Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses.

Dhanush Haspula,Namrata Tomar,Brian R Hoffmann,Andrew K Vallejos,Timothy M Moore,Ranjan K Dash

doi:10.3389/fphys.2019.00558

Abstract

Hyperglycemia is a critical factor in the development of endothelial dysfunction in type 2 diabetes mellitus (T2DM). Whether hyperglycemic states result in a disruption of similar molecular mechanisms in endothelial cells under both diabetic and non-diabetic states, remains largely unknown. This study aimed to address this gap in knowledge through molecular and functional characterization of primary rat cardiac microvascular endothelial cells (RCMVECs) derived from the T2DM Goto-Kakizaki (GK) rat model in comparison to control Wistar-Kyoto (WKY) in response to a normal (NG) and hyperglycemic (HG) microenvironment. GK and WKY RCMVECs were cultured under NG (4.5 mM) and HG (25 mM) conditions for 3 weeks, followed by tandem mass spectrometry (MS/MS), qPCR, tube formation assay, microplate based fluorimetry, and mitochondrial respiration analyses. Following database matching and filtering (false discovery rate ≤ 5%, scan count ≥ 10), we identified a greater percentage of significantly altered proteins in GK (7.1%, HG versus NG), when compared to WKY (3.5%, HG versus NG) RCMVECs. Further stringent filters (log2ratio of > 2 or < –2, p < 0.05) followed by enrichment and pathway analyses of the MS/MS and quantitative PCR datasets (84 total genes screened), resulted in the identification of several molecular targets involved in angiogenic, redox and metabolic functions that were distinctively altered in GK as compared to WKY RCMVECs following HG exposure. While the expression of thirteen inflammatory and apoptotic genes were significantly increased in GK RCMVECs under HG conditions (p < 0.05), only 2 were significantly elevated in WKY RCMVECs under HG conditions. Several glycolytic enzymes were markedly reduced and pyruvate kinase activity was elevated in GK HG RCMVECs, while in mitochondrial respiratory chain activity was altered. Supporting this, TNFα and phorbol ester (PMA)-induced Reactive Oxygen Species (ROS) production were significantly enhanced in GK HG RCMVECs when compared to baseline levels (p < 0.05). Additionally, PMA mediated increase was the greatest in GK HG RCMVECs (p < 0.05). While HG caused reduction in tube formation assay parameters for WKY RCMVECs, GK RCMVECs exhibited impaired phenotypes under baseline conditions regardless of the glycemic microenvironment. We conclude that hyperglycemic microenvironment caused distinctive changes in the bioenergetics and REDOX pathways in the diabetic endothelium as compared to those observed in a healthy endothelium.

Highlights

Cardiovascular disease (CVD) is one of the most significant pathophysiological phenotypes associated with the increase of diabetes worldwide
54 out of 2489 proteins (2.2%) were uniquely present in either groups under baseline conditions (NG). This is suggestive of a low percentage of dissimilar proteins in GK normal glucose treated RCMVECS (GK NG) when compared to WKY normal glucose treated RCMVECS (WKY NG) rat cardiac microvascular endothelial cells (RCMVECs)
While 199 out of 2792 proteins (7.1%) were uniquely altered in GK high glucose treated RCMVECS (GK HG) versus NG treated RCMVECs (Figure 2C), only 88 out of 2513 proteins (3.5%) were uniquely altered in WKY high glucose treated RCMVECS (WKY HG) versus NG treated RCMVECs (Figure 2B). This suggests that the proteins in GK HG versus NG treated RCMVECs were less similar than the proteins in WKY HG when compared to WKY NG

Summary

Introduction

Cardiovascular disease (CVD) is one of the most significant pathophysiological phenotypes associated with the increase of diabetes worldwide. The unprecedented rise in the number of newly diagnosed diabetes cases, and disproportionately higher CVD mortality in individuals with diabetes than those without (Centers for Disease Control and Prevention, 2014), are both alarming statistics. This spike in a multitude of CVD risk factors roughly parallels the rise in consumption of sugar-sweetened or artificiallysweetened beverages (Hu and Malik, 2010). Previous work in our laboratory and others have shown that hyperglycemic conditions alone displayed significant reduction in endothelial functions (Tesfamariam et al, 1990; Popov, 2010; Prisco et al, 2014), Elevation in the levels of superoxide production, potentially resulting in pro-inflammatory states and altered expression of vasodilatory mediators, are deemed as essential mechanisms of hyperglycemia-induced vascular damage (Nishikawa et al, 2000; Giacco and Brownlee, 2010; Sogawa et al, 2010)

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