Hypercholesterolemia Induces Endothelial Dysfunction Through Impaired Kir2.1 Channel Activity Upstream of NO Production

Abstract

Hypercholesterolemia‐induced endothelial dysfunction is largely attributed to a loss of nitric oxide production/release, however the underlying mechanisms are unclear. Our recent study demonstrated that endothelial inwardly‐rectifying K+ channels (Kir2.1) are essential for flow‐induced activation of eNOS, NO production and flow‐induced vasodilation (FIV) in murine mesenteric resistance arteries. Our group also showed earlier that Kir channels are strongly suppressed by the elevation of cellular cholesterol. Therefore, we hypothesize that under hypercholesterolemic conditions cholesterol‐induced inhibition of Kir2.1, the major mesenteric EC Kir channel, would result in endothelial dysfunction (i.e. reduced dilations to flow) ultimately by preventing NO production. To test this hypothesis, we first determined the impact of clinically relevant concentrations of LDL on Kir current in human aortic ECs. We show that at concentrations considered to be at risk for atherosclerosis and heart disease we observed decreased Kir current density (10.8 ± 1.0 pA/pF for Control vs. 5.1 ± 0.3 pA/pF for 150 mg/dL LDL vs. 2.5 ± 0.4 pA/pF for 250 mg/dL LDL), an effect significantly reversed by the presence of HDL (5.1 ± 0.7 pA/pF for 150 mg/dL LDL vs. 10.2 ± 2.3 pA/pF for 50 mg/dL HDL + LDL). We also show that while Kir currents in endothelial cells freshly‐isolated from mesenteric arteries of WT mice are clearly shear‐stress sensitive, sensitivity of the channels to shear is lost in dyslipidemic ApoE−/− mice. Furthermore, the loss of FIV in pressurized and preconstricted 1st order mesenteric arteries (100–150 μm lumen diameters) isolated from dyslipidemic ApoE−/− mice can be attributed to the loss of endothelial Kir function. Specifically, in WT mice exposure to an intralumenal shear stress of ~30 dyn/cm2 results in an average dilation of 93.1% of the maximal diameter while ApoE−/− mice exhibit dilations of only 36.4%. In Kir2.1‐deficient mice (Kir2.1+/−), maximal dilation is reached at 42.2% and no further decrease is observed in ApoE−/−/Kir2.1+/− double knock‐out mice suggesting that impaired Kir channels are responsible for the decrease in FIV in hypercholesterolemic conditions. Consistent with these observations, Ba2+, a Kir channel blocker, can reduce dilations to flow in arteries of WT mice but has little to no effect in arteries from ApoE−/− mice. Additionally, NO production in arteries of ApoE−/− and ApoE−/− × Kir2.1+/− mice exposed to internal flow for 30 minutes is decreased compared to WT arteries. Most importantly, we found that overexpressing Kir2.1 specifically in mesenteric endothelium using adenoviral transfection driven by the VE‐Cadherin promoter recovered both FMV and flow‐induced NO production in ApoE−/− or ApoE−/− × Kir2.1+/− arteries strongly confirming the role for EC Kir2.1 impairment in hypercholesterolemia.Support or Funding InformationNIH RO1 HL073965American Heart Association Postdoctoral Award 16POST27000011