Activating P2Y1 receptors improves function in arteries with repressed autophagy

Rationale: Shear-stress -induced nitric oxide (NO) generation by human arterial endothelial cells (HAECs) is prevented by pharmacological or genetic autophagy disruption. The response is restored by activating purinergic 2Y1 receptor (P2Y1-R) signaling to endothelial cell (EC) NO synthase (eNOS). Here we determined the translational relevance of these findings. Objective: Test the hypotheses that : (i) older humans and mice display blunted autophagy initiation and EC NO generation evoked by functional hyperemia; and that impaired intraluminal flow-mediated vasodilation displayed by : (ii) older mice with repressed EC autophagy; (iii) adult mice with pharmacological autophagy inhibition; and (iv) adult mice with EC specific autophagy-related gene 3 (Atg3) depletion, is rejuvenated by activating P2Y1-Rs. Methods and results: Rhythmic handgrip exercise elevated radial artery shear rate similarly in adult and older males. Compared to baseline, autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs from adult but not older subjects. Relative to adult (7-month) mice, older (24-month) animals exhibited : (i) repressed EC mRNA and arterial protein indexes of autophagy and p-eNOSS1177 at baseline; (ii) blunted activation of EC autophagy and p-eNOSS1177 in response to treadmill-running; and (iii) impaired flow mediated arterial vasodilation examined ex vivo. Age-associated reductions in vasodilatory capacity were recapitulated in arteries from adult mice by : (i) NOS inhibition; (ii) autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) P2Y1-R blockade; and (v) germline depletion of P2Y1-Rs. P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) restored shear-induced p-eNOSS1177 and NO generation in HAECs that was otherwise attenuated by: (i) 3-MA; or (ii) Atg3 knockdown. Importantly, 2-Me-ADP improved intraluminal flow-mediated vasodilation in arteries from : (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals. Conclusions: Arterial dysfunction concurrent with repressed EC autophagy is improved by activating P2Y1-Rs.