The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.
Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.
The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.
Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.
microRNAs; Epithelial-mesenchymal transition; Pulmonary fibrosis; Alveolar type II cells; Lung single cell separation
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Fibrosis is the final common pathway in virtually all forms of chronic organ failure, including lung, liver, and kidney, and is a leading cause of morbidity and mortality worldwide. Fibrosis results from the excessive activity of fibroblasts, in particular a differentiated form known as myofibroblast that is responsible for the excessive and persistent accumulation of scar tissue and ultimately organ failure. Idiopathic Lung Fibrosis (IPF) is a chronic and often rapidly fatal pulmonary disorder of unknown origin characterized by fibrosis of the supporting framework (interstitium) of the lungs. Given the poor prognosis of IPF patients, new insights into the biology of (myo)fibroblasts is of major interest to develop new therapeutics aimed at reducing (myo)fibroblast activity to slow or even reverse disease progression, thereby preserving organ function and prolonging life. MicroRNAs (miRNAs), a class of non-coding RNA recently identified, are associated with normal cellular processes; and deregulation of miRNAs plays a causative role in a vast array of complex diseases. In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF.
Fibrotic skin disorders may be debilitating and impair quality of life. There are few effective treatment options for cutaneous fibrotic diseases. In this review, we discuss our current understanding of the role of microRNAs (miRNAs) in skin fibrosis. MiRNAs are a class of small, noncoding RNAs involved in skin fibrosis. These small RNAs range from 18 to 25 nucleotides in length and modify gene expression by binding to target messenger RNA (mRNA), causing degradation of the target mRNA or inhibiting the translation into proteins. We present an overview of the biogenesis, maturation and function of miRNAs. We highlight miRNA’s role in key skin fibrotic processes including: transforming growth factor (TGF)-beta signaling, extracellular matrix (ECM) deposition, and fibroblast proliferation and differentiation. Some miRNAs are profibrotic and their upregulation favors these processes contributing to fibrosis, while antifibrotic miRNAs inhibit these processes and may be reduced in fibrosis. Finally we describe the diagnostic and therapeutic significance of miRNAs in the management of skin fibrosis. The discovery that miRNAs are detectable in serum, plasma, and other bodily fluids, and are relatively stable, suggests miRNAs may serve as valuable biomarkers to monitor disease progression and response to treatment. In the treatment of skin fibrosis, antifibrotic miRNAs may be upregulated using mimics and viral vectors. Conversely, profibrotic miRNAs may be downregulated by employing anti-miRNAs, sponges, erasers and masks. We believe that miRNA-based therapies hold promise as important treatments and may transform the management of fibrotic skin diseases by physicians.
skin fibrosis; microRNA; miRNA; collagen; therapeutics
Fibroblasts are responsible for producing the majority of collagen and other extracellular matrix (ECM) proteins in tissues. In the injured tissue, transforming growth factor-β (TGF-β)-activated fibroblasts or differentiated myofibroblasts synthesize excessive ECM proteins and play a pivotal role in the pathogenesis of fibrosis in heart, kidney and other organs. Recent studies suggest that fibroblast-like cells, derived from endothelial cells by endothelial-to-mesenchymal transition (EndMT), contribute to the pathogenesis of cardiac fibrosis. The molecular basis of EndMT, however, is poorly understood. Here, we investigated the molecular basis of EndMT in mouse cardiac endothelial cells (MCECs) in response to TGF-β2. MCECs exposed to TGF-β2 underwent EndMT as evidenced by morphologic changes, lack of acetylated–low density lipoprotein (Ac-LDL) uptake, and the presence of alpha-smooth muscle actin (α-SMA) staining. Treatment with SB431542, a small molecule inhibitor of TGF-β-receptor I (TβRI) kinase, but not PD98059, a MEK inhibitor, completely blocked TGF-β2-induced EndMT. The transcript and protein levels of α-SMA, Snail and β-catenin as well as acetyltransferase p300 (ATp300) were elevated in EndMT derived fibroblast-like cells. Importantly, microRNA (miRNA) array data revealed that the expression levels of specific miRNAs, known to be dysregulated in different cardiovascular diseases, were altered during EndMT. The protein level of cellular p53, a bonafide target of miR-125b, was downregulated in EndMT-derived fibroblast-like cells. Here, we report for the first time, the differential expression of miRNAs during cardiac EndMT. These results collectively suggest that TβRI serine-threonine kinase-induced TGF-β signaling and microRNAs, the epigenetic regulator of gene expression at the posttranscriptional level, are involved in EndMT and promote profibrotic signaling in EndMT-derived fibroblast-like cells. Pharmacologic agents that restrict the progression of cardiac EndMT, a phenomenon that is found in adults only in the pathological conditions, in targeting specific miRNA may be helpful in preventing and treating cardiac fibrosis.
Cardiac EndMT; TβRI kinase; microRNA; ATp300; Epigenetics; Fibrosis
MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix–associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)–β1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-β1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-β1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-β1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.
miR-29; pulmonary fibrosis; basement membrane; profibrotic genes; TGF-β1
Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs.
In this work, microRNA expression profiling of the lungs from treated C57BL/6J mice, relative to that of untreated controls, was undertaken to determine which alterations in microRNAs could in part regulate the fibrosis phenotype induced by bleomycin delivered through mini-osmotic pumps. We identified 11 microRNAs, including miR-21 and miR-34a, to be significantly differentially expressed (P < 0.01) in lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. In situ hybridization of both miR-21 and miR-34a indicated that they were expressed in alveolar macrophages. Using a previously reported gene expression profile, we identified 195 genes to be both predicted targets of the 11 microRNAs and of altered expression in bleomycin-induced lung disease of C57BL/6J mice. Pathway analysis with these 195 genes indicated that altered microRNA expression may be associated with hepatocyte growth factor signaling, cholecystokinin/gastrin-mediated signaling, and insulin-like growth factor (IGF-1) signaling, among others, in fibrotic lung disease. The relevance of the IGF-1 pathway in this model was then demonstrated by showing lung tissue of bleomycin treated C57BL/6J mice had increased expression of Igf1 and that increased numbers of Igf-1 positive cells, predominantly in macrophages, were detected in the lungs.
We conclude that altered microRNA expression in macrophages is a feature which putatively influences the insulin-like growth factor signaling component of bleomycin-induced pulmonary fibrosis.
Pulmonary fibrosis; microRNA; Bleomycin; Insulin-like growth factor; Pathway analysis; Mouse model
Progressive reduction in kidney function in patients following myocardial infarction (MI) is associated with an increase in circulating uremic toxins levels leading to increased extracellular matrix deposition. We have recently reported that treatment with uremic toxin adsorbent AST-120 in rats with MI inhibits serum levels of uremic toxin indoxyl sulfate (IS) and downregulates expression of cardiac profibrotic cytokine transforming growth factor beta (TGF-β1). In this study, we examined the effect of uremic toxins post-MI on cardiac microRNA-21 and microRNA-29b expression, and also the regulation of target genes and matrix remodeling proteins involved in TGFβ1 and angiotensin II signaling pathways. Sixteen weeks after MI, cardiac tissues were assessed for pathological and molecular changes. The percentage area of cardiac fibrosis was 4.67 ± 0.17 in vehicle-treated MI, 2.9 ± 0.26 in sham, and 3.32 ± 0.38 in AST-120-treated MI, group of rats. Compared to sham group, we found a twofold increase in the cardiac expression of microRNA-21 and 0.5-fold decrease in microRNA-29b in heart tissue from vehicle-treated MI. Treatment with AST-120 lowered serum IS levels and attenuated both, cardiac fibrosis and changes in expression of these microRNAs observed after MI. We also found increased mRNA expression of angiotensin-converting enzyme (ACE) and angiotensin receptor 1a (Agtr1a) in cardiac tissue collected from MI rats. Treatment with AST-120 attenuated both, expression of ACE and Agtr1a mRNA. Exposure of rat cardiac fibroblasts to IS upregulated angiotensin II signaling and altered the expression of both microRNA-21 and microRNA-29b. These results collectively suggest a clear role of IS in altering microRNA-21 and microRNA-29b in MI heart, via a mechanism involving angiotensin signaling pathway, which leads to cardiac fibrosis.
AST-120; indoxyl sulfate; microRNA 21; microRNA 29b; myocardial infarction; uremic toxin
MicroRNAs (miRNAs) are a family of small, noncoding RNAs that regulate gene expression in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. As a result, miRNAs emerged as major area of biomedical research with relevance to kidney fibrosis. Fibrosis is characterized by the excess deposition of extracellular matrix (ECM) components, which is the end result of an imbalance of metabolism of the ECM molecule. Recent evidence suggests that miRNAs participate in the fibrotic process in a number of organs including the heart, kidney, liver, and lung. Epithelial mesenchymal transition (EMT) and endothelial mesenchymal transition (EndMT) programs play vital roles in the development of fibrosis in the kidney. A growing number of the extracellular and intracellular molecules that control EMT and EndMT have been identified and could be exploited in developing therapeutics for fibrosis. This review highlights recent advances on the role of miRNAs in the kidney diseases; diabetic nephropathy especially focused on EMT and EndMT program responsible for the development of kidney fibrosis. These miRNAs can be utilized as a potential novel drug target for the studying of underlying mechanism and treatment of kidney fibrosis.
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.
Objectives: To determine changes in expression and role of microRNAs in IPF.
Methods: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-β. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.
Measurements and Main Results: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-β down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and α-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.
Conclusions: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis.
Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
epithelial–mesenchymal transition; HMGA2 (high-mobility group AT-hook 2); microRNA; transforming growth factor-β
Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition.
To identify the mechanism of Arsenic trioxide’s (ATO)’s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen.
Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO’s down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H2O2 and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression.
In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.
Arsenic trioxide; IPF; TGF-β1; Pulmonary fibrosis; PML; Bleomycin
Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure. Although dysregulation of microRNA (miRNA) is involved in various pathophysiological processes in the heart, the role of miRNA in fibrosis regulation after MI is not clear. Previously we observed the correlation between fibrosis and the miR-24 expression in hypertrophic hearts, herein we assessed how miR-24 regulates fibrosis after MI. Using qRT-PCR, we showed that miR-24 was down-regulated in the MI heart; the change in miR-24 expression was closely related to extracellular matrix (ECM) remodelling. In vivo, miR-24 could improve heart function and attenuate fibrosis in the infarct border zone of the heart two weeks after MI through intramyocardial injection of Lentiviruses. Moreover, in vitro experiments suggested that up-regulation of miR-24 by synthetic miR-24 precursors could reduce fibrosis and also decrease the differentiation and migration of cardiac fibroblasts (CFs). TGF-β (a pathological mediator of fibrotic disease) increased miR-24 expression, overexpression of miR-24 reduced TGF-β secretion and Smad2/3 phosphorylation in CFs. By performing microarray analyses and bioinformatics analyses, we found furin to be a potential target for miR-24 in fibrosis (furin is a protease which controls latent TGF-β activation processing). Finally, we demonstrated that protein and mRNA levels of furin were regulated by miR-24 in CFs. These findings suggest that miR-24 has a critical role in CF function and cardiac fibrosis after MI through a furin–TGF-β pathway. Thus, miR-24 may be used as a target for treatment of MI and other fibrotic heart diseases.
myocardial infarction; cardiac fibrosis; microRNA-24; furin; TGF-β; telocyte
The progression of liver fibrosis and cirrhosis is associated with the persistence of an injury causing agent, leading to changes in the extracellular environment and a disruption of the cellular homeostasis of liver resident cells. Recruitment of inflammatory cells, apoptosis of hepatocytes, and changes in liver microvasculature are some examples of changing cellular environment that lead to the induction of stress responses in nearby cells. During liver fibrosis, the major stresses include hypoxia, oxidative stress, and endoplasmic reticulum stress. When hepatic stellate cells (HSCs) are subjected to such stress, they modulate fibrosis progression by induction of their activation toward a myofibroblastic phenotype, or by undergoing apoptosis, and thus helping fibrosis resolution. It is widely accepted that microRNAs are import regulators of gene expression, both during normal cellular homeostasis, as well as in pathologic conditions. MicroRNAs are short RNA sequences that regulate the gene expression by mRNA destabilization and inhibition of mRNA translation. Specific microRNAs have been identified to play a role in the activation process of HSCs on the one hand and in stress-responsive pathways on the other hand in other cell types (Table 2). However, so far there are no reports for the involvement of miRNAs in the different stress responses linked to HSC activation. Here, we review briefly the major stress response pathways and propose several miRNAs to be regulated by these stress responsive pathways in activating HSCs, and discuss their potential specific pro-or anti-fibrotic characteristics.
miRNAs; hepatic stellate cells; fibrosis; ER stress; hypoxia; oxidative stress
MicroRNAs (miRNAs) are endogenous short non-coding RNAs that regulate most of important cellular processes by inhibiting gene expression through the post-transcriptional repression of their target mRNAs. In kidneys, miRNAs have been associated in renal development, homeostasis, and physiological functions. Results from clinical and experimental animal studies demonstrate that miRNAs play essential roles in the pathogenesis of various renal diseases. Chronic kidney diseases (CKD) is characterized by renal fibrosis. Transforming growth factor beta (TGF-β) is recognized as a major mediator of renal fibrosis because it is able to stimulate the accumulation of extracellular matrix (ECM) proteins to impair normal kidney function. Recently, emerging evidence demonstrate the relationship between TGF-β signaling and miRNAs expression during renal diseases. TGF-β regulates expression of several microRNAs, such as miR-21, miR-192, miR-200, miR-433, and miR-29. MiR-21, miR-192, and miR-433 which are positively induced by TGF-β signaling play a pathological role in kidney diseases. In contrast, members in both miR-29 and miR-200 families which are inhibited by TGF-β signaling protect kidneys from renal fibrosis by suppressing the deposition of ECM and preventing epithelial-to-mesenchymal transition, respectively. Clinically, the presence of miRNAs in blood and urine has been examined to be early biomarkers for detecting renal diseases. From experimental animal studies of CKD, targeting microRNAs also provides evidence about therapeutic potential of miRNAs during renal diseases. Now, it comes to the stage to examine the exact mechanisms of miRNAs during the initiation and progression of renal diseases. Therefore, determining the function of miRNAs in renal fibrosis may facilitate the development of both early diagnosis and treatment of renal diseases.
microRNAs; kidney diseases; renal fibrosis; TGF-β signaling; biomarkers
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.
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.
Osteopontin (OPN) is a pleiotropic cytokine, which has been shown to be a pivotal factor in myofibroblast activation in cardiac fibrosis, thereby acting as a strong driver of heart failure development in humans. MicroRNAs (miRNAs) are under intense investigation as powerful regulators of various diseases. First phase I and II clinical trials using miRNA inhibitors have been initiated. We here show, that OPN is essential in the activation of AP-1 and subsequent transcription of miR-21 in cardiac fibrosis related to Ang II. OPN null mice are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation. In the future, these findings may result in miRNA therapeutic approaches to treat patients with cardiac remodelling, in which levels of OPN and miR-21 are increased.
Osteopontin; Cardiac fibrosis; microRNA; Angiotensin II; miR-21
Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition, as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-β1), chemokines (MCP-1, MIP-1β), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin–angiotensin–aldosterone system (ANG II) have been identified as important regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our current understanding of the cellular and molecular mechanisms of fibrogenesis.
fibrosis; myofibroblast; collagen; wound healing; liver; lung
Idiopathic pulmonary fibrosis (IPF) is a fatal disorder resulting from the progressive remodeling of lungs, with no known effective treatment. Although transforming growth factor (TGF)-β has a well-established role in lung fibrosis, clinical experience with neutralizing antibodies to TGF-β has been disappointing, and strategies to directly suppress TGF-β1 secretion are needed. In this study we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs involved in TGF-β1 regulation and to validate the role of miR-326 in pulmonary fibrosis.We show that hsa-miR-326 regulates TGF-β1 expression and that hsa-miR-326 levels are inversely correlated to TGF-β1 protein levels in multiple human cell lines. The increase in TGF-β1 expression during the progression of bleomycin-induced lung fibrosis in mice was associated with loss of mmu-miR-326. Restoration of mmu-miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response. Moreover, human IPF lung specimens had markedly diminished miR-326 expression as compared with nonfibrotic lungs. Additional targets of miR-326 controlling TGF-β signaling and fibrosis-related pathways were identified, and miR-326 was found to down-regulate profibrotic genes, such as Ets1, Smad3, and matrix metalloproteinase 9, whereas it up-regulates antifibrotic genes, such as Smad7. Our results suggest for the first time that miR-326 plays a key role in regulating TGF-β1 expression and other profibrotic genes and could be useful in developing better therapeutic strategies for alleviating lung fibrosis.
idiopathic pulmonary fibrosis; microRNAs; transforming growth factor-β signaling
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; mimic; miR-29; pulmonary fibrosis; therapeutics
In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.
Rationale: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17∼92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression.
Objectives: We investigated the miR-17∼92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue.
Methods: Expression and DNA methylation patterns of miR-17∼92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17∼92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5′-aza-2′-deoxycytidine.
Measurements and Main Results: Compared with control samples, miR-17∼92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17∼92 promoter was increased. Several miRNAs from the miR-17∼92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17∼92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5′-aza-2′-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17∼92 cluster expression, and attenuated pulmonary fibrosis.
Conclusions: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17∼92 and DNMT-1 in lung fibrosis.
microRNA; miR-17∼92; pulmonary fibrosis; DNA methylation; DNMT-1
Previous studies suggest that transforming growth factor- beta (TGF-∃) provokes cardiac hypertrophy and myocardial fibrosis; however, it is unclear whether the deleterious effects of TGF-∃ signaling are conveyed through SMAD-dependent or SMAD-independent signaling pathways.
Methods and Results
To determine the contribution of SMAD dependent signaling to cardiac remodeling, we performed transaortic constriction (TAC) in SMAD3 null (SMAD3−/−) and littermate control mice (age 10–12 weeks). Cumulative survival 20 days post-TAC was significantly less in the SMAD3 −/− mice when compared to littermate controls (43.6% vs 90.9%, p<0.01). TAC resulted in a significant increase in cardiac hypertrophy in the SMAD3 −/− mice, denoted by an increase in the heart-weight-to-tibial length ratio and increased myocyte cross-sectional area. Loss of SMAD3 signaling also resulted in a significant 60% decrease in myocardial fibrosis (p < 0.05). A microRNA microarray showed that 55 microRNAs were differentially expressed in littermate and SMAD3−/− mice, and that 10 of these microRNAs were predicted to bind to genes that regulate the extracellular matrix. Of these 10 candidate microRNAs, both miR-25 and miR-29a were sufficient to decrease collagen gene expression when transfected into isolated cardiac fibroblasts in vitro.
The results suggest that SMAD3 signaling plays dual roles in the heart: one beneficial role by delimiting hypertrophic growth, and the other deleterious by modulating myocardial fibrosis, possibly through a pathway that entails accumulation of microRNAs that decrease collagen gene expression.
Fibrosis; microRNA; pressure overload; hypertropy
Purpose of review
MicroRNAs (miRNAs) are short noncoding RNAs that inhibit gene expression in plants and animals. miRNAs have emerged as key players in virtually all aspects of mammalian biology. Aberrant miRNA expression is observed in numerous human diseases such as diabetes, hypercholesterolemia, cancer, and tissue fibrosis. Therefore, approaches to correct miRNA expression represent the novel therapeutic strategies for these diseases.
miRNAs are essential for kidney development and homeostasis. Aberrant miRNA expression is observed in the mouse models of kidney fibrosis. Three TGF-β-regulated miRNA families, miR-21, miR-200, and miR-29 have been shown to modulate renal fibrosis. miR-21, through a feed-forward loop, amplifies TGF-β signaling and promotes fibrosis. Conversely, miR-200 and miR-29 reduce fibrosis by inhibiting epithelial-tomesenchymal transition and preventing the deposition of extracellular matrix, respectively. Inhibition of miR-21 expression or augmenting miR-29 expression prevents kidney fibrosis in mice.
Aberrant miRNA expression perturbs signaling pathways that lead to progression of kidney fibrosis. Thus, miRNAs represent novel biomarkers and therapeutic targets in the treatment of kidney fibrosis.
kidney fibrosis; microRNA; miR-200; miR-21; miR-29
MicroRNAs are implicated in many biological and pathological processes and are emerging as key actors in lung health and disease. Specific patterns of dysregulated microRNAs have been found in idiopathic pulmonary fibrosis (IPF), an untreatable interstitial lung disease of unknown etiology. IPF is characterized by dramatic and extensive phenotypic changes in the lung that include alveolar cell hyperplasia, fibroblast proliferation and formation of myofibroblast foci, deposition of extracellular matrix, and changes in lung transcriptional programming. Here, we discuss the latest insights about the role of microRNAs in lung fibrosis with a focus on the contribution of animal models of disease to the derivation of these insights.
Non-alcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease and is considered to be a causative factor of cryptogenic cirrhosis and hepatocellular carcinoma. microRNAs (miRNAs) are small non-coding RNAs that negatively regulate messenger RNA (mRNA). Recently, it was demonstrated that the aberrant expression of certain miRNAs plays a pivotal role in liver disease. The aim of the present study was to evaluate changes in miRNA profiles associated with metformin treatment in a NASH model. Eight-week-old male mice were fed a methionine- and choline-deficient (MCD) diet alone or with 0.08% metformin for 15 weeks. Metformin significantly downregulated the level of plasma transaminases and attenuated hepatic steatosis and liver fibrosis. The expression of miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p was enhanced among the 71 upregulated miRNAs, and the expression of miRNA-122, miRNA-194, miRNA-101b and miRNA-705 was decreased among 60 downregulated miRNAs in the liver of MCD-fed mice when compared with control mice. Of note, miRNA profiles were altered following treatment with metformin in MCD-fed mice. miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p were down-regulated, but miRNA-122, miRNA-194, miRNA-101b and miRNA-705 were significantly upregulated in MCD-fed mice treated with metformin. miRNA profiles were altered in MCD-fed mice and metformin attenuated this effect on miRNA expression. Therefore, miRNA profiles are a potential tool that may be utilized to clarify the mechanism behind the metformin-induced improvement of hepatic steatosis and liver fibrosis. Furthermore, identification of targetable miRNAs may be used as a novel therapy in human NASH.
MCD; hepatic steatosis; liver fibrosis
Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers.
miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.
Transforming growth factor β; fibrosis; miR 19b; biomarker; hepatic stellate cell
MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = −0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.