Alain van Mil

MicroRNA-1 and -499 Regulate Differentiation and Proliferation in Human-Derived Cardiomyocyte Progenitor Cells

Objective—To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. Cardiomyocyte progenitor cells (CMPCs) are a promising cell population, easily expanded and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for stem cell maintenance and differentiation via translational repression. We hypothesize that miRNAs are also involved in proliferation/differentiation of the human CMPCs in vitro. Methods and Results—Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression profiling demonstrated that muscle-specific miR-1 and miR-499 were highly upregulated in differentiated cells. Transient transfection of miR-1 and -499 in CMPC reduced proliferation rate by 25% and 15%, respectively, and enhanced differentiation into cardiomyocytes in human CMPCs and embryonic stem cells, likely via the repression of histone deacetylase 4 or Sox6. Histone deacetylase 4 and Sox6 protein levels were reduced, and small interference RNA (siRNA)-mediated knockdown of Sox6 strongly induced myogenic differentiation. Conclusion—miRNAs regulate the proliferation of human CMPC and their differentiation into cardiomyocytes. By modulating miR-1 and -499 expression levels, human CMPC function can be altered and differentiation directed, thereby enhancing cardiomyogenic differentiation.

Inhibition of miR-25 improves cardiac contractility in the failing heart

Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.

Early assessment of acute coronary syndromes in the emergency department: the potential diagnostic value of circulating microRNAs

Previous studies investigating the role of circulating microRNAs in acute coronary syndrome (ACS) were based on small patient numbers, performed no comparison with established markers of cardiac injury and did not have appropriate controls. We determined the potential diagnostic value of circulating microRNAs as novel early biomarkers in 332 suspected ACS patients on presentation to the emergency department (ED) in a prospective single‐centre study including cardiac miRNAs (miR‐1, ‐208a and ‐499), miR‐21 and miR‐146a. Levels of all miRs studied were significantly increased in 106 patients diagnosed with ACS, even in patients with initially negative high‐sensitive (hs) troponin or symptom onset <3 h. MiR‐1, miR‐499 and miR‐21 significantly increased the diagnostic value in all suspected ACS patients when added to hs‐troponin T (AUC 0.90). These three miRs were strong predictors of ACS independent of clinical co‐variates including patient history and cardiovascular risk factors. Interestingly, the combination of these three miRs resulted in a significantly higher AUC of 0.94 than hs‐troponin T (0.89). Circulating microRNAs hold great potential as novel early biomarkers for the management of suspected ACS patients.

MicroRNA-214 inhibits angiogenesis by targeting Quaking and reducing angiogenic growth factor release

AIMS Angiogenesis is a critical component of many pathological conditions in adult tissues and is essential for embryonic development. MicroRNAs are indispensable for normal vascular development, but their exact role in regulating angiogenesis remains unresolved. Previously, we have observed that miR-214 is differentially expressed in compensatory arteriogenesis. Here, we investigated the potential role of miR-214 in the process of angiogenesis. METHODS AND RESULTS miR-214 is expressed in all major vascular cell types, and modulation of miR-214 levels in endothelial cells significantly affected tubular sprouting. In vivo silencing of miR-214 enhanced the formation of a perfused vascular network in implanted Matrigel plugs and retinal developmental angiogenesis in mice. miR-214 directly targets Quaking, a protein critical for vascular development. Quaking knockdown reduced pro-angiogenic growth factor expression and inhibited endothelial cell sprouting similar to miR-214 overexpression. In accordance, silencing of miR-214 increased the secretion of pro-angiogenic growth factors, including vascular endothelial growth factor, and enhanced the pro-angiogenic action of the endothelial cell-derived conditioned medium, whereas miR-214 overexpression had the opposite effect. CONCLUSION Here, we report a novel role for miR-214 in regulating angiogenesis and identify Quaking as a direct target of miR-214. The anti-angiogenic effect of miR-214 is mediated through the down-regulation of Quaking and pro-angiogenic growth factor expression. This study presents miR-214 as a potential important target for pro- or anti-angiogenic therapies.

MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1.

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16.

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA‐155 (miR‐155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre‐miR‐155, anti‐miR‐155 or control‐miR (ctrl‐miR), followed by scratch‐ and transwell‐ assays. These functional assays displayed that miR‐155 over‐expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR‐155 transfected cells was collected and zymography analysis showed a significant decrease in MMP‐2 and MMP‐9 activities. The predicted 3′‐UTR of MMP‐16, an activator of MMP‐2 and ‐9, was cloned into the pMIR‐REPORT vector and luciferase assays were performed. Introduction of miR‐155 significantly reduced luciferase activity which could be abolished by cotransfection with anti‐miR‐155 or target site mutagenesis. By using MMP‐16 siRNA to reduce MMP‐16 levels or by using an MMP‐16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP‐16, miR‐155 efficiently inhibits cell migration via a reduction in MMP‐2 and ‐9 activities. Our study shows that miR‐155 might be used to improve local retention of hCMPCs after intramyocardial delivery.

microRNA-1 enhances the angiogenic differentiation of human cardiomyocyte progenitor cells

Instigated by the discovery of adult cardiac progenitor cells, cell replacement therapy has become a promising option for myocardial repair in the past decade. We have previously shown that human-derived cardiomyocyte progenitor cells (hCMPCs) can differentiate into cardiomyocyte-, endothelial-, and smooth muscle-like cells in vitro, and in vivo after transplantation in a mouse model of myocardial infarction, resulting in preservation of cardiac function. However, to allow successful repopulation of the injured myocardium, it is of key importance to restore myocardial perfusion by the formation of new vasculature. Several studies have shown that microRNAs regulate vascular differentiation of different stem/progenitor cells. Here, we show that miR-1 is upregulated in hCMPCs during angiogenic differentiation. Upregulation of miR-1 enhanced the formation of vascular tubes on Matrigel and within a collagen matrix, and also increased hCMPC motility, as shown by planar and transwell migration assays. By western blot, qRT-PCR and luciferase reporter assays, miR-1 was found to directly target and inhibit the expression of sprouty-related EVH1 domain-containing protein 1 (Spred1). Knocking down Spred1 phenocopies the functional effect seen for miR-1 upregulation. Using a systems biology approach, we found that in hCMPCs, miR-1 is proposed to control a network of genes predominantly involved in angiogenesis-related processes, including the Spred1 pathway. Our data shows that by upregulation of miR-1, the angiogenic differentiation of hCMPCs can be enhanced, which may be used as a new therapeutic approach to improve the efficiency of cell-based therapy for cardiac regeneration by enhancing the formation of new vasculature.

MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear‐and radial wall‐stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR‐132/212 expression was significantly upregulated after occlusion of the femoral artery. miR‐132/212 knockout (KO) mice display a slower perfusion recovery after hind‐limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR‐132/212 KO mice compared to WT, it can be stimulated with exogenous miR‐132, a dominant member of the miR‐132/212 family. Moreover, in in vitro pericyte‐endothelial co‐culture cell assays, overexpression of miR‐132 and mir‐212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR‐132/212 may exert their effects by enhancing the Ras‐Mitogen‐activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR‐132/212 cluster promotes arteriogenesis by modulating Ras‐MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.

The Potential of Modulating Small RNA Activity In Vivo

Small RNAs have shown to be ubiquitous, useful, post-transcriptional gene silencers in a diverse array of living organisms. As a result of homologous sequence interactions, these small RNAs repress gene expression. Through a process called RNA interference (RNAi), double strand RNA molecules are processed by an enzyme called Dicer, which cleaves RNA duplexes into 21-23 base pair oligomers. Depending on their end-point functions, these oligomers are named differently, the two most common being small interfering RNAs (siRNAs) and microRNAs (miRNAs). These small RNAs are the effector molecules for inducing RNAi, leading to post-transcriptional gene silencing by guiding the RNAi-induced silencing complex (RISC) to the target mRNA. By exploiting these small RNAs, it is possible to regulate the expression of genes related to human disease. The knockdown of such target genes can be achieved by transfecting cells with synthetically engineered small RNAs or small RNA expressing vectors. Within recent years, studies have also shown the important role of miRNAs in different diseases. By using several chemically engineered anti-miRNA oligonucleotides, disease related miRNAs can be specifically and effectively silenced. Since RNAi has developed into an everyday method for in vitro knockdown of any target gene of interest, the next step is to further explore its potential in vivo and the unique opportunities it holds for the development of novel therapeutic strategies. This review explores the various applications of small RNA technology in in vivo studies, and its potential for silencing genes associated with various human diseases. We describe the latest development in small RNA technology for both gene knockdown, and the inhibition of translational silencing in animal studies. A variety of small RNA formulations and modifications will be reviewed for their improvement on stability and half-life, their safety and off-target effects, and their efficiency and specificity of gene silencing.

MicroRNA 214 Is a Potential Regulator of Thyroid Hormone Levels in the Mouse Heart Following Myocardial Infarction, by Targeting the Thyroid-Hormone-Inactivating Enzyme Deiodinase Type III

Cardiac thyroid-hormone signaling is a critical determinant of cellular metabolism and function in health and disease. A local hypothyroid condition within the failing heart in rodents has been associated with the re-expression of the fetally expressed thyroid-hormone-inactivating enzyme deiodinase type III (Dio3). While this enzyme emerges as a common denominator in the development of heart failure, the mechanism underlying its regulation remains largely unclear. In the present study, we investigated the involvement of microRNAs (miRNAs) in the regulation of Dio3 mRNA expression in the remodeling left ventricle (LV) of the mouse heart following myocardial infarction (MI). In silico analysis indicated that of the miRNAs that are differentially expressed in the post-MI heart, miR-214 has the highest potential to target Dio3 mRNA. In accordance, a luciferase reporter assay, including the full-length 3′UTR of mouse Dio3 mRNA, showed a 30% suppression of luciferase activity by miR-214. In the post-MI mouse heart, miR-214 and Dio3 protein were shown to be co-expressed in cardiomyocytes, while time-course analysis revealed that Dio3 mRNA expression precedes miR-214 expression in the post-MI LV. This suggests that a Dio3-induced decrease of T3 levels is involved in the induction of miR-214, which was supported by the finding that cardiac miR-214 expression is down regulated by T3 in mice. In vitro analysis of human DIO3 mRNA furthermore showed that miR-214 is able to suppress both mRNA and protein expression. Dio3 mRNA is a target of miR-214 and the Dio3-dependent stimulation of miR-214 expression in post-MI cardiomyocytes supports the involvement of a negative feedback mechanism regulating Dio3 expression.