Hyperglycemia‐Induced Inhibition of the Angiotensin II Type 1 Receptor Pathway in the Vascular Endothelium

Abstract

Hyperglycemia, associated with diabetes, is known to damage blood vessels and contribute to negative cardiovascular health outcomes. However, the etiology behind the negative influence of the exposure to prolonged elevated glucose on endothelial cells is not yet fully understood. Our laboratories’ previous study identified changes in endothelial cell surface glycosylation resulting from hyperglycemia. One such modified protein was the Angiotensin II type 1 receptor (AT1R), which was N‐glycosylated adjacent to the active site. Activation of AT1R by angiotensin II (AngII) at subpressor doses is associated with positive endothelial cell growth, proliferation, and tube formation. However, AngII binding to the Angiotensin II type 2 receptor (AT2R), has been shown to elicit opposing effects. The objective of this study was to further investigate the effects of this hyperglycemia‐induced glycosylation of AT1R. A two‐week high glucose treatment (25 mM) of rat cardiac microvascular endothelial cells (RCMVECs) displayed impaired function by tube formation analysis (TFA) in comparison to a normal glucose treatment (4.5 mM). The decrease tube formation was further exacerbated by the addition of a subpressor dose of AngII. To further test the influence of hyperglycemia‐induced AT1R N‐glycosylation on signaling we treated RCMVECs with the PNGaseF enzyme for N‐glycosylation removal at the cell surface in combination with functional assays and AngII treatment under varying glycemic conditions. The efficacy of this treatment was tested through TFA and measurements of ERK1/2 activation through phosphorylation (known contributor to AT1R signaling). We observed a decrease in tube length, tube area, and number of branch points in a high glucose environment plus Ang‐II and were able to recover functionality with the addition of PNGaseF. While normal glucose treated cells had shown ERK1/2 activation, no observable increases in ERK1/2 were observed in response to AngII in the high glucose conditions. In the high glucose plus AngII treatment group we also observed a significant decrease in Vegfr2 gene expression compared to the normal glucose plus AngII treatment group. Further analysis is currently underway to further validate this mechanism of AT1R signaling impairment, but the data we have so far lead us to believe that hyperglycemia‐induced glycosylation blocks the AT1R active site to decrease functionality. It is possible that the hyperglycemia‐induced glycosylation of AT1R inhibits AngII binding, allowing for a higher rate of binding in the AT2R to further impair endothelial cell functionality.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University.