Hyperglycemia-Induced Defects in Angiogenesis in the Chicken Chorioallantoic Membrane Model

Abstract

Diabetes is associated with abnormal angiogenesis. Increased angiogenesis contributes to severe forms of diabetic retinopathy, but angiogenesis is decreased in response to myocardial ischemia in diabetic patients. We evaluated the direct effect of hyperglycemia on angiogenesis in the chicken chorioallantoic membrane assay, a model of active neoangiogenesis. Hyperglycemia, lasting up to 7 days, was induced in 7-day-old chick embryos by a single intravitellus glucose injection. Control embryos received either water (volumic control) or mannitol (osmotic control). Hyperglycemia decreased angiogenesis in this model from the 5th day on. The pattern and expression level of the main vascular growth factors' genes were not altered by hyperglycemia, as assessed by in situ hybridization and semiquantitative RT-PCR. As early as 2 days after hyperglycemia was induced, an increased apoptosis of endothelial cells and pericytes was detected by transferase-mediated deoxyuridine triphosphate nick-end labeling assay and electron microscopy. In the meantime, endothelial cell proliferation was decreased, as assessed by incorporation of bromo-deoxyuridine. Hyperglycemia can therefore impair angiogenesis without altering the expression level of vascular growth factors through induction of apoptosis and decreased proliferation of endothelial cells. Diabetes 53:752-761, 2004

Shell-Less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformations in Mammalian Embryos

“…We adjusted the osmolarity of glucose solutions by adding distilled water [21] to match the plasma osmolarity of the chick, i.e. 280 mOsmoles/l [22]. Consequently, isosmotic glucose solutions were applied to the embryos.…”

Receptor for Advanced Glycation End Products Is Involved in Impaired Angiogenic Response in Diabetes

“…Our data also show that the decrease in PCNA-positive cells in diabetes is also dependent on the presence of RAGE. Although the effect of diabetes on endothelial cell proliferation is often presented on the basis of what is observed in the late phase of diabetic retinopathy, recent observations suggest that hyperglycemia or AGEs suppresses angiogenesis even in retina in vivo (8,37). Larger et al (37) directly measured endothelial cell proliferation in the chicken chorioallantoic membrane assay, which is suppressed by hyperglycemia.…”

“…Although the effect of diabetes on endothelial cell proliferation is often presented on the basis of what is observed in the late phase of diabetic retinopathy, recent observations suggest that hyperglycemia or AGEs suppresses angiogenesis even in retina in vivo (8,37). Larger et al (37) directly measured endothelial cell proliferation in the chicken chorioallantoic membrane assay, which is suppressed by hyperglycemia. Regarding smooth muscle cells in major arteries, such as mouse arterial injury model, RAGE appears to be involved in increased smooth muscle cell proliferation in vivo (38,39).…”

Angiogenesis Associated With Visceral and Subcutaneous Adipose Tissue in Severe Human Obesity

“…Angiogenesis was quantified on CAM 5-7 days later (days 13-15 of incubation) with a MZ FLIII Leica stereomicroscope (ϫ8). Angiographies were performed as described previously (28). For VEGF inhibition, PTK 787/ZK222584 (Novartis Pharma, Basel, Switzerland), an inhibitor of the tyrosine kinase of the VEGF receptor active in human and chicken (27), was added to the adipose tissue samples at 200 mol/l twice daily for 48 h. Bevacizumab (Genentech, San Francisco, CA), a monoclonal antibody that specifically inhibits human VEGF with no cross-reaction with other species (29), was added at 320 g/ml with the same protocol.…”