Abstract 010: Similar Increases in Arterial Shear-rate Evoked by Handgrip Exercise Activate Autophagy and Enos to a Lesser Extent in Primary Endothelial Cells From Older vs. Adult Subjects

Abstract

We reported that genetic or pharmacological suppression of autophagy in bovine and human arterial endothelial cells (ECs) prevents shear-stress induced p-eNOS S1177 and NO generation (Bharath et al., Arterioscler Thromb Vasc Biol , 2017). To date, one human study indicates an aging-associated repression of EC autophagy is concurrent with arterial dysfunction (LaRocca et al., J Physiol , 2012). We tested the hypothesis that a physiological elevation of arterial shear-rate activates autophagy and p-eNOS S1177 to a lesser extent in primary ECs from older vs. adult participants. After familiarization with laboratory procedures, flow-mediated vasodilation (%FMD) was observed to be lower (p<0.05) in 4 older (68±5 yr; 3.9±1.3 %) vs. 5 adult (23±3 yr; 7.9±1.1 %) male subjects. Within 1-week, a catheter was placed into the radial artery (RA). ECs were collected 30-min later via j-wire from the RA. Next, subjects performed rhythmic handgrip exercise (RHE) for 60-min that elevated (p<0.05) arterial shear-rate similarly (2.6 ± 0.2-fold) between groups from pre-exercise values. Shear-rate [8V mean /brachial artery (BA) diameter] was obtained by assessing BA diameter, BA blood flow velocity (V mean ), and BA blood flow [(V mean π (vessel diameter / 2) 2 x 60)] at 5-10 min intervals using Doppler ultrasound. Compared to pre-exercise values, RHE did not alter heart rate, stroke volume, cardiac output, or arterial pressure (plethysmography) in either group. Immediately following RHE ECs were collected from the RA. Using quantitative immunofluorescence, primary ECs (75 ECs per endpoint per subject) were identified by positive co-staining for VE-cadherin and DAPI via confocal microscopy. Immortalized human arterial ECs processed and stained in parallel served as fluorescence intensity controls. Relative to pre-exercise, ECs from adult but not old subjects at 60-min displayed increased expression of microtubule associated protein light chain 3 (p<0.04), lysosomal associated membrane protein 2a (p<0.01), autophagy-related gene 3 (p=0.06), and p-eNOS S1177 (p<0.02), and decreased expression of the adaptor protein p62 (p<0.02). These data indicate aging limits shear-induced EC autophagy and eNOS activation in response to functional hyperemia.