Oxidative modifications of LDL are a major risk factor in the development of vascular disease and are known to induce endothelial dysfunction, one of the earliest manifestations of atherosclerosis (1, 2). Our studies focus on the oxidized LDL (oxLDL)-induced impact on endothelial biomechanics and its role in vascular dysfunction.Our recent studies showed that the stiffness of aortic endothelial cells (ECs) is significantly increased by exposing the cells to oxLDL in vitro or by dyslipidemia in the dietinduced porcine atherosclerosis model in vivo (3, 4). An increase in endothelial stiffness was accompanied by an increase in endothelial contractile forces generated on the cell-substrate interface and an enhanced ability of ECs to form branching networks in 3D cultures (3, 4), which is considered a prerequisite of angiogenesis (5). Moreover, earlier studies demonstrated a correlation between increased endothelial force and network formation across several endothelial subtypes (6). We proposed, therefore, that oxLDL-induced endothelial stiffening may lead to increased angiogenic activity of ECs during the development of atherosclerotic plaques. This process is expected to be of major clinical importance because neovascularization of the plaques is increasingly recognized as a critical process and a major risk factor for plaque vulnerability (7). The goal of this study is to elucidate the mechanism of oxLDL-induced endothelial stiffening and evaluate a link between this effect and the ability of ECs to form functional capillaries. Abstract Endothelial biomechanics is

BackgroundHypercholesterolemia‐induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K+ (Kir) channels and that Kir2.1 is an upstream mediator of flow‐induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐induced NO production and flow‐induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions.Methods and ResultsKir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient (Apoe −/−) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe −/−mice, Kir2.1 +/− /Apoe −/− exhibit the same blunted FIV and flow‐induced NO response as Apoe −/−or Kir2.1 +/− alone, but while FIV in Apoe −/− mice can be rescued by cholesterol depletion, in Kir2.1 +/− /Apoe −/− mice cholesterol depletion has no effect on FIV. Endothelial‐specific overexpression of Kir2.1 in arteries from Apoe −/− and Kir2.1 +/− /Apoe −/− mice results in full rescue of FIV and NO production in Apoe −/− mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect. Kir2.1 +/− /Apoe −/−mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta.ConclusionsWe conclude that hypercholesterolemia‐induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐induced NO production, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu-oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27). Both Cu-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

Objective: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial inwardly rectifying K+ (Kir) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type–Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. Conclusions: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.

The vascular endothelium is an important regulator of vascular tone. Acute changes in mechanical stressors, such as blood flow (fluid shear) and pressure, are converted into chemical signals by the endothelium to control vasomotor function. We recently showed that endothelial Kir2.1 channels are critical to the vasodilatory response to increases in blood flow through eNOS and NO production. It is well established that obesity causes endothelial dysfunction likely through impairment of nitric oxide signaling/bioavailability. Furthermore, obesity impairs the vasculature of visceral adipose depots while arteries in the subcutaneous adipose appear unaffected. Therefore, we tested the role of endothelial Kir2.1 in obesity‐induced endothelial dysfunction in vascular beds of spatially distinct adipose depots in obese humans and in a diet‐induced obese mouse model. Mice were fed a high fat, high cholesterol diet for 4 or 8 weeks prior to analysis of vascular function via response to flow in an ex vivo arterial preparation. Endothelial function of arteries from both subcutaneous and visceral adipose depots were tested. After 4 weeks of high fat feeding, no differences were observed in vascular function between arteries from either adipose depot nor when compared to control mice fed a normal chow diet. However, after 8 weeks visceral arteries from high fat fed mice had a significantly blunted response to flow, while subcutaneous arteries retained a full dilatory response. Overexpressing endothelial Kir2.1 using adenoviral transduction with expression driven by the VE‐Cadherin promoter in visceral arteries of diet‐induced obese mice rescued the response to flow suggesting that Kir2.1 channel function or expression may be impaired exclusively in arteries of visceral adipose of obese mice. Importantly, we observed similar findings in obese human arteries from visceral and subcutaneous adipose biopsies collected during bariatric surgery.Support or Funding InformationNIH R01 HL073965 (IL)American Heart Association 16POST27000011 (ISF)NIH 5T32HL007829‐24 (ISF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dyslipidemia is a modification in lipid composition that potentially causes adverse cardiovascular outcomes. An increase in oxidized low density lipoprotein (oxLDL), a common dyslipidemic condition, is considered to be detrimental. We show here that pathological levels of oxLDL induces endothelial cell (EC) stiffening and this stiffening is RhoA dependent. Furthermore, we tested the immediate downstream effector of RhoA, Rho kinase (ROCK). ROCK inhibition caused a complete abrogation of oxLDL induced EC stiffening. 7‐ketocholesterol (7KC), an important oxysterol component of oxLDL, has previously been shown to induce EC stiffening. We now extend our study to the inhibition of RhoA, which abolished the stiffening effect of 7KC. Previous studies have observed that oxLDL increases network formation in vitro. Here we now demonstrate that oxLDL increases migration and proliferation in vitro, and capillary formation in vivo. Similarly to oxLDL induced EC stiffening, the effects of oxLDL on angiogenesis were also inhibited by blocking RhoA and ROCK signaling. Additionally, PGPC and POVPC, components of oxLDL, were tested on capillary formation. We propose that oxLDL induced EC stiffening may play a potential role in angiogenesis under dyslipidemic conditions.

BACKGROUNDObesity is a major risk factor for cardiovascular disease. Our previous studies demonstrated that microvascular flow‐induced dilation (FID) is reduced in visceral adipose tissue (VAT) compared to subcutaneous AT (SAT) in morbidly obese subjects. Recent studies showed that VAT accumulation is more predictive of impaired vascular function than SAT. In the current study, we examine the differential effect of aerobic exercise (AE) training on improving the FID in SAT versus VAT microvessels in morbid obesity.METHODSWe obtained SAT and VAT biopsies from obese subjects undergoing bariatric surgery (n=6 and recruitment is ongoing; age: 35±3 yrs; BMI: 46±3 kg/m2; SBP: 114±6 mmHg; DBP: 80±7 mmHg) who were randomized to two groups (n=3 each): (1) AE training group (75% HRmax, 60 min/d, 2–3 d/wk, 4 wks) and (2) non‐exercising (control) group. Arterioles were isolated from SAT and VAT biopsies and cannulated for reactivity measurements in response to flow (pressure gradients of Δ10–Δ100 cmH2O) with and without the nitric oxide (NO) synthase (L‐NAME; 10−4 mol/L), PEGylated catalase (PEG‐Cat; 500 U/ml), or the superoxide dismutase (SOD) mimetic, Tempol (10−5 mol/L). NO and reactive oxygen species (ROS) generation were measured in the arterioles using NO detection dye and cell‐permeant ROS indicator (H2DCFDA).RESULTSFollowing training the AE group demonstrated reductions in BMI (−3%, p=0.03) and resting systolic BP (−4%, p=0.1). FID was higher in the AE than the control group across all pressure gradients (20–35% higher, p<0.05). The FID of VAT arterioles was reduced compared to SAT arterioles in both the AE and control groups. L‐NAME and PEG‐Cat reduced the FID (% of dilation at Δ60 pressure gradient) in the SAT arterioles in the AE group (L‐NAME: −18%, p=0.1; PEG‐Cat: −44%, p<0.001) and to a relatively lower extent in the control group (L‐NAME: −11%, p=0.1; PEG‐Cat: −29%, p=0.01). FID in the VAT arterioles was reduced in response to L‐NAME and PEG‐Cat (−14%, p=0.004; −38%, p=0.01, respectively) in the AE group however, no changes were detected in VAT arterioles from the control group. Tempol improved FID in the SAT and VAT arterioles; a higher magnitude of increase was noted in the control group (49%, p=0.01) compared to the AE group (16%, p=0.046) and in the VAT arterioles (49%, p=0.01) compared to the SAT arterioles (14%, p=0.02). Furthermore, the sensitivity of VAT arterioles to PEG‐Cat was induced by Tempol. PEG‐Cat reduced the FID in Tempol‐treated VAT arterioles by 47% and 35% (p<0.001) in the control and AE groups, respectively indicating that Tempol‐induced FID improvements in VAT arterioles may indicate an increased H2O2 production. Under flow conditions, NO‐fluorescence was greater and ROS generation lower in SAT arterioles compared to VAT arterioles and in the AE group compared to the control group.CONCLUSIONOur results suggest that 1) VAT arterioles display reduced vasodilator reactivity to flow compared to SAT arterioles and 2) AE training improves the FID in both SAT and VAT arterioles via NO and H2O2 dependent mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.