Age‐associated Arterial Dysfunction is Rejuvenated by Targeted Reactivation of Protein Kinase Cδ

Abstract

Genetic repression of endothelial cell (EC) autophagy impairs EC glycolysis and EC ATP production (Bharath et al., ATVB, 2017). This limits purinergic 2Y1 receptor mediated activating phosphorylation of EC nitric oxide (NO) synthase (p‐eNOSS1177) via protein kinase C δ (PKCδT505). A consequence of this impaired signaling pathway is compromised shear‐stress ‐ induced NO generation. We sought to determine whether reactivating PKCδT505 in the context of repressed EC autophagy restores EC function. First, we hypothesized that bryostatin‐1 (Bry‐1) rescues shear‐induced p‐eNOSS1177 and NO generation in human arterial ECs (HAECs) with genetic autophagy compromise. Bry‐1 binds to the regulatory C1 domain of the PKC isoforms. As anticipated, laminar shear stress x 45‐min elevated (p<0.05) p‐PKCδT505, p‐eNOSS1177, and NO generation in HAECs with intact autophagy but not in HAECs after Atg3 knockout (+Atg3KO; n=6). All responses were restored in HAECs + Atg3KO by incubation with Bry‐1, without affecting autophagy, cell death, or apoptosis. Next, we hypothesized that reactivating PKCδ restores vasodilatory function in arteries from aged mice with physiological repression of vascular autophagy. Lower (p<0.05) mRNA and protein expression of autophagy indexes was confirmed in primary arterial ECs and vascular homogenates from ~24 month (old; O) vs. ~7 month (adult, A) old male mice (n=5). Further, basal, glucose‐stimulated, and oligomycin‐stimulated extracellular acidification rate was suppressed (p<0.05) in primary arterial ECs from O vs. A mice, indicating impaired EC glycolysis. Femoral arteries from the same mice were examined ex vivo. Vasodilatory responses to intraluminal flow evoked by pressure gradients of 6, 12, 18, 24, and 30 mmHg (x 3‐min each @ 60 mmHg) was blunted (p<0.05) in O vs. A mice (n=5). As hypothesized, Bry‐1 restored intraluminal flow‐mediated vasodilation (FMD) in arteries from O mice to values observed in A mice (n=5). Vascular smooth muscle function, evaluated by vasodilatory responses to sodium nitroprusside, was not different between groups. In a separate cohort of vessels, nitric oxide synthase inhibition attenuated intraluminal FMD to a greater extent (p<0.05) in arteries from A vs. O mice (n=4). Finally, we hypothesized that Bry‐1 improves PKCδT505 and p‐eNOSS1177 in primary arterial ECs from aged humans. As background, earlier we reported p‐PKCδT505 and p‐eNOSS1177 was lower (p<0.05) in primary ECs obtained from the radial artery of older (~ 68 y) vs. adult (~ 23 y) males that also displayed impaired brachial artery FMD peak (Park et al, FASEB J, 2019). To test this, radial artery ECs were obtained from older males (n=4) via sterile j‐wire. Treatment of non‐fixed ECs with Bry‐1 elevated (p<0.05) PKCδT505 and p‐eNOSS1177 vs. non‐fixed ECs treated with vehicle. Here, we provide proof of concept that activating p‐PKCδT505 using Bry‐1 in the context of impaired vascular autophagy has potential to rejuvenate EC function.Support or Funding InformationJMC (UU Graduate Research Fellowship); SKP (17POST33670663); JDS (NIH RO1HL141540)