Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function

Extracellular vesicles and insulin‐mediated vascular function in metabolic syndrome

Abstract Metabolic Syndrome (MetS) raises cardiovascular disease risk. Extracellular vesicles (EVs) have emerged as important mediators of insulin sensitivity, although few studies on vascular function exist in humans. We determined the effect of insulin on EVs in relation to vascular function. Adults with MetS (n = 51, n = 9 M, 54.8 ± 1.0 years, 36.4 ± 0.7 kg/m2, ATPIII: 3.5 ± 0.1 a.u., VO2max: 22.1 ± 0.6 ml/kg/min) were enrolled in this cross‐sectional study. Peripheral insulin sensitivity (M‐value) was determined during a euglycemic clamp (40 mU/m2/min, 90 mg/dl), and blood was collected for EVs (CD105+, CD45+, CD41+, TX+, and CD31+; spectral flow cytometry), inflammation, insulin, and substrates. Central hemodynamics (applanation tonometry) was determined at 0 and 120 min via aortic waveforms. Pressure myography was used to assess insulin‐induced arterial vasodilation from mouse 3rd order mesenteric arteries (100–200 μm in diameter) at 0.2, 2 and 20 nM of insulin with EVs from healthy and MetS adults. Adults with MetS had low peripheral insulin sensitivity (2.6 ± 0.2 mg/kg/min) and high HOMA‐IR (4.7 ± 0.4 a.u.) plus Adipose‐IR (13.0 ± 1.3 a.u.). Insulin decreased total/particle counts (p < 0.001), CD45+ EVs (p = 0.002), AIx75 (p = 0.005) and Pb (p = 0.04), FFA (p < 0.001), total adiponectin (p = 0.006), ICAM (p = 0.002), and VCAM (p = 0.03). Higher M‐value related to lower fasted total EVs (r = −0.40, p = 0.004) while higher Adipose‐IR associated with higher fasted EVs (r = 0.42, p = 0.004) independent of VAT. Fasting CD105+ and CD45+ derived total EVs correlated with fasting AIx75 (r = 0.29, p < 0.05) and Pb (r = 0.30, p < 0.05). EVs from MetS participants blunted insulin‐induced vasodilation in mesenteric arteries compared with increases from healthy controls across insulin doses (all p < 0.005). These data highlight EVs as potentially novel mediators of vascular insulin sensitivity and disease risk

Endothelial Pannexin 3 - B Cell Lymphoma-6 Interactions Protect Against Oxidative Stress

Pannexin channel isoforms (Panx1-3) are thought to release nucleotides into the extracellular milieu and have been shown to effect vascular hemodynamics. For this reason, we examined their mRNA and protein expression in hypertensive humans and genetically-inbred hypertensive mice. In both mouse and humans, we found a significant reduction in Panx3 expression in resistance artery endothelium. Thus, we hypothesized Panx3 may be a regulator of vascular function. In en face endothelial preparations from 3rd order mesenteric arteries, we localized Panx3 to the Golgi Apparatus as opposed to Panx1 which localized to the plasma membrane. Next, we generated an inducible, endothelial cell Panx3 knockout mouse (Panx3ECKO ). Radiotelemetry revealed a renin-independent spontaneous hypertension, with unremarkable immune infiltration in the kidney. There was no change in cytoplasmic or released ATP. To understand how Panx3 may regulate blood pressure, we examined whether Panx3 interacted with B Cell Lymphoma 6 (BCL6), a potential binding partner. En face proximity ligation assays demonstrated an interaction between Panx3 and BCL6 in the Golgi. Panx3ECKO mice exhibited significantly decreased BCL6 protein, hinting that Panx3 may stabilize BCL6 by binding at the BCL6 ubiquitin sites. In silico threading of the Panx3 sequence onto the cryo-EM structure of Panx1 confirmed this site of interaction. BCL6 is a NFκB repressor, thus its degradation in Panx3ECKO mice caused an increase in NFκB activity with IκBα and p100 significantly upregulated. A novel mimetic peptide designed to block Panx3-BCL6 interactions was administered into C57Bl/6J mice, which recapitulated these results. In addition, Panx3ECKO mice had increased endothelial NOX4 (but not NOX1 or NOX2), likely due to increased NFκB activity-this correlated with a significant increase in plasma H2 O2 , nitrotyrosine (NT3), and 4-hydroxynonenal (4-HNE). In line with this observation, 3rd order mesenteric arteries from Panx3ECKO mice constricted (not dilated) to acetylcholine, which was rescued with the H2 O2 -scavenger catalase (1000U/mL), suggesting that vascular oxidative stress drives hypertension in Panx3ECKO mice. Interestingly, Panx3ECKO mice also exhibit a significant increase in IL-4 receptors on endothelium, and increased IL-4 cytokines in bone marrow lysates. Because IL-4 can drive BCL6 expression in other cell types, we suggest a possible homeostatic immune-endothelial signaling axis. These data elucidate a novel Golgi-localized oxidative signaling pathway in endothelium with a potential immune-derived negative feedback loop