Sandor Batkai

Cardiac fibroblast–derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands (star miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target.

The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy

Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.

Long noncoding RNA Chast promotes cardiac remodeling

Inhibition of the long noncoding RNA Chast prevents pressure overload–induced cardiac remodeling in mice. The missing lnc in cardiac hypertrophy RNA that does not code for a protein comprises a large portion of the human genome. These so-called noncoding RNAs are emerging as important players in disease pathogenesis, yet their functional roles are not always well known. Viereck et al. have discovered a new long noncoding RNA (lncRNA) that promotes cardiac remodeling and hypertrophy in mice, which could one day be targeted with therapeutics to treat human cardiovascular diseases. The identified lncRNA, which the authors named Chast (for “cardiac hypertrophy–associated transcript”), was discovered to be up-regulated in hypertrophic mouse hearts. When mouse and human heart cells expressed Chast, they tended to be larger than their normal counterparts. By silencing Chast with antisense oligonucleotides, mice either did not develop hypertrophy or were rescued from established disease. In a step toward translation, the authors discovered a human homolog, CHAST, that similarly caused cells in a dish to enlarge. Additional investigation in patients will confirm the relevance of this lncRNA in human disease and whether it is indeed a promising target for treating cardiac hypertrophy and heart failure. Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload–induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy–associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)–operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell–derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain–containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: A comprehensive review from an analytical and biological perspective☆

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by β- and ω-oxidation pathways.

MicroRNAs in Hypertension: Mechanisms and Therapeutic Targets

Hypertension is a complex, multifactorial disease, and its development is determined by a combination of genetic susceptibility and environmental factors. Several mechanisms have been implicated in the pathogenesis of hypertension: increased activity of the sympathetic nervous system, overactivation of the renin-angiotensin aldosterone system (RAAS), dysfunction of vascular endothelium, impaired platelet function, thrombogenesis, vascular smooth muscle and cardiac hypertrophy, and altered angiogenesis. MicroRNAs are short, noncoding nucleotides regulating target messenger RNAs at the post-transcriptional level. MicroRNAs are involved in virtually all biologic processes, including cellular proliferation, apoptosis, and differentiation. Thus, microRNA deregulation often results in impaired cellular function and disease development, so microRNAs have potential therapeutic relevance. Many aspects of the development of essential hypertension at the molecular level are still unknown. The elucidation of these processes regulated by microRNAs and the identification of novel microRNA targets in the pathogenesis of hypertension is a highly valuable and exciting strategy that may eventually led to the development of novel treatment approaches for hypertension. This article reviews the potential role of microRNAs in the mechanisms associated with the development and consequences of hypertension and discusses advances in microRNA-based approaches that may be important in treating hypertension.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis

Aims Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. Methods and results OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. Conclusion In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.

miR-21 promotes fibrosis in an acute cardiac allograft transplantation model

AIMSnCardiac transplantation is the only curative therapy for end-stage heart failure. Fibrosis is one of the major causes for impaired function of cardiac allografts. MicroRNAs, a class of small non-coding RNAs, play a critical role in the development of cardiovascular disease, but the role of microRNAs in cardiac allograft failure is not well understood.nnnMETHODS AND RESULTSnTo uncover a role of microRNAs during cardiac graft fibrosis, we generated global microRNA profiles in allogeneic (BALB/c in C57BL/6N) and isogeneic (C57BL/6N in C57BL/6N) murine hearts after transplantation. miR-21 together with cardiac fibrosis was increased in cardiac allografts compared with isografts. Likewise, patients with cardiac rejection after heart transplantation showed increased cardiac miR-21 levels. miR-21 was induced upon treatment with IL-6 in a monocyte cell line. Overexpression of miR-21 in this monocyte cell line activated a fibrotic gene programme and promoted monocyte-to-fibrocyte transition together with activation of chemokine (C-C) motif ligand 2 (monocyte chemoattractant protein 1) via the phosphatase and tensin homologue/activator protein 1 regulatory axis. In vivo, both genetic and pharmacological inhibition of miR-21 successfully reduced fibrosis and fibrocyte accumulation in cardiac allografts.nnnCONCLUSIONnThus, inhibition of miR-21 is a novel strategy to target fibrosis development in cardiac allografts.

Inhibition of the Cardiac Fibroblast-Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic Dysfunction

Rationale: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. Objective: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. Methods and Results: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-&bgr; I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-&bgr; I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. Conclusions: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.

Activation of the miR-17 family and miR-21 during murine kidney ischemia-reperfusion injury.

BACKGROUNDnIschemia-reperfusion (I/R) is the main cause of acute kidney injury (AKI) in patients. We investigated renal microRNA (miRNA) expression profiles and the time course of changes in selected miRNA expressions after renal I/R to characterize the miRNA network activated during development and recovery from AKI.nnnMETHODS AND RESULTSnOne day after lethal (30 minutes) and sublethal (20 minutes) renal ischemia, AKI was verified by renal histology (tubular necrosis, regeneration), blood urea nitrogen (BUN) level, renal mRNA expression, and plasma concentration of neutrophil gelatinase-associated lipocalin (NGAL) in C57BL/6J mice. On the first day after 30-minute, lethal I/R miR-21, miR-17-5p, and miR-106a were elevated out of the 21 miRNAs successfully profiled on the Luminex multiplex assay. After 20-minute, sublethal I/R, renal miR-17-5p and miR-106a expressions were elevated on the first and second days of reperfusion, while miR-21 expression increased later and lasted longer. Renal miR-17-5p and miR-21 expressions correlated with each other. Renal function returned to normal on the fourth day after sublethal I/R.nnnCONCLUSIONSnOur results demonstrate that besides miR-21, miR-17-5p, and miR-106a are additionally activated during the maintenance and recovery phases of renal I/R injury. Furthermore, a correlation between renal miR-17-5p and miR-21 expressions warrants further investigation of how they may influence each other and the outcome of renal ischemia-reperfusion injury.

MicroRNA-Mediated Epigenetic Silencing of Sirtuin1 Contributes to Impaired Angiogenic Responses

Rationale: Transforming growth factor (TGF)-&bgr; was linked to abnormal vessel function and can mediate impairment of endothelial angiogenic responses. Its effect on microRNAs and downstream targets in this context is not known. Objective: To study the role of microRNAs in TGF-&bgr;–mediated angiogenic activity. Methods and Results: MicroRNA profiling after TGF-&bgr; treatment of endothelial cells identified miR-30a-3p, along with other members of the miR-30 family, to be strongly silenced. Supplementation of miR-30a-3p restored function in TGF-&bgr;–treated endothelial cells. We identified the epigenetic factor methyl-CpG-binding protein 2 (MeCP2) to be a direct and functional target of miR-30a-3p. Viral overexpression of MeCP2 mimicked the effects of TGF-&bgr;, suggesting that derepression of MeCP2 after TGF-&bgr; treatment may be responsible for impaired angiogenic responses. Silencing of MeCP2 rescued detrimental TGF-&bgr; effects on endothelial cells. Microarray transcriptome analysis of MeCP2-overexpressing endothelial cells identified several deregulated genes important for endothelial cell function including sirtuin1 (Sirt1). In vivo experiments using endothelial cell–specific MeCP2 null or Sirt1 transgenic mice confirmed the involvement of MeCP2/Sirt1 in the regulation of angiogenic functions of endothelial cells. Additional experiments identified that MeCP2 inhibited endothelial angiogenic characteristics partly by epigenetic silencing of Sirt1. Conclusions: TGF-&bgr; impairs endothelial angiogenic responses partly by downregulating miR-30a-3p and subsequent derepression of MeCP2-mediated epigenetic silencing of Sirt1.