Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis.
MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice.
MicroRNAs (miRNAs) play key regulatory roles in diverse biological processes and are frequently dysregulated in human diseases. Thus, miRNAs have emerged as a class of promising targets for therapeutic intervention. Here, we describe the current strategies for therapeutic modulation of miRNAs and provide an update on the development of miRNA-based therapeutics for the treatment of cancer, cardiovascular disease and hepatitis C virus (HCV) infection.
Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI.
Pulmonary endothelial cell apoptosis is a transient, yet defining pathogenic event integral to the onset of many pulmonary vascular diseases such as pulmonary hypertension (PH). However, there is a paucity of information concerning the molecular pathway(s) that control pulmonary arterial endothelial cell apoptosis. Here, we introduce a molecular axis that when functionally active seems to induce pulmonary arterial endothelial cell apoptosis in vitro and PH in vivo. In response to apoptotic stimuli, human pulmonary arterial endothelial cells exhibited robust induction of a programmed cell death 4 (PDCD4)/caspase-3/apoptotic pathway that was reversible by direct PDCD4 silencing. Indirectly, this pathway was also repressed by delivery of a microRNA-21 mimic. In vivo, genetic deletion of microRNA-21 in mice (miR-21(-/-) mice) resulted in functional activation of the PDCD4/caspase-3 axis in the pulmonary tissues, leading to the onset of progressive PH. Conversely, microRNA-21-overexpressing mice (CAG-microRNA-21 mice) exhibited reduced PDCD4 expression in pulmonary tissues and were partially resistant to PH in response to chronic hypoxia plus SU 5416 injury. Furthermore, direct PDCD4 knockout in mice (PDCD4(-/-) mice) potently blocked pulmonary caspase-3 activation and the development of chronic hypoxia plus SU 5416 PH, confirming its importance in disease onset. Broadly, these findings support the existence of a microRNA-21-responsive PDCD4/caspase-3 pathway in the pulmonary tissues that when active serves to promote endothelial apoptosis in vitro and PH in vivo.
Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-? signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-? blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-? signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.
MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion.
The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.
Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease.
Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI.
Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown.
Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood.
Originally identified as moderate biological modifiers, microRNAs have recently emerged as powerful regulators of diverse cellular processes with especially important roles in disease and tissue remodeling. The rapid pace of studies on microRNA regulation and function necessitates the development of suitable techniques for measuring and modulating microRNAs in different model systems. This review summarizes experimental strategies for microRNA research and highlights the strengths and weaknesses of different approaches. The development of more specific and sensitive assays will further illuminate the biology behind microRNAs and will advance opportunities to safely pursue them as therapeutic modalities.
MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3 untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent cardiac hypertrophy and fibrosis in rodents in response to pressure overload. In contrast, we have shown here that miR-21-null mice are normal and, in response to a variety of cardiac stresses, display cardiac hypertrophy, fibrosis, upregulation of stress-responsive cardiac genes, and loss of cardiac contractility comparable to wild-type littermates. Similarly, inhibition of miR-21 through intravenous delivery of a locked nucleic acid-modified (LNA-modified) antimiR oligonucleotide also failed to block the remodeling response of the heart to stress. We therefore conclude that miR-21 is not essential for pathological cardiac remodeling.
Lung cancer is the leading cause of cancer-related deaths in the world, and non-small-cell lung cancer (NSCLC) accounts for 80% of cases. MicroRNA-21 (miR-21) expression is increased and predicts poor survival in NSCLC. Although miR-21 function has been studied in vitro with cancer cell lines, the role of miR-21 in tumor development in vivo is unknown. We utilize transgenic mice with loss-of-function and gain-of-function miR-21 alleles combined with a model of NSCLC to determine the role of miR-21 in lung cancer. We show that overexpression of miR-21 enhances tumorigenesis and that genetic deletion of miR-21 partially protects against tumor formation. MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.
microRNAs are small non-coding RNAs that regulate gene expression at the posttranscriptional level by either inhibiting mRNA translation or inducing mRNA degradation. These regulatory mechanisms occur in a sequence-specific manner through the direct binding of the microRNA to complementary reverse sequences in the 3 UTR of target mRNAs. The sequence-specific nature of microRNAs allows for the regulation of numerous target mRNAs, which often are related genes, resulting in the robust regulation of entire pathways. Previous studies have identified expression signatures of microRNAs during various pathological settings, including those of cardiovascular disease. As evident through gain- and loss-of-function studies in mice, it is apparent microRNAs play specific and essential roles during cardiac hypertrophy, fibrosis, angiogenesis, apoptosis, and contractility. The powerful effects of altering microRNA levels genetically have resulted in the rapid progression of oligo-based regulation of microRNAs as a new class of cardiovascular therapeutics. Here we summarize the current oligo-based technologies in use to regulate microRNA levels in vivo and how these technologies have been applied to multiple microRNAs during cardiovascular disease.
Ischemic heart disease is a form of congestive heart failure that is caused by insufficient blood supply to the heart, resulting in a loss of viable tissue. In response to the injury, the non-ischemic myocardium displays signs of secondary remodeling, like interstitial fibrosis and hypertrophy of cardiac myocytes. This remodeling process further deteriorates pump function and increases susceptibility to arrhythmias. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression in a sequence-dependent manner. Recently, several groups identified miRNAs as crucial gene regulators in response to myocardial infarction (MI) and during post-MI remodeling. In this review, we discuss how modulation of these miRNAs represents a promising new therapeutic strategy to improve the clinical outcome in ischemic heart disease.
Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied.
Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.
Cardiac and skeletal muscle development are controlled by evolutionarily conserved networks of transcription factors that coordinate the expression of genes involved in muscle growth, morphogenesis, differentiation, and contractility. In addition to regulating the expression of protein-coding genes, recent studies have revealed that myogenic transcription factors control the expression of a collection of microRNAs, which act through multiple mechanisms to modulate muscle development and function. In some cases, microRNAs fine-tune the expression of target mRNAs, whereas in other cases they function as on-off switches. MicroRNA control of gene expression appears to be especially important during cardiovascular and skeletal muscle diseases, in which microRNAs participate in stress-dependent remodeling of striated muscle tissues. We review findings that point to the importance of microRNA-mediated control of gene expression during muscle development and disease, and consider the potential of microRNAs as therapeutic targets.
In recent years, prominent roles for microRNAs (miRNAs) have been uncovered in several cardiovascular disorders. The ability to therapeutically manipulate miRNA expression and function through systemic or local delivery of miRNA inhibitors, referred to as antimiRs, has triggered enthusiasm for miRNAs as novel therapeutic targets. Here, we focus on the pharmacokinetic and pharmacodynamic properties of current antimiR designs and their relevance to cardiovascular indications, and evaluate the opportunities and obstacles associated with this new therapeutic modality.
Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective: We assessed the role of miR-145 in PAH.
Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.
Chemical biology promises discovery of new and unexpected mechanistic pathways, protein functions and disease targets. Here, we probed the mechanism-of-action and protein targets of 3,5-disubstituted isoxazoles (Isx), cardiomyogenic small molecules that target Notch-activated epicardium-derived cells (NECs) in vivo and promote functional recovery after myocardial infarction (MI). Mechanistic studies in NECs led to an Isx-activated G(q) protein-coupled receptor (G(q)PCR) hypothesis tested in a cell-based functional target screen for GPCRs regulated by Isx. This screen identified one agonist hit, the extracellular proton/pH-sensing GPCR GPR68, confirmed through genetic gain- and loss-of-function. Overlooked until now, GPR68 expression and localization were highly regulated in early post-natal and adult post-infarct mouse heart, where GPR68-expressing cells accumulated subepicardially. Remarkably, GPR68-expressing cardiomyocytes established a proton-sensing cellular "buffer zone" surrounding the MI. Isx pharmacologically regulated gene expression (mRNAs and miRs) in this GPR68-enriched border zone, driving cardiomyogenic and pro-survival transcriptional programs in vivo. In conclusion, we tracked a (micromolar) bioactive small molecules mechanism-of-action to a candidate target protein, GPR68, and validated this target as a previously unrecognized regulator of myocardial cellular responses to tissue acidosis, setting the stage for future (nanomolar) target-based drug lead discovery.
Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca²? during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca²? handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca²? overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca²? influx; and to repression of several downstream effectors of Ca²? signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca²? homeostasis and survival during cardiac injury.
Rarely a new research area has gotten such an overwhelming amount of attention as have microRNAs. Although several basic questions regarding their biological principles still remain to be answered, many specific characteristics of microRNAs in combination with compelling therapeutic efficacy data and a clear involvement in human disease have triggered the biotechnology community to start exploring the possibilities of viewing microRNAs as therapeutic entities. This review serves to provide some general insight into some of the current microRNAs targets, how one goes from the initial bench discovery to actually developing a therapeutically useful modality, and will briefly summarize the current patent landscape and the companies that have started to explore microRNAs as the next drug target.
Until recently, microRNAs (miRNAs) were considered to be relatively small players in biological systems by having a balancing function through moderate effects on gene expression levels. However, it has become appreciated that miRNAs are actually much more relevant during both development and disease, which is underscored by the attention they have been receiving. The goal of this thematic review series is to highlight current knowledge of miRNA function during cardiovascular development, their dysregulation under disease conditions and the disease modifying functions they have been shown to exert in the cardiovascular system. These reviews, in addition to discussing the recent advancements in using miRNAs as circulating biomarkers or therapeutic modalities, will hopefully be able to provide a strong basis for future research to further expand our insights into miRNA function in cardiovascular biology.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.