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).
Project Narrative "Angiogenesis" means growth of blood vessels anywhere in the body, and this process is crucial for normal development and healing of wounds and injuries. However, if this process becomes excessive it can cause chronic bleeding and a non-healing wound. Tumors also need blood vessels to grow and invade the surrounding tissues, so they secrete substances (growth factors) that promote the growth of vasculature around them. We have found that one of the small cellular RNAs produced within the vascular cells can inhibit the excessive vessel growth in response to the above growth factors and can be used to modulate the tendency of blood vessels to grow excessively in cancer and chronic wounds.