Integrative Glycoproteomic and Phosphoproteomic Analyses to reveal Vascular Alterations in Response to a Hyperglycemic Microenvironment

Abstract

Hyperglycemia‐induced vascular complications are preeminent factors for a decreased life expectancy in patients with diabetes. At a molecular level, increased concentrations of blood glucose is known to accelerate endothelial dysfunction through several negative intracellular signaling events. We hypothesize that these changes are not independent of spontaneous cell surface glycosylation. The cytosolic phosphoproteome and cell surface glycoproteome (both N‐and O‐glycoproteome) were isolated from rat microvascular endothelial cells (RMVECs) cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks. This was followed by tandem mass spectrometry (MS/MS) and integrative bioinformatics analysis. Functional assays, namely tube formation, ROS production, and proliferation assays were performed on RMVECs in the presence or absence of HG conditions. The deleterious impact of hyperglycemic conditions on RMVEC function was confirmed by their inability to form robust tubes in vitro (n=3, p<0.05), and a diminished response to acetylcholine ex vivo (n=3, p<0.05). Hyperglycemic states resulted in differential glycosylation of several receptors, ion channels and cell surface antigens that are critical for endothelial functioning. Tandem MS/MS analysis revealed 87 proteins significantly increased in N‐glycosylation (65 unique) and 143 proteins significantly increased in O‐glycosylation (78 unique) in HG treated versus NG RMVECs (p<0.05). Targets included AT1R (N‐glycosylation: Unique HG, p<0.05), JAM‐A (O‐glycosylation: 2.05 fold, p<0.05) and CD59 (N‐glycosylation: 2.02 fold, p<0.05 and O‐glycosylation: Unique HG, p<0.05). Phosphopeptide pathway enrichment analysis revealed a 25% decrease in phosphorylation status, when filtered for scans (≥ 5) and p‐value (<0.05), in HG treated versus NG RMVECs. Pronounced alterations were observed in several components of insulin (INSR: 1.1 fold, p<0.05 and PKCβ: 2.3 fold, p<0.05), angiogenic (S1PR2: unique HG, p<0.05), and apoptotic (BCL2: −3.75 fold, p<0.05) signaling pathways. Furthermore, elimination of glucose moieties by the treating HG RMVECs with glycosidases, resulted in a normalization of several endothelial functional phenotypes. This study identified a multitude of changes in the glycosylation and phosphorylation status of several surface receptors and cytosolic proteins that are integral to vascular homeostasis. Since glycosidases reversed several of the impairments brought about by a hyperglycemic insult on the endothelium, reversion of dysfunctional endothelial cells to a healthy phenotype by negating a build‐up of glucose moieties on cell surface proteins may be a potential new avenue for the treatment of progressive cardiovascular complications in diabetes.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, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.