Genes associated with angiogenic response to Ang-1 or VEGF
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ABSTRACT: 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
Project description: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 âonâ these quiescent rings producing florid angiogenesis. Conversely, Ang-1 potentiates an existing angiogenic response, but is unable to turn the quiescent system âonâ. 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 âangiogenesis inducer genesâ 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. Experiment Overall Design: 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). Experiment Overall Design: 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. Experiment Overall Design: 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. Experiment Overall Design: All samples were incubated for 18 hours post VEGF and Ang-1 treatment and then RNA was harvested. Experiment Overall Design: 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. Experiment Overall Design: 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. Experiment Overall Design: Total RNA will be extracted from each aortic ring using the Trizol reagent (Invitrogen) protocol. RNA will be stored at â80C 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.
Project description:Angiogenesis in cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrys to examine the effects of TNF-alpha on gene expression in both fibrin and collagen gels during the first 48 hours or culture. Rat aortic rings were cultured in either collagen or fibrin maticies. Half of the cultures from each matrix group were treated with 10ng/ml recombinant rat TNF-alpha, and half were left untreated. These cultures were used to prepare total RNA
Project description:Angiogenesis in collagen gel cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrays to detail the pattern of gene expression underlying initial 24 hours of growth, prior to the sprouting of visible neovessles, and identified distinct classes of up-regulated genes during this process. Either freshly harvested aortic rings, representing day 0, or collagen gel cultures of rat aorta were grown in serum free medium and used to prepare total RNA.
Project description:Angiogenesis in cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrys to examine the effects of TNF-alpha on gene expression in both fibrin and collagen gels during the first 48 hours or culture.
Project description:Angiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked-to tumorigenesis, rheumatoid arthritis and heart disease. Here we show pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RI? subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as 'redox-dead' Cys17Ser RI? knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth. Disulphide-activation of PKA represents a new therapeutic target in diseases with aberrant angiogenesis. The aim of this study was to identify unique differences in gene expression that may account for decreased angiogenesis in C42S PKARI knock-in mice compared with their littermate wild-types observed in cell and whole animal studies Freshly isolated mouse aortic vessels from 3 C42S PKARI knock-in or 3 littermate wild-types were used in this study. Each vessel was divided in two rings and either left untreated or treated with VEGF.
Project description:Cold triggers VEGF dependent but hypoxia independent angiogenesis in adipose tissues and anti-VEGF agents modulate adipose metabolism; The molecular mechanisms of angiogenesis in relation to adipose tissue metabolism remain poorly understood. Here we show that exposure of mice to cold led to conversion of white adipose tissue (WAT) to brown-like adipose tissue, accompanying the switch of an active angiogenic phenotype. Gene expression profile analysis showed VEGF was upregulated via most likely hypoxia-independent PGC-1 transcriptional activation. Intriguingly, VEGFR2 blockage abolished the cold-induced angiogenesis, significantly impaired nonshivering thermogenesis capacity, and markedly reduced adipose metabolism. Unexpectedly, VEGFR1 blockage resulted in opposite effects by increasing adipose vascularity and metabolism. These findings demonstrate that VEGFR2 and VEGFR1 mediate polarized activities in modulating adipose angiogenesis and metabolism. Taken together, our findings have conceptual implications in applying angiogenesis modulators for the treatment of obesity and metabolic disorders. Experiment Overall Design: Mice were exposed to cold and white addipose tissue was collected at different time points
Project description:RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.