The goal of this exploratory proposal is to define the role of microRNAs (miRNA) in mechanisms of arterial muscularization and hypercontractility in pulmonary arterial hypertension (PAH). The major hypothesis is that miRNA expression and patterns of miRNA-mediated gene silencing change dynamically in endothelial, myofibroblast, smooth muscle and progenitor cells during vessel remodeling that occurs in PAH. Altered gene silencing is proposed to contribute significantly to arterial muscularization and to formation of obstructive vascular lesions. To test this hypothesis expression of miRNAs in pulmonary blood vessels will be assayed in two rat models of PAH - rats exposed to chronic hypoxia plus SU-5146, a VEGF receptor antagonist, and rats treated with monocrotaline. The three Specific Aims are: 1. Define the time-dependent changes in miRNA expression in pulmonary artery during development of PAH. miRNA expression will be assayed at 0, 7, 21 and 35 days in large (>1mm) and small (200-400 5m) pulmonary arteries in normal rats and rats exposed to hypoxia plus SU-5146. miRNA expression will be assayed at 0 and 21d in rats treated with monocrotaline. miRNAs differentially expressed at these times will be verified by quantitative real-time PCR. Cellular localization of PCR-verified miRNAs will be assessed by in situ hybridization in fixed lung samples from humans with PAH. 2. Compare miRNAs regulated by TGFbeta and bone morphogenetic proteins (BMP) to miRNAs altered in PAH. miRNAs regulated by TGF beta1, TGFbeta3 and BMP-4 will be assayed in cultured rat pulmonary artery tissues and human pulmonary artery smooth muscle cells in culture. Time- and concentration- dependent patterns of miRNA expression will be compared to miRNAs that change dynamically during development of PAH in the rat models and in human lung tissues. Selected miRNAs will then be tested for functional effects on pulmonary artery smooth muscle. 3. To test the sufficiency of miRNAs to alter smooth muscle phenotype primary miRNA transcripts and antisense miRNAs will be expressed in cultured rat and human pulmonary artery smooth muscle cells. Smooth muscle contractile proteins, cell contraction, cell proliferation and cell migration will be measured as readouts of the contractile vs proliferating phenotypes. The results will establish the physiological significance of selected miRNAs in vascular smooth muscle, and will identify candidate miRNAs that could be developed as therapeutic agents to reverse vessel remodeling in severe PAH.
Exciting new developments in studies of small ribonucleic acids called microRNAs have changed our understanding of how organs develop and how chronic diseases might be treated. We are proposing an important novel role for microRNAs in determining the structure and function of cells in the pulmonary vasculature. Discovering how these molecules control the function of pulmonary blood vessels could lead to new treatments of pulmonary hypertension.
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