MiR-1 is a Critical Regulator of VEGF-Induced Angiogenesis

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): Project Summary VEGF (Vascular Endothelial Growth Factor) is one of the main angiogenic factors involved in the development and maintenance of blood vessels and is shown to play a central role in various pathological processes such as wound healing, carcinogenesis and metastasis. MicroRNAs are a recently recognized group of non-coding small RNA molecules that regulate gene expression by inhibiting the translation of mRNAs or facilitating their degradation. We characterized the lung microRNA profile of the CC10-rtTA-VEGF transgenic mice in microarray experiments and confirmed our findings by stem-loop RT-PCR. The level of miR-1 was quantified in (CD45-, CD31+) and (CD45-, CD105+) cells separated from total lung by FACS sorting. We measured the level of miR- 1 in primary mouse lung endothelial cells in vitro after stimulation with VEGF. The effect of miR-1 supplementation on VEGF-induced proliferation and cord formation was studied in cell culture. The in vivo effect of miR-1 on angiogenesis after intranasal delivery was characterized by staining of the trachea from VEGF transgenic mice with CD31. We found that the levels of miR-1 were consistently diminished in the total lung RNAs from CC10-rtTa- VEGF transgenic mice, endothelial cells of these mice separated by FACS sorting, and mouse primary lung endothelial cells stimulated by VEGF in vitro, each compared to appropriate controls. MiR-1 supplementation downregulated VEGF-induced endothelial cell proliferation and cord formation in vitro and intranasal delivery of miR-1 inhibited angiogenesis in the bronchial circulation in vivo. Hypothesis: MiR-1 is a critical regulator of VEGF-induced endothelial cell responses To test the validity of this hypothesis we propose to: Aim #1: Characterize the spectrum of VEGF-induced endothelial cell responses regulated by miR-1 in vitro. Aim #2: Characterize the effect of miR-1 over-expression on VEGF responses in vivo (by A. direct delivery and B. transgenic modeling). Future Directions: Define the mechanism of miR-1 effects (by A. characterizing the effect of miR-1 on signaling and, B: identifying mRNAs recruited to RNA Induced Silencing complex (RISC).