Type 2 Diabetes Mellitus and Ex Vivo High Glucose Exposure Induce a Switch in the Mechanism of Microvascular Dilation That is Rescued by Activation of Autophagy

Abstract

Flow-mediated dilation, (FMD) manifests as arteriolar vasodilation in response to shear stress in an endothelium-dependent manner. Nitric oxide (NO) is the primary mediator of FMD in health, while hydrogen peroxide (H2O2) predominates in chronic disease states such as coronary artery disease (CAD). We have previously demonstrated that autophagy, a cell recycling process, is critical in mediating the switch in dilator mechanism in CAD. Type 2 Diabetes Mellitus (T2DM) is an independent risk factor in the development of cardiovascular disease, with hyperglycemia a widely-acknowledged hallmark of the disease. We sought to examine if presence of T2DM or high glucose exposure induce a switch in the mechanism of microvascular dilation to shear stress that is dependent upon autophagic flux. Human arterioles from discarded adipose tissue from T2DM subjects and healthy controls were isolated and prepared for videomicroscopy. Arterioles from healthy subjects were exposed to ex vivo high glucose (HG; 20 mM D-glucose) and exposed to an autophagy activator (trehalose, 10 mM). Shear-induced autophagic flux was measured using Lysotracker Red DND-99 in the presence and absence of the lysosomal inhibitor bafilomycin A (BAFA1). Internal diameters were measured after graded increases in intraluminal pressure gradients (flow) in the presence/absence of a NOS inhibitor (L-NAME; 100μM) or H2O2 scavenger (Peg-Catalase, Peg-Cat; 500 units/mL). Data are expressed as fold change in fluorescence (shear-induced autophagy) and % maximal dilation (FMD). Significance was defined as P<0.05. Shear-induced autophagy is repressed in arterioles from T2DM subjects vs. controls as addition of BAFA1 did not change Lysotracker Red DND-99 fluorescence intensity, while addition of BAFA1 to arterioles from healthy subjects repressed Lysotracker intensity. Presence of T2DM and exposure to ex vivo HG induces a switch in the mechanism of dilation from NO to H2O2, as the magnitude of dilation is reduced in the presence of the H2O2 scavenger Peg-Cat (T2DM control: 62.0±8.0% vs. 16.1±4.0%, P<0.05; HG: 66.7±8.9% vs. 17.8±9.5%, P<0.05), but not L-NAME. Activation of autophagy with trehalose preserved NO as the primary mechanism of dilation as evidenced by a reduction in magnitude of vasodilation in the presence of L-NAME (T2DM: 80.3±5.5% vs. 36.9±9.4%*, P<0.05; HG: 85.7±3.9% vs. 27.5±12.2%, but not Peg-Cat. Our data demonstrate that T2DM, an independent risk factor for cardiovascular disease, and hyperglycemia, a hallmark of T2DM, result in a pathological switch in the mechanism of microvascular vasodilation from NO to H2O2. In both cases, this disease-associated response is a result of decreased autophagic flux, and activation of autophagic flux preserves NO as the primary mechanism of dilation.