Augmentation of Hyperglycemia‐Induced Damage in a Diabetic Rat Endothelium is Potentially Regulated by the Nfκβ Signaling Pathway

Abstract

Endothelial dysfunction is a key factor for the development of organ failure in type 2 diabetes mellitus (T2DM). While hyperglycemia is a well‐established factor in the development of microvascular complications in T2DM, specific mechanisms of endothelial damage remains equivocal. In a previous study, we observed that hyperglycemic states resulted in distinct alterations in proteins involved in metabolic and angiogenic processes in endothelial cells isolated from diabetic when compared to non‐diabetic rats. In the present study, we identified and validated diverse NFκβ‐regulated cellular functions that are involved in augmenting hyperglycemia‐induced damage in the diabetic endothelium. Bioinformatics and functional studies were carried out in the primary cardiac microvascular endothelial cells (RCMVECs) derived from the spontaneously T2DM Goto‐Kakizaki (GK) rat model in comparison to the control Wistar Kyoto (WKY) rat model under normal and hyperglycemic conditions. GK and WKY RCMVECs were cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks, followed by tandem mass spectrometry (MS/MS), qPCR, western blotting, microplate‐based cell proliferation, metabolic and REDOX assays. Evidence of hyperglycemia‐induced cytotoxicity in GK RCMVECs was determined by employing various microplate‐based assays, wherein a 20% reduction in cell proliferation (p<0.05) and a 33% increase in PMA‐induced ROS production (p<0.05) was observed. Enrichment and pathway analyses of the proteomic tandem MS/MS and qPCR datasets, revealed induction of inflammatory, metabolic and apoptotic pathways, which are tightly regulated by NFκβ, in GK RCMVECs (HG vs NG). Platelet‐activating factor receptor (LogFC = 3.5, p <0.01) and beta‐catenin (LogFC= 1.3, p <0.01), both key inductors of NFκβ signaling, were significantly upregulated in GK RCMVECs (HG vs NG). Western blotting analysis revealed significant upregulation of NFκβ in GK RCMVECs (HG vs NG) (FC=1.6, p<0.05) and in GK NG vs WKY NG RCMVECs (FC=2.5, p<0.05), suggesting a potential increase in NFκβ activity in the diabetic endothelium. Additionally, the activity of hexokinase‐2, which is reported to be a transcriptional target of NFκβ, was observed to be reduced in GK NG vs WKY NG RCMVECs (20% reduction, p<0.05). Furthermore, qPCR analysis identified an upregulation of several inflammatory transcripts that are that are regulated by NFκβ such as CXCR4 (FC=7.5, p<0.01) and NLRP3 (FC=5.8, p<0.01) expression in GK RCMVECs (HG vs NG). By the integration of high‐throughput tandem MS/MS analysis, bioinformatics analyses and in vitro functional assays, we identified a potential induction of NFκβ signaling in GK (HG vs NG) RCMVECs, resulting in metabolic reprogramming and a heightened inflammatory and apoptotic response.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.