Characterization of Rat Aortic Fragment within Collagen Gel as an Angiogenesis Model; Capillary Morphology may Reflect the Action Mechanisms of Angiogenesis Inhibitors.

Abstract

Angiogenesis, the formation of new blood vessels from pre-existing vessels, occurs physiologically during embryonal development, tissue injury and inflammation. It is also well known to play an important role in several pathological conditions, such as cancer, diabetic retinopathy, rheumatoid arthritis and atherosclerosis.1) An understanding of the angiogenic process may lead to new therapies for the above diseases, and some angiogenesis inhibitors are already under clinical study as anti-cancer drugs.2)The formation of new capillaries is a complex biological process whose regulatory mechanisms are incompletely understood. The initiation of angiogenesis involves focal reduction of intercellular interactions and of interactions between endothelial cells and the surrounding extracellular matrix (ECM). Cell migration, proliferation and capillary tube formation then follow. It is clear that complex orchestration of many processes, some overlapping, is required in order for angiogenesis to proceed. Various types of angiogenic growth factors such as vascular endothelial growth factor (VEGF), 3) basic fibroblast growth factor (bFGF), 4) platelet-derived growth factor (PDGF) 5) and hepatocyte growth factor (HGF) 6) stimulate both the endothelial cells and mesenchymal cells. ECM-degrading proteolytic enzymes, including matrix metalloprotease (MMP) and plasminogen activator (PA), are secreted by endothelial cells in response to angiogenic factors and/or cytokines. 7) Cell adhesion molecules such as integrins, cadherin and PECAM also regulate the angiogenic process.8) There are some reports on the interactions of the angiogenic factors mentioned above; for example, bFGF up-regulates VEGF production 9); the production of proteolytic enzymes and integrin alpha subunits is induced by VEGF, 10) bFGF 11) and PDGF 12); MMP-2 associates with integrin avb3 and growth factor receptor.13) Therefore, an in vitro model of angiogenesis should retain, as far as possible, the in vivo interactions of angiogenic factors and the regulatory mechanisms.Fragments of rat thoracic aorta within type I collagen gel provide a good model of the overall angiogenic process, including cell migration, proliferation and capillary tube formation, for 1 to 2 weeks. The aortic fragments contain several types of cells, such as endothelial cells, smooth muscle cells and fibroblasts, which co-ordinate to form capillary vessels in the collagen gel. It was reported that aortic fragments endogenously produce bFGF, 14) and exogenous growth factors such as PDGF, insulin-like growth fator (IGF) and VEGF enhance capillary formation. 15)In this report, we show that VEGF, its receptor and proteolytic enzymes (MMP, PA) are endogenously expressed in the rat aorta model and gradually increase during the process of angiogenesis. When the action of these endogenous factors was blocked by inhibitors, capillary tube formation was clearly inhibited, demonstrating that these endogenous factors play key roles in angiogenesis in this model. Interestingly, the formed capillaries a...

Overproduction of N.EPSILON.-(Carboxymethyl)lysine-Induced Neovascularization in Cultured Choroidal Explant of Streptozotocin-Diabetic Rat

“…13) There are additional evidences that VEGF differentiates CD34 Ϫ , VE-cadherin Ϫ multipotential adult progenitor cell into CD34 ϩ , VE-cadherin + cell in vitro 38) and that VEGF increases to initiate the process of vascularization by stimulating chemoattraction and proliferation of CD34 ϩ cells such as angioblast and endothelial cell although VEGF alone is not sufficient to direct blood vessel organization and/or maturation. [39][40][41] These evidences suggest that VEGF and TNFa play a role for CML-facilitated proliferation of CD34 ϩ cells during the course of diabetes.…”

Regulation of TGFβ1-mediated growth inhibition and apoptosis by RUNX2 isoforms in endothelial cells

“…Ectopic expression of RUNX2 supports these observations since RUNX2 overexpression increased cell growth, DNA synthesis, in vitro cell sprouting, and resistance to TGFb 1 in BAEC transfectants relative to control cells. EC within aortic sprouts secrete plasminogen activators and metalloproteinases, mediate degradation of the ECM during vascular sprouting, and respond to angiogenic cytokines such as VEGF (Nicosia and Ottinetti, 1990;Hiraoka et al, 1998;Hata-Sugi et al, 2002). Previous observations from our laboratory showed that the expression of uPA and MT1-MMP was downregulated in EC transfected with dominant-negative Runt, which inhibited endogenous RUNX2 transcriptional activity (Sun et al, 2001).…”

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

“…Quantitative assessment used an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium) assay to measure the relative number of viable cells growing from the aorta rings. After removal of the ring of aorta, 300 ml freshly prepared solution containing 100 mg MTS and 1 mg PMS (phenazine methosulfate) in DPBS was added to each well and incubated for 24 h at 37 C. The medium used for this study preferentially selects for endothelial out-growth, but cell number will include some fibroblasts and smooth muscle cells (Hata-Sugi et al 2002).…”