Genes associated with angiogenic response to Ang-1 or VEGF

Specific Aims To identify novel transcriptional events associated with angiogenesis in VEGF and Ang-1 stimulated rat aortic rings. Our studies take advantage of the capacity of rat aortic rings to generate new vessels in collagen gels. Rat aortic rings embedded in collagen gel immediately after excision from the animal produce a self-limited angiogenic response under serum-free conditions and in the absence of exogenous stimuli. This angiogenic response can be dose-dependently promoted by vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), which are critical regulators of the angiogenic process during embryonal development and postnatal angiogenesis. Aortic rings lose their capacity to spontaneously generate new vessels if embedded in collagen gels 10-14 days after excision. VEGF has the capacity to turn back “on” these quiescent rings producing florid angiogenesis. Conversely, Ang-1 potentiates an existing angiogenic response, but is unable to turn the quiescent system “on”. Since VEGF-mediated induction of angiogenic sprouting occurs 1-2 days of treatment, we hypothesize that this process is regulated by a unique set of “angiogenesis inducer genes” that are activated by VEGF and not by Ang-1. Identification of the proteins encoded by these genes may advance our understanding of the molecular mechanisms that regulate the earliest stages of the angiogenic cascade. Keywords: Response to growth factors Rat thoracic aortic rings will be excised from 1 month old Fisher 344 rats and placed into suspension culture. 15-20 individual rings will be isolated from each animal and cultured in suspension in serum free medium. The rings will be divided into three groups as described below. (Assay is described in; Nicosia et al., 1997 Am.J.Pathol, 151). All individual aortic rings isolated from a single animal will be split into three groups, VEGF treated, Ang-1 treated, and untreated controls. Rings dissected from proximal and distal portions of the aorta will be mixed so that positional bias will not be carried through to the RNA preparation. 13 days post isolation, one set of aortic rings will be treated with 10ng/ml of recombinant human VEGF in serum free EBM media, a second set of rings will be treated with 100ng/ml of human recombinant Ang-1 (R&D Systems 923-AN) in conjunction with 5ug/ml poly-His antibody (R&D Systems MAB050), and a third set of rings will be incubated in serum free medium alone. All samples were incubated for 18 hours post VEGF and Ang-1 treatment and then RNA was harvested. With the number of aortic rings typically obtained, we anticipate having 4-6 individual rings from each animal for each experimental condition. Furthermore, 10-100ng of total RNA will be collected per aortic ring, corresponding to 400-600ng of total RNA for each experimental condition per animal. In order to have a minimum of three independent samples from each experimental condition, three animals were sacrificed, each of which gave rise to three individual RNA samples. The resulting 9 individual RNA samples to be compared with respect to transcriptional profiles. Total RNA will be extracted from each aortic ring using the Trizol reagent (Invitrogen) protocol. RNA will be stored at –80C until all 9 individual samples have been prepared. Trizol extracted total RNA will be further purified/concentrated using the MicroRNAEasy Kit (Qiagen) with DNAseI digestion.