cellular apoptosis during myocardial ischaemia–reperfusion injury. However, considering the fact that microRNAs might exert a cell‐specific role, it is highly interesting to determine the role of miR‐19b in cardiac fibroblasts. Here, we found that miR‐19b was able to promote cardiac fibroblast proliferation and migration. However, miR‐19b mimics and inhibitors did not modulate the expression level of collagen I. Pten was identified as a target gene of miR‐19b, which was responsible for the effect of miR‐19b in controlling cardiac fibroblast proliferation and migration. Our data suggest that the role of miR‐19b is cell specific, and systemic miR‐19b targeting in cardiac remodelling might be problematic. Therefore, it is highly needed and also urgent to investigate the role of miR‐19b in cardiac remodelling in vivo.

miR‐149 controls non‐alcoholic fatty liver by targeting FGF‐21

Non‐alcoholic fatty liver disease (NAFLD), a lipid metabolism disorder characterized by the accumulation of intrahepatic fat, has emerged as a global public health problem. However, its underlying molecular mechanism remains unclear. We previously have found that miR‐149 was elevated in NAFLD induced by high‐fat diet mice model, whereas decreased by a 16‐week running programme. Here, we reported that miR‐149 was increased in HepG2 cells treated with long‐chain fatty acid (FFA). In addition, miR‐149 was able to promote lipogenesis in HepG2 cells in the absence of FFA treatment. Moreover, inhibition of miR‐149 was capable of inhibiting lipogenesis in HepG2 cells in the presence of FFA treatment. Meanwhile, fibroblast growth factor‐21 (FGF‐21) was identified as a target gene of miR‐149, which was demonstrated by the fact that miR‐149 could negatively regulate the protein expression level of FGF‐21, and FGF‐21 was also responsible for the effect of miR‐149 inhibitor in decreasing lipogenesis in HepG2 cells in the presence of FFA treatment. These data implicate that miR‐149 might be a novel therapeutic target for NAFLD.

Desregulated microRNAs in aging-related heart failure

Heart failure is the major cause of death in the western world. Despite the development and use of standard evidence-based therapeutic strategies for heart failure like inhibition of the activity of the β-adrenergic signaling and renin-angiotensin-aldosterone system, the prevalence of heart failure is still increasing, while morbidity and mortality have not been satisfactorily improved (Hofmann and Frantz, 2013). Growing evidence has indicated that the rising incidence of heart failure is substantially associated with age. In the United States, a high proportion of the estimated 5 million heart failure patients are older people, and a vast majority of heart failure-related hospitalization and death occurred in patients over 65 years old (Go et al., 2014). With the tendency of global aging, it is necessary to go deeper in exploring the aging-related heart failure. Cardiac aging is characterized by a series of complex events of ventricle and valvular changes involving left ventricular hypertrophy, diastolic dysfunction, increased risk of atrial fibrillation, valvular degeneration and fibrosis, and decreased maximal exercise capacity. These changes make the aged heart more susceptible to stress, leading to a high prevalence of cardiovascular diseases and heart failure (Correia et al., 2002; Dai et al., 2012a). The mechanisms of progression to heart failure in the aged heart have been previously described. The oxidative stress and mitochondrial damage are responsible for triggering the increased cardiomyocyte death including necrosis, apoptosis and autophagy, accompanied by hypertrophy of remaining cells and impaired structure of extracellular matrix (ECM), thus leading to ventricular remodeling and reduced cardiac contractility (Nadal-Ginard et al., 2003; Sarkar et al., 2004; Lindsey et al., 2006; Dai et al., 2012b; Venkataraman et al., 2013). Meanwhile, cardiac hypertrophy leads to a mismatch in oxygen supply and demand, which contributes to endothelial dysfunction and angiogenesis (Shiojima et al., 2005; Izumiya et al., 2006; Heineke et al., 2007). In response to these chronic stress, the aged heart undergoes a complex pathophysiological changes and finally progresses to symptomatic heart failure (Foo et al., 2005; Dai et al., 2012a). MicroRNAs (miRNAs, miRs) are a novel class of small non-coding RNAs with approximately 20-24 length of base, which function as endogenous suppressors of gene expression through mRNA degradation and/or translational inhibition mainly by binding to 3′-untranslated region (3′-UTR) of target mRNAs (Lim et al., 2005; Van Rooij, 2011). Nowadays over 2000 miRNAs have been identified in human genome and each miRNA can modulate numerous target genes and build complex signaling networks (Kim and Nam, 2006; Liang, 2009). As a center player of gene regulation, many essential biological processes are regulated by miRNAs, including proliferation, apoptosis, necrosis, autophagy, differentiation, and stress responses (Bartel, 2004). Due to these multiple roles, miRNAs are critically involved in the development of multifarious heart diseases, such as heart hypertrophy, arrhythmia, acute myocardial infarction, and heart failure (Latronico and Condorelli, 2009; Xiao et al., 2012, 2014; Fu et al., 2013; Vickers et al., 2014). Several microarray studies have revealed expression profiles of specific miRNAs that are aberrantly expressed in heart failure. MiR-1, -29, -30, -133, and -150 were found to be downregulated in heart failure, whereas miR-21, -23, -27, -125, -132, -146, -195, -199, -214, -223, and 342 were upregulated (Van Rooij et al., 2006; Cheng et al., 2007; Ikeda et al., 2007; Sayed et al., 2007; Tatsuguchi et al., 2007; Thum et al., 2007; Sucharov et al., 2008; Matkovich et al., 2009; Naga Prasad et al., 2009). In addition, several circulating miRNAs including miR-423-5P have been considered as putative biomarkers for heart failure (Tijsen et al., 2010). Some distinguished reviews have summarized it in detail (Elzenaar et al., 2013; Kumarswamy and Thum, 2013; De Rosa et al., 2014; Harada et al., 2014). As we know, cardiac aging is among the predominant risk factors for the development of heart failure (Correia et al., 2002; Dai et al., 2012a). Recent advances suggest that miRNAs may also play a role in the regulation of gene expression in cardiovascular aging processes (Zhang et al., 2012; Olivieri et al., 2013; Menghini et al., 2014). It has been previously demonstrated that 65 miRNAs were differentially expressed in the old versus young mouse adult hearts, approximately half of which belong to 11 miRNA clusters, indicating that these clusters contribute to the complex regulation of gene expression during heart aging (Zhang et al., 2012). In addition, miR-22 was shown to be involved in aging-related cardiac fibrosis, whose overexpression contributed to cellular senescence and migration of cardiac fibroblasts (Jazbutyte et al., 2013). More recently, it was demonstrated that aging-induced expression of miR-34a and inhibition of its target PNUTs lead to increased cardiomyocyte death and reduced cardiac contractility function, by inducing telomere shortening and DNA damage responses (Boon et al., 2013). However, the role of miRNAs in aging-related heart failure is far from elucidated. A previous study showed that the members of miR-17-92 cluster, including miR-18a, -19a, and -19b, were all downregulated in failure-prone heart of aged mice as well as in cardiac biopsies of idiopathic cardiomyopathy patients at old age with severely impaired cardiac function (ejection fraction, EF<30%), accompanied by increased expression of the ECM proteins connective tissue growth factor (CTGF) and thrombospondin-1 (TSP-1). Furthermore, the in vitro studies showed that these expression changes were specific in aged cardiomyocytes but not in cardiac fibroblasts, and the inhibition of miR-18/19 in cardiomyocytes contributed to collagen synthesis (Collagen 1A1 and 1A3) via the regulation of pro-fibrotic CTGF and TSP-1. Although the mechanisms underlying these regulations are still unknown, it provides a close relationship between miR-18/19 and aging-induced cardiac remodeling and heart failure (Van Almen et al., 2011). With the development of the research for roles of miRNAs in aging-related heart failure, its cellular and molecular mechanisms as well as pathophysiological changes will be further clarified, which will help develop novel miRNA-targeted therapeutic strategies for heart failure in aged people.

MicroRNA-221 is Required for Proliferation of Mouse Embryonic Stem Cells via P57 Targeting

Factors responsible for the rapid proliferative properties of embryonic stem (ES) cells are largely unknown. MicroRNA-221/222 (miR-221/222) regulate proliferation in many somatic cells, however, their roles in proliferation of ES cells are unclear. In this study, E14 mouse ES cells proliferation was determined by total cell counting, Cell Counting Kit (CCK-8), size of colonies and cell cycle analysis, while apoptosis and necrosis using Annexin V and propidium iodide staining. miR-221 inhibitor decreased proliferation of ES cells without inducing apoptosis and necrosis. miR-221 mimic, miR-222 mimic and miR-222 inhibitor did not affect ES cells proliferation. The expression level of miR-221 remained unchanged upon embryoid body (EB) formation. ES cells with miR-221 inhibition maintained an undifferentiated state, as indicated by unchanged alkaline phosphatase enzyme activity and Sox2, Nanong, and Oct4 expressions. P57 was post-transcriptionally regulated by miR-221 in ES cells. P57 knockdown completely abolished the inhibition effects of ES cells proliferation observed in miR-221 reduction, further indicating that miR-221 inhibition is likely to mediate its antiproliferative effects via P57 expression. To exclude that the function of miR-221 in ES cells is E14 specific, the effects of miR-221 mimic and inhibitor in size of colonies and cell cycle of R1 mouse ES cells were also determined and similar effects in inhibiting proliferation were achieved with miR-221 inhibition. Therefore, miR-221 is required for mouse ES cells proliferation via P57 targeting. This study indicates that miR-221 is among the regulators that control ES cells proliferation and might be used to influence the fate of ES cells.