Zhiyong Lei

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.

Targeted delivery of miRNA therapeutics for cardiovascular diseases: opportunities and challenges.

Dysregulation of miRNA expression has been associated with many cardiovascular diseases in animal models, as well as in patients. In the present review, we summarize recent findings on the role of miRNAs in cardiovascular diseases and discuss the opportunities, possibilities and challenges of using miRNAs as future therapeutic targets. Furthermore, we focus on the different approaches that can be used to deliver these newly developed miRNA therapeutics to their sites of action. Since siRNAs are structurally homologous with the miRNA therapeutics, important lessons learned from siRNA delivery strategies are discussed that might be applicable to targeted delivery of miRNA therapeutics, thereby reducing costs and potential side effects, and improving efficacy.

miR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects against Myocardial Ischemia-Reperfusion Injury

Limited microRNAs (miRNAs, miRs) have been reported to be necessary for exercise-induced cardiac growth and essential for protection against pathological cardiac remodeling. Here we determined members of the miR-17-92 cluster and their passenger miRNAs expressions in two distinct murine exercise models and found that miR-17-3p was increased in both. miR-17-3p promoted cardiomyocyte hypertrophy, proliferation, and survival. TIMP-3 was identified as a direct target gene of miR-17-3p whereas PTEN was indirectly inhibited by miR-17-3p. Inhibition of miR-17-3p in vivo attenuated exercise-induced cardiac growth including cardiomyocyte hypertrophy and expression of markers of myocyte proliferation. Importantly, mice injected with miR-17-3p agomir were protected from adverse remodeling after cardiac ischemia/reperfusion injury. Collectively, these data suggest that miR-17-3p contributes to exercise-induced cardiac growth and protects against adverse ventricular remodeling. miR-17-3p may represent a novel therapeutic target to promote functional recovery after cardiac ischemia/reperfusion.

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.

ULTRASOUND AND MICROBUBBLE-INDUCED LOCAL DELIVERY OF MICRORNA-BASED THERAPEUTICS

MicroRNAs are involved in many pathologic processes and are a promising target for therapeutic intervention. However, successful, localized delivery of microRNA-based therapeutics is lacking. In this study, cationic ultrasound-responsive microbubbles (MBs) were used to deliver microRNA blockers and mimics in vitro and in vivo. Cationic MBs successfully delivered microRNA blockers to human endothelial cells on ultrasound (US) exposure in vitro. This in vitro US protocol did not successfully deliver microRNA mimics to skeletal muscle of mice, whereas an US protocol that is routinely used for contrast imaging did. Additionally, we used cationic MBs and US to locally deliver antimiR and antagomiR molecules with US causing inertial cavitation. Delivery of antimiR to the extracellular compartments of the muscle was only slightly increased, whereas delivery of antagomiR to the capillaries, myocytes and extracellular space was significantly increased. AntagomiR seems to be a more suitable microRNA blocker than antimiR for use in combination with MBs and US for local delivery.

MicroRNA Therapeutics for Cardiac Regeneration

It is estimated that a typical myocardial infarction results in the loss of approximately one billion functional cardiomyocytes, which are replaced by a non-contractile fibrous scar, eventually leading to heart failure. The currently available surgical, drug, and device-based therapies cannot reverse the loss of functional myocardium, which is the fundamental cause of the problem. As a result of this lack of an available medical solution, heart failure has evolved into a global epidemic. Therefore, the development of regenerative therapeutic strategies to halt the progression of ischemic heart disease to advanced heart failure has become one of the most urgent medical needs of this century. This review first addresses the extremely limited endogenous regenerative capacity of the mammalian heart, and the benefits and limitations of stem cell-based therapies for cardiac repair. Then it discusses the known roles of microRNAs after cardiac injury and the possibility of employing microRNAs to enhance cardiac regeneration.

Engineering CRISPR / Cpf1 with tRNA promotes genome editing capability in mammalian systems

CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3′-to-5′ exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.

Dgcr8 is Indispensable for Cardiac Lineage Specification in Embryonic Stem Cells

Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.

MMISH : Multicolor microRNA in situ hybridization for paraffin embedded samples

Highlights • A robust, sensitive and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections can be combined with both immunohistochemical and immunofluorescent staining.• Usage of urea in our buffers which enhances the target-probe affinity by preventing intermolecular interaction within miRNAs or individual probes, and by reversing the EDC fixation induced epitope loss by denaturing the antigens, less toxic compared to toxic formamide.• Second, it can be combined with immunofluorescent stainings, which allows one to analyze the expression and precise (sub)cellular location of the miRNA of interest.

Genetic and Epigenetic Regulation Networks: Governing from Cardiovascular Development to Remodeling.

1Cardiac Regeneration and Ageing Lab, Experimental Center of Life Sciences, School of Life Science, Shanghai University, Shanghai 200444, China 2Victor Babes National Institute of Pathology, 050096 Bucharest, Romania 3Division of Cellular and Molecular Biology and Histology, Carol Davila University of Medicine and Pharmacy, 050474 Bucharest, Romania 4Laboratory of Experimental Cardiology, University Medical Centre Utrecht, 3508 GA Utrecht, Netherlands 5Cardiovascular Division of the Massachusetts General Hospital and Harvard Medical School, Boston, MA 02215, USA 6Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China