Autophagy is a process whereby damaged cellular cargo is delivered to the lysosome for degradation and eventual recycling or removal. Knowledge concerning the importance of autophagy to endothelial cell (EC) metabolism is evolving. Previously we reported that 180‐min × 20 dyne · cm 2 shear‐stress initiates autophagy, activates EC nitric oxide (NO) synthase (eNOS) at serine 1177 (p‐eNOSS1177), and generates NO in immortalized bovine and human arterial endothelial cells (ECs) (Bharath et al., Arterioscler Thromb Vasc Biol, 2017). Importantly, when EC autophagy was repressed using genetic or pharmacological procedures, the ability of shear‐stress to generate NO was prevented. Here we report data from the first step to eventually determine whether these findings obtained in vitro can be translated to primary human ECs. After familiarization with laboratory procedures (visit 1), five male subjects (23±3 yr) completed a maximal voluntary contraction (MVC) test and a flow‐mediated vasodilation (FMD) assessment during a second visit (visit 2). Within 1‐week, a 20 g catheter was placed into the radial artery (RA) during visit 3. Next, ECs were collected via j‐wire biopsy from the RA catheter, transferred to dissociation buffer, recovered by washing and centrifugation, placed onto previously prepared glass slides, and frozen at −80°C. Thirty‐min after collecting pre‐exercise ECs, subjects performed rhythmic handgrip exercise for 60‐min at a contraction intensity that elevated (p<0.05) arterial shear‐rate 2.7 ± 0.3‐fold from baseline. Shear‐rate [8Vmean/brachial artery (BA) diameter] was obtained by direct and continuous assessment of BA diameter, BA blood flow velocity (Vmean), and BA blood flow [(Vmeanπ (vessel diameter/2)2 × 60)] using Doppler ultrasound. Heart rate (HR, beats/min; 3‐lead ECG), stroke volume (SV, ml/beat), cardiac output (CO, L/min), and mean arterial pressure (MAP, mmHg; automated plethysmography), respectively, were not different when pre‐exercise values (56±1, 101±6, 5.6±0.3, and 91±3) were compared to 60‐min values (60±2, 104±6, 6.2±0.4, and 94±2). At 60‐min of handgrip exercise, ECs were collected from the RA, prepared, and stored as described. Using quantitative immunofluorescence, primary ECs (75 ECs per endpoint) were identified by positive co‐staining for VE‐Cadherin and DAPI via confocal microscopy. Immortalized human arterial endothelial cells (P4–6) were processed and stained in parallel using identical procedures to serve as fluorescence intensity controls. Relative to pre‐exercise values, ECs obtained at 60‐min displayed increased expression of beclin‐1 (p<0.01), microtubule associated protein light chain 3 (LC3 II; p=0.05), autophagy‐related gene 3 (Atg3; p=0.06), lysosomal associated membrane protein 2a (LAMP2a; p<0.004), and p‐eNOSS1177 (p<0.02), and decreased expression (i.e., enhanced degradation) of the adapter protein p62/SQSTM1 (p<0.02). Taken together, these findings provide novel evidence in primary human ECs that elevated shear‐rate evoked by functional hyperemia initiates autoph...

Dietary inorganic nitrate (nitrate) is a promising adjunctive treatment to reduce blood pressure and improve vascular function in hypertension. However, it remains unknown if the efficacy of nitrate is dependent upon an elevated blood pressure or altered by medication in patients with hypertension. Therefore, blood pressure and vascular function, measured by passive leg movement (PLM) and flow-mediated dilation (FMD), were assessed following 3 days of placebo (nitrate-free beetroot juice) and nitrate (nitrate-rich beetroot juice) administration in 13 patients (age: 53 ± 12 yr) with hypertension taking antihypertensive medications ( study 1) and in 14 patients (49 ± 13 yr) with hypertension not taking antihypertensive medications ( study 2). In study 1, plasma nitrite concentration was greater for nitrate than placebo (341 ± 118 vs. 308 ± 123 nmol/L, P < 0.05), yet blood pressure and vascular function were unaltered. In study 2, plasma nitrite concentration was greater for nitrate than placebo (340 ± 102 vs. 295 ± 93 nmol/L, P < 0.01). Systolic (136 ± 16 vs. 141 ± 19 mmHg), diastolic (84 ± 13 vs. 88 ± 12 mmHg), and mean (101 ± 12 vs. 106 ± 13 mmHg) blood pressures were lower ( P < 0.05), whereas the PLM change in leg vascular conductance (6.0 ± 3.0 vs. 5.1 ± 2.6 mL·min−1·mmHg−1) and FMD (6.1 ± 2.4% vs. 4.1 ± 2.7%) were greater ( P < 0.05) for nitrate than placebo. The changes in systolic blood pressure ( r = −0.60) and FMD ( r = −0.48) induced by nitrate were inversely correlated ( P < 0.05) to the respective baseline values obtained in the placebo condition. Thus, the efficacy of nitrate to improve blood pressure and vascular function in hypertension appears to be dependent on the degree of blood pressure elevation and vascular dysfunction and not antihypertensive medication status, per se. NEW & NOTEWORTHY Dietary nitrate (nitrate) is a promising intervention to improve blood pressure and vascular function in hypertension. We demonstrate that these beneficial effects of nitrate are inversely related to the baseline value in a continuous manner with no distinction between antihypertensive medication status. Thus, the efficacy of nitrate to improve blood pressure and vascular function in hypertension appears to be dependent on the degree of blood pressure elevation and vascular dysfunction and not antihypertensive mediation status.

Dietary salt restriction is a well-established approach to lower blood pressure and reduce cardiovascular disease risk in hypertensive individuals. However, little is currently known regarding the effects of salt restriction on central and peripheral hemodynamic responses to exercise in those with hypertension. Therefore, this study sought to determine the impact of salt restriction on the central and peripheral hemodynamic responses to static-intermittent handgrip (HG) and dynamic single-leg knee extension (KE) exercise in individuals with hypertension. Twenty-two subjects (14 men and 8 women, 51 ± 10 yr, 173 ± 11 cm, 99 ± 23 kg) forewent their antihypertensive medication use for at least 2 wk before embarking on a 5-day liberal salt (LS: 200 mmol/day) diet followed by a 5-day restricted salt (RS: 10 mmol/day) diet. Subjects were studied at rest and during static intermittent HG exercise at 15, 30, and 45% of maximal voluntary contraction and KE exercise at 40, 60, and 80% of maximum KE work rate. Salt restriction lowered resting systolic blood pressure (supine: −12 ± 12 mmHg, seated: −17 ± 12 mmHg) and diastolic blood pressure (supine: −3 ± 9 mmHg, seated: −5 ± 7 mmHg, P < 0.05). Despite an ~8 mmHg lower mean arterial blood pressure during both HG and KE exercise following salt restriction, neither central nor peripheral hemodynamics were altered. Therefore, salt restriction can lower blood pressure during exercise in subjects with hypertension, reducing the risk of cardiovascular events, without impacting central and peripheral hemodynamics during either arm or leg exercise. NEW & NOTEWORTHY This is the first study to examine the potential blood pressure-lowering benefit of a salt-restrictive diet in individuals with hypertension without any deleterious effects of exercising blood flow. While mean arterial pressure decreased by ~8 mmHg following salt restriction, these findings provide evidence for salt restriction to provide protective effects of reducing blood pressure without inhibiting central or peripheral hemodynamics required to sustain arm or leg exercise in subjects with hypertension.

Exaggerated blood pressure and diminished limb hemodynamics during exercise in patients with hypertension often are not resolved by antihypertensive medications. We hypothesized that, independent of antihypertensive medication status, dietary nitrate supplementation would increase limb blood flow, decrease mean arterial pressure (MAP), and increase limb vascular conductance during exercise in patients with hypertension. Patients with hypertension either abstained from (n=14, Off-Meds) or continued (n=12, On-Meds) antihypertensive medications. Within each group, patients consumed (cross-over design) nitrate-rich or nitrate-depleted (placebo) beetroot juice for 3-days before performing handgrip (HG) and knee-extensor exercise (KE). Blood flow and MAP were measured using Doppler ultrasound and an automated monitor, respectively. Dietary nitrate increased plasma-[nitrite] Off-Meds and On-Meds. There were no significant effects of dietary nitrate on blood flow, MAP, or vascular conductance during HG in Off-Meds or On-Meds. For KE, dietary nitrate decreased MAP (mean±SD across all three exercise intensities, 118±14 vs. 122±14 mmHg, p=0.024) and increased vascular conductance (26.2±6.1 vs. 24.7±7.0 ml/min/mmHg, p=0.024), but did not affect blood flow for Off-Meds, with no effects On-Meds. Dietary nitrate-induced changes in blood flow (r=-0.67, p<0.001), MAP (r=-0.43, p=0.009), and vascular conductance (r=-0.64, p<0.001) during KE, but only vascular conductance (r=-0.35, p=0.039) during HG, were significantly related to the magnitude of placebo values, with no differentiation between groups. Thus, the effects of dietary nitrate on limb hemodynamics and MAP during exercise in patients with hypertension are dependent on the values at baseline, independent of antihypertensive medication status, and dependent on whether exercise was performed by the forearm or quadriceps.

The purpose of this study was to determine the effects of exercise in hot, cold, and temperate environments on plasma interleukin-6 (IL-6). Eleven recreationally trained males (age 5 25 ¡ 4 years, height 5 178 ¡ 5 cm, weight 5 79.4 ¡ 13.5 kg, body fat 5 14.7 ¡ 3.6%, VO 2 peak 5 54.6 ¡ 11.5 ml kg 21 min 21) performed a 1 hr cycling bout in hot (33˚C), cold (7˚C), and temperate (20˚C) environments at 60% of W max followed by 3 hr of supine recovery in temperate conditions. Expired gases were measured every 15 min during exercise and once every hour during recovery. Heart rate was continuously measured throughout the trials. Blood samples were obtained from the antecubital vein pre-exercise, immediately post-exercise, and 3 hr post-exercise. Blood samples were analyzed for plasma concentrations of IL-6 using a commercial ELISA kit. Plasma IL-6 concentrations were significantly higher immediately post-exercise (14.8 ¡ 1.6 pg ml 21 , p 5 0.008) and 3 hr post-exercise (14.8 ¡ 0.9 pg ml 21, p 5 0.018) compared to pre-exercise (11.4 ¡ 2.4 pg ml 21), across all trials. There were no differences in plasma IL-6 concentrations (p 5 0.207) between temperature conditions. Oxygen consumption and heart rate were higher and respiratory exchange ratio was lower in the hot compared to other conditions (p , 0.05). These data indicate that the temperature in which exercise occurs does not affect acute plasma IL-6 response despite differences in metabolic state.

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Aim The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signaling to eNOS. Here we illustrate the translational and functional significance of these findings. Methods and Results First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperemia evoked by RHE elevated radial artery shear rate similarly from baseline in adult and older subjects for 60-min. Compared to baseline, indexes of autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Further, 20 dyne • cm2 laminar shear stress x 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOSS1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOSS1177in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by : (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y1-receptor (P2Y1-R) blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. Conclusions Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.

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