Evidence for an Age‐Associated Impairment of Exercise‐Induced Autophagy and eNOS Activation in Primary Arterial Endothelial Cells from Humans

Abstract

Evidence that age‐associated arterial dysfunction is concurrent with repressed basal endothelial cell (EC) autophagy in humans is extremely limited. Earlier we documented that genetic or pharmacological suppression of autophagy in bovine and human arterial ECs prevents shear‐stress induced p‐eNOSS1177 and NO generation, and exaggerates shear‐induced O2•− production. The impact of physiological suppression of EC autophagy via aging on these responses has not been examined in primary arterial ECs from humans. We tested the hypothesis that elevated arterial shear‐rate activates autophagy, p‐eNOSS1177, and NO generation to a lesser extent, and O2•− production to a greater extent, in primary arterial ECs from older vs. adult males. ECs were collected using a sterile j‐wire inserted through a radial artery (RA) catheter of 6 older (68±2 y) and 6 adult (23±1 y) male subjects twice i.e., before (pre) and following (post) 60‐min rhythmic handgrip exercise (HG). Primary ECs (75 ECs per endpoint per subject) were identified by positive co‐staining for VE‐cadherin and DAPI via confocal microscopy using quantitative immunofluorescence. Commercial human arterial ECs processed and stained in parallel served as fluorescence intensity controls. Indices of autophagy, p‐eNOSS1177, and NO generation (DAF) were lower (p<0.01), whereas O2•− production (DHE) was higher (p<0.01), in ECs collected from older vs. adult subjects pre‐HG. Shear‐rate was calculated throughout the procedure by assessing brachial artery (BA) diameter, BA blood flow velocity, and BA blood flow using Doppler ultrasound. Compared to pre‐HG, HG elevated (p<0.05) shear‐rate similarly (2.7 ± 0.2–fold) in adult and older subjects, but did not alter heart rate, stroke volume, cardiac output, or arterial pressure (plethysmography) in either group. Relative to pre‐HG, ECs from adults at 60‐min HG displayed: (i) increased (p<0.05) expression of microtubule associated protein light chain 3B, lysosomal associated membrane protein 2a, autophagy‐related gene 3, and BECLIN‐1; (ii) decreased (p<0.05) expression of the adaptor protein p62; and (iii) increased p‐eNOSS1177, NO generation, and O2•− production. Conversely, ECs from older participants were refractory to elevated shear rate concerning autophagy, p‐eNOSS1177, NO generation, and O2•− production. Age‐associated reductions in basal and shear‐induced EC autophagy might contribute importantly to the age‐associated decline in arterial function.Support or Funding InformationAHA17POST33670663 (SKP); UU GRF (JMC); NIHR01DK098646‐01A1, NIHR01DK099110, AHA 16GRNT30990018 (SB); VA IK2RX001215 CDA2, AHA14SDG1850039 (JDT); AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540 (JDS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.