Pulmonary arterial hypertension (PAH) is a chronic progressive disease with significant morbidity and mortality. The disease is characterized by vascular remodeling that includes increased muscularization of distal blood vessels and vessel stiffening associated with changes in extracellular matrix deposition. In humans, chronic hypoxia causes PAH, and hypoxia-induced rodent models of PAH have been used for years to study the disease. With the development of single-cell RNA sequencing technology, it is now possible to examine hypoxia-dependent transcriptional changes in vivo at a cell-specific level. In this study, we used single-cell RNA sequencing to compare lungs from wild-type (Wt) mice exposed to hypoxia for 28 days to normoxia-treated control mice. We additionally examined mice deficient for <i>Notch3</i>, a smooth muscle-enriched gene linked to PAH. Data analysis revealed that hypoxia promoted cell number changes in immune and endothelial cell types in the lung, activated the innate immunity pathway, and resulted in specific changes in gene expression in vascular cells. Surprisingly, we found limited differences in lungs from mice deficient for <i>Notch3</i> compared to Wt controls. These findings provide novel insight into the effects of chronic hypoxia exposure on gene expression and cell phenotypes in vivo and identify unique changes to cells of the vasculature.
Cardiac fibrosis is associated with many types of cardiovascular diseases and is characterized by the deposition of excess extracellular matrix by the cardiac fibroblasts. Previous studies demonstrated that microRNA miR‐145 acts as an inhibitor of fibrosis by targeting the profibrotic TGFβ pathway. Our preliminary data suggest that within the TGFβ pathway, miR‐145 specifically targets the p38 mitogen‐activated protein (MAP) kinase signaling, which is known to activate cardiac fibroblasts and drive the fibrotic response. Therefore, I hypothesize that miR‐145 targets the p38 MAP kinase pathway to regulate cardiac fibroblast activation and suppress fibrosis. Human cardiac fibroblasts (hCFs) were transfected with a miR‐145 mimic or a control mimic and TGFβ, a fibrotic agonist, was added to activate fibroblasts. Cells were processed for RNA and quantitative‐PCR was used to measure relative expression of genes within the p38 MAP kinase pathway. My findings show that MAP2K3 and MAP4K4 expression are inhibited by miR‐145 in activated hCFs. Further, TGFβ 3 and RGS2, both negative regulators of fibrosis, were induced in miR‐145 treated cells. These data indicate that miR‐145 modulates components of the p38 MAP kinase signaling pathway, which may contribute to its ability to regulate cardiac fibrosis. Ongoing studies will examine if overexpression of miR‐145 in a mouse model will affect the expression of these p38 MAP kinase mediators and contribute to a reduction in cardiac fibrosis.Support or Funding InformationThis project was supported by the American Physiological Society‐Undergraduate Research Excellency Fellowship, the American Heart Association Summer Undergraduate Research Fellowship, and the National Institute of Health grant R01‐HL‐135657 (to B. Lilly).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Summary MicroRNAs are modulators of cellular phenotypes and their functions contribute to development, homeostasis, and disease. miR‐145 is a conserved microRNA that has been implicated in regulating an array of phenotypes. These include supporting smooth muscle differentiation, repression of stem cell pluripotency, and inhibition of tumor growth and metastasis. Previously, our lab demonstrated that miR‐145 acts to suppress cardiac fibrosis through inhibition of the TGF‐β signaling pathway. The range of effects that miR‐145 has on different cell types makes it an attractive microRNA for further study. Here we describe the generation of transgenic mice that conditionally express miR‐145 through Cre recombinase‐mediated activation. Characterization of individual founder lines indicates that overexpression of miR‐145 in the developing cardiovascular system has detrimental effects, with three independent miR‐145 transgenic lines exhibiting Cre‐dependent lethality. Expression analysis demonstrates that the transgene is robustly expressed and our analysis reveals a novel downstream target of miR‐145, Tnnt2. The miR‐145 transgenic mice represent a valuable tool to understand the role of miR‐145 in diverse cell types and to address its potential as a therapeutic mediator for the treatment of disease.
Background Both downregulation and elevation of microRNA miR ‐ 145 has been linked to an array of cardiopulmonary phenotypes, and a host of studies suggest that it is an important contributor in governing the differentiation of cardiac and vascular smooth muscle cell types. Methods and results To better understand the role of elevated miR ‐ 145 in utero within the cardiopulmonary system, we utilized a transgene to overexpress miR ‐ 145 embryonically in mice and examined the consequences of this lineage‐restricted enhanced expression. Overexpression of miR ‐ 145 has detrimental effects that manifest after birth as overexpressor mice are unable to survive beyond postnatal day 18. The miR ‐ 145 expressing mice exhibit respiratory distress and fail to thrive. Gross analysis revealed an enlarged right ventricle, and pulmonary dysplasia with vascular hypertrophy. Single cell sequencing of RNA derived from lungs of control and miR ‐ 145 transgenic mice demonstrated that miR ‐ 145 overexpression had global effects on the lung with an increase in immune cells and evidence of leukocyte extravasation associated with vascular inflammation. Conclusions These data provide novel findings that demonstrate a pathological role for miR ‐ 145 in the cardiopulmonary system that extends beyond its normal function in governing smooth muscle differentiation.
Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5'-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5'-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.
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