Circulating Extracellular Vesicles Are Elevated with Prediabetes and Adversely Affect Cerebral Endothelial Nitric Oxide and Endothelin-1 Production

Abstract

Experimental and clinical studies indicate that a proatherogenic endothelial phenotype develops in the prediabetic, hyperglycemic state providing the platform for accelerated vascular disease with prediabetes and, in turn, type 2 diabetes. Prediabetes (fasting plasma glucose concentrations: 100-125 mg/dL) is associated with an increased risk of stroke. Circulating endothelial cell-derived microvesicles (EMVs) are now recognized as both a biomarker and mediator of vascular health and disease. In fact, EMVs have been shown to be involved in the pathogenesis of cerebrovascular dysfunction leading to increased stroke risk. Circulating EMVs are elevated in adults with prediabetes; whether EMVs associated with the prediabetic state promote endothelial abnormalities known to increase stroke risk is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with prediabetes on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 20 mid-life adults (age:44-69 yr): 10 normoglycemic (NG; 8M/2F; age: 55±2 yr; BMI: 25.7±0.8 kg/m2; fasting plasma glucose: 87±2 mg/dL) and 10 prediabetic (PreD: 8M/2F; 55±2 yr; 27.1±1.5 kg/m2; 107±2 mg/dL). Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Expression of intracellular proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~65%) in PreD vs NG (180±25 vs 110±13 EMV/μL) and significantly associated with plasma glucose concentrations (r=0.48; P=0.03). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (47.6±2.6 vs 58.6±3.5 AU; P=0.02) and p-eNOS (Thr495) higher (82.6±4.4 vs 31.1±2.5 AU; p<0.001) in cells treated with EMVs from PreD vs NG. Concordantly, NO production was ~20% lower (6.0±0.2 vs 7.2±0.4 μmol/L; p<0.05) in cells treated with EMVs from PreD adults. PreD-associated EMVs also significantly increased (~25-70%) the expression of Big ET-1 (804.4±47.7 vs 656.7±30.7 AU) and endothelin converting enzyme (290.0±24.6 vs 169.6±19.0 AU) as well as ET-1 production (30.2±4.3 vs 24.2±2.0 pg/mL) in hCMECs. In summary, EMVs harvested from adults with PreD reduced endothelial nitric oxide synthase (eNOS) activation and NO production and increased ET-1 synthesis and release in brain endothelial cells in vitro. Increased risk and incidence of stroke associated with prediabetes may be mediated by circulating EMVs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.