MicroRNAs (miRNAs) are short (;22nt) noncoding RNAs, that have emerged as important regulators of genes involved in the control of multiple physiological pathways, and often function to modulate or fine-tune cellular phenotypes such as development and differentiation. The role of miRNAs in specific processes involving vasculature patterning and defining miRNA targets in the vasculature system is an exciting, emerging new area with potential promise for novel therapeutic approaches in vascular related diseases. The miRNA17- 92 cluster (miR17-92) has been initially linked to oncogenic activity as demonstrated by its amplification in human B-cell lymphomas. A link between this miRNA cluster and angiogenesis was first indicated by a paracrine effect of tumor-derived miR17-92 on promoting angiogenesis. In this case, specific components of the cluster miR19 and miR18 were demonstrated to cause reduction of anti-angiogenic molecules suggesting a pro-angiogenic role for this cluster. In contrast, it has recently been proposed that this cluster has a cell intrinsic anti-angiogenic activity in endothelial cells in vitro. Importantly, we have shown that components of the miR17-92 can regulate positively aspects of VEGF induced cell growth and morphogenesis of endothelial cells (EC) in vitro. Herein, we propose to present the first in vivo platform to study the function of endothelial-derived miR17-92. The work proposed in this application will contribute to identifying the role of EC derived miRNA17-92 cluster in both physiological and pathophysiological angiogenesis in vivo. In addition we will characterize the mechanisms by which miR17-92 regulates angiogenesis by identifying novel targets for this cluster in the context of EC biology. We hypothesize that the miR17-92 cluster is a key regulator in vascular patterning and remodeling and that mice conditionally lacking miR17-92 in endothelial cells will exhibit abnormal angiogenic responses. Recent work by Tyler Jack's group has generated mice conditionally lacking the miR17-92 cluster. A major tool in this proposal is the generation of mice conditionally lacking the miR17-92 cluster in ECs. We have generated these mice and have exciting preliminary data indicating a potential developmental role for this cluster in regulating the timing and organization of the retinal vasculature. We will specifically examine the following: 1) The in vivo role of endothelial derived miR17-92 in models of angiogenesis 2) Identify and characterize the role of the EC miR17- 92 cluster as well as its individual "seed family" derivatives in in vitro aspects of angiogenesis. 3) Identify putative targets of the miR17-92 cluster in EC using SILAC analysis.
New strategies to target blood vessel growth, is an ongoing and exciting field of research highly relevant to the clinical treatment of cancer, ocular and cardiovascular disease. Given the mounting evidence that anti-VEGF treatment, the gold standard for anti- angiogenic therapy, causes resistance , alternative lines of treatment are highly demanded. Germane to this proposal, the emerging fields of microRNAs, as modulators of blood vessel development, provide a potential new frontier in targeting blood vessel growth .