The microRNA-130/301 family controls vasoconstriction in pulmonary hypertension.
ABSTRACT: Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.
Project description:Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPAR? with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.
Project description:Purpose: Guided by an in silico combination of microRNA (miRNA) target prediction, analysis of transcriptomic changes in 137 human diseases, and advanced gene network modeling, we predicted the miR-130/301 family of miRNAs as a shared regulator of a fibrotic gene network across human diseases, thus orchestrating broad control over disease manifestation. The goals of this study are to compare the lung mRNA profile of mouse model of Pulmonary hypertension, one of the most fibrotic pathology uncovered by our in silico prediction, treated with an inhibitor of miR-130/301 (Short-130) to mice treated with a control inhibitor (Short-NC). Methods: Eight-week-old mice (C57BL/6) were injected with SU5416 (20 mg/kg/dose; Sigma-Aldrich), followed by exposure to normobaric hypoxia (10% O2; OxyCycler chamber, Biospherix Ltd.) for 2 weeks. After 2 weeks and confirmation of PH development in 5 mice (right heart catheterization), mice were further treated with 3 intrapharyngeal injections (every 4 days) of control or miR-130/301 shortmer oligonucleotides, designed as fully modified antisense oligonucleotides complementary to the seed sequence of the miR-130/301 miRNA family (10 mg/kg/dose; Regulus). Specifically, the control and miR-130/301 shortmer oligonucleotides were nontoxic, lipid-permeable, high-affinity oligonucleotides. The miR-130/301 shortmer carried a sequence complementary to the active site of the miR-130/301 miRNA family, containing a phosphorothioate backbone and modifications (fluoro-, methoxyethyl, and bicyclic sugar) at the sugar 2’ position. Three days after the last injection, right heart catheterization was performed followed by harvesting of lung tissue for RNA extraction. Lung mRNA profiles of those mice or control mice (Normoxia+SU5416) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the gene-level count. The gene level counts were then normalized with the R/Bioconductor package limma using the voom /variance stabilization method. The data were quality controlled for outliers using principal component analysis (PCA). Differential expression analysis between transcriptome profiles of experimental groups was performed using the R / Bioconductor package limma. Results: Transcriptomic analyses of whole lung from mice with hypoxia+SU5416-induced PH revealed a generalized de-repression of miR-130/301 targets by Short-130 treatment. Importantly, although whole lung transcriptomics likely captured only a subset of the miR-130/301 targets affecting the diseased pulmonary vasculature, pathway enrichment nonetheless revealed pronounced representation of several pathways known to be involved in fibrosis. Thus, the miR-130/301 family indeed induces a programmatic shift at the molecular level toward the fibrotic pathophenotype in vivo Overall design: Whole lung mRNA profiles of Normoxia (Control) and hypoxia+SU5416-induced PH mice treated with Short-NC or Short-130 were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
Project description:Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.
Project description:Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPAR?-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease.
Project description:The molecular origins of fibrosis affecting multiple tissue beds remain incompletely defined. Previously, we delineated the critical role of the control of extracellular matrix (ECM) stiffening by the mechanosensitive microRNA-130/301 family, as activated by the YAP/TAZ co-transcription factors, in promoting pulmonary hypertension (PH). We hypothesized that similar mechanisms may dictate fibrosis in other tissue beds beyond the pulmonary vasculature. Employing an in silico combination of microRNA target prediction, transcriptomic analysis of 137 human diseases and physiologic states, and advanced gene network modeling, we predicted the microRNA-130/301 family as a master regulator of fibrotic pathways across a cohort of seemingly disparate diseases and conditions. In two such diseases (pulmonary fibrosis and liver fibrosis), inhibition of microRNA-130/301 prevented the induction of ECM modification, YAP/TAZ, and downstream tissue fibrosis. Thus, mechanical forces act through a central feedback circuit between microRNA-130/301 and YAP/TAZ to sustain a common fibrotic phenotype across a network of human physiologic and pathophysiologic states. Such re-conceptualization of interconnections based on shared systems of disease and non-disease gene networks may have broad implications for future convergent diagnostic and therapeutic strategies.
Project description:Induced pluripotent stem cells (iPSCs) can be generated by overexpression of Oct4, Sox2 and Klf4 in murine fibroblasts. By conducting a microRNA (miRNA) library screen, we identified a set of miRNAs critically regulating iPSC formation. We revealed a new miRNA family (miR-130/301/721) as an important regulator of iPSC induction by targeting the homeobox transcription factor Meox2 (also known as Gax). Meox2-specific silencing mimicked the effects of this miRNA family on reprogramming. Mechanistically, miRNA-resistant Meox2 overexpression abrogated effects of miR-130/301/721 on reprogramming. In conclusion, the miRNA family miR-130/301/721 enhances iPSC generation via repression of Meox2.
Project description:Pulmonary hypertension (PH) is a progressive and often fatal disorder whose pathogenesis involves pulmonary artery smooth muscle cell (PASMC) proliferation. Although modern PH therapies have significantly improved survival, continued progress rests on the discovery of novel therapies and molecular targets. MicroRNA (miR)-21 has emerged as an important non-coding RNA that contributes to PH pathogenesis by enhancing vascular cell proliferation, however little is known about available therapies that modulate its expression. We previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR?) agonists attenuated hypoxia-induced HPASMC proliferation, vascular remodeling and PH through pleiotropic actions on multiple targets, including transforming growth factor (TGF)-?1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a validated target of miR-21. We therefore hypothesized that antiproliferative effects conferred by PPAR? activation are mediated through inhibition of hypoxia-induced miR-21 expression. Human PASMC monolayers were exposed to hypoxia then treated with the PPAR? agonist, rosiglitazone (RSG,10 ?M), or in parallel, C57Bl/6J mice were exposed to hypoxia then treated with RSG. RSG attenuated hypoxic increases in miR-21 expression in vitro and in vivo and abrogated reductions in PTEN and PASMC proliferation. Antiproliferative effects of RSG were lost following siRNA-mediated PTEN depletion. Furthermore, miR-21 mimic decreased PTEN and stimulated PASMC proliferation, whereas miR-21 inhibition increased PTEN and attenuated hypoxia-induced HPASMC proliferation. Collectively, these results demonstrate that PPAR? ligands regulate proliferative responses to hypoxia by preventing hypoxic increases in miR-21 and reductions in PTEN. These findings further clarify molecular mechanisms that support targeting PPAR? to attenuate pathogenic derangements in PH.
Project description:Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and ?-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein ? (C/EBP?) and peroxisome proliferator-activated receptor ? (PPAR?)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBP?, and PPAR? compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.
Project description:The contributions of altered post-transcriptional gene silencing to the development of metabolic disorders remain poorly understood thus far. The objective of this study was to evaluate the roles of miR-181a in the regulation of hepatic glucose and lipid metabolism. MiR-181a is abundantly expressed in the liver, and we found that blood and hepatic miR-181a levels were significantly increased in patients and dairy cows with non-alcoholic fatty liver disease, as well as in high-fat diet and ob/ob mice. We determined that sirtuin1 is a target of miR-181a. Moreover, we found that hepatic sirtuin1 and peroxisome proliferator-activated receptor-? coactivator-1? expression levels are downregulated, and acetylated peroxisome proliferator-activated receptor-? coactivator-1? expression levels are upregulated in patients and dairy cows with non-alcoholic fatty liver disease, as well as in high-fat diet and ob/ob mice. MiR-181a overexpression inhibits the sirtuin1-peroxisome proliferator-activated receptor-? coactivator-1? pathway, reduces insulin sensitivity, and increases gluconeogenesis and lipid synthesis in dairy cow hepatocytes and HepG2 cells. Conversely, silencing of miR-181a over-activates the sirtuin1-peroxisome proliferator-activated receptor-? coactivator-1? pathway, increases insulin sensitivity and glycogen content, and decreases gluconeogenesis and lipid synthesis in hepatocytes, even under non-esterified fatty acids treatment conditions. Furthermore, miR-181a overexpression or sirtuin1 knockdown in mice increases lipid accumulation and decreases insulin sensitivity and glycogen content in the liver. Taken together, these findings indicate that increased hepatic miR-181a impairs glucose and lipid homeostasis by silencing sirtuin1 in non-alcoholic fatty liver disease.
Project description:BACKGROUND:Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16?-hydroxyestrone (16?OHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16?OHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. METHODS AND RESULTS:MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16?OHE 1.25 ?g/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator-activated receptor-? and CD36 in those mice exposed to 16?OHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti-miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti-miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti-miR-29 treatment. CONCLUSIONS:16?OHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.