Bharath, Leena P;
Ruan, Ting;
Babu, PVA;
Boudina, Sihem;
Graham, Timothy E;
Symons, J David
Intact Autophagy is Requisite for Shear‐Stress‐Induced Nitric Oxide Generation by Endothelial Cells via a Novel Glucose‐Dependent Purinergic Signaling Pathway
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Medientyp:
E-Artikel
Titel:
Intact Autophagy is Requisite for Shear‐Stress‐Induced Nitric Oxide Generation by Endothelial Cells via a Novel Glucose‐Dependent Purinergic Signaling Pathway
Beteiligte:
Bharath, Leena P;
Ruan, Ting;
Babu, PVA;
Boudina, Sihem;
Graham, Timothy E;
Symons, J David
Beschreibung:
<jats:p>Impaired intracellular autophagy has been implicated as a cause of vascular dysfunction in aging human arteries. Earlier we reported that shear‐stress (180‐min × 20 dyne•cm<jats:sup>2</jats:sup>) induces autophagy that is concurrent with phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at serine (S) 1177 (p‐eNOS<jats:sup>S1177</jats:sup>) and NO generation in bovine aortic endothelial cells (ECs). Genetic inhibition of autophagy via Atg3 or Atg5 siRNA suppresses shear‐induced p‐eNOS<jats:sup>S1177</jats:sup> and NO generation, increases reactive oxygen species generation, and unleashes inflammatory/adhesion responses, suggesting strongly that autophagy is critical for maintaining EC function. Here we report one mechanism through which autophagy regulates shear‐induced p‐eNOS<jats:sup>S1177</jats:sup> and NO production in ECs. Tumor suppressor and metabolism regulator, p53, is strongly upregulated by Atg3 siRNA, causing reduced shear‐induced Glut‐1 expression, glucose transport, glycolysis, and ATP production in ECs. As expected, AMPK is activated by the energy deficit, but does not restore p‐eNOS<jats:sup>S1177</jats:sup> and NO generation in this setting. However, shear‐evoked p‐eNOS<jats:sup>S1177</jats:sup> and NO generation can be restored in autophagy‐impaired ECs by (i) adding extracellular ADP or by (ii) overexpressing Glut‐1. These findings prompted a further investigation of purinergic signaling to eNOS in the context of suppressed EC autophagy. As predicted, P2Y<jats:sub>1</jats:sub> receptor blockade (MRS2179) and P2Y<jats:sub>1</jats:sub> siRNA can recapitulate the Atg3 phenotype i.e., shear‐induced p‐eNOS<jats:sup>S1177</jats:sup> and NO generation are prevented. Furthermore, shear‐induced ATP/ADP release increases p‐PKCδ<jats:sup>Thr505</jats:sup>, a downstream effector of P2Y<jats:sub>1</jats:sub> and activator of p‐eNOS<jats:sup>S1177</jats:sup>. As would be anticipated by these findings, shear‐induced p‐eNOS<jats:sup>S1177</jats:sup> and NO generation can be restored in autophagy‐impaired ECs by (i) the PKCδ activator bryostatin or by (ii) PKCδ overexpression, whereas shear‐induced p‐eNOS<jats:sup>S1177</jats:sup> and NO generation are prevented when autophagy‐intact ECs are transfected with PKCδ siRNA. Ongoing experiments are determining the extent to which this mechanism is responsible for impaired endothelial function that we have observed in arteries from old mice, that is concurrent with lower Atg3 protein expression, compromised autophagy, and attenuated p‐eNOS<jats:sup>S1177</jats:sup> : total eNOS, vs. arteries from adult mice. Collectively, targeted activation of purinergic signaling and/or PKCδ may present a new strategy for treating arterial dysfunction that exists with impaired EC autophagy in the context of healthy aging.</jats:p><jats:p><jats:bold>Support or Funding Information</jats:bold></jats:p><jats:p>UU Diabetes and Metabolism Center; UU Center on Aging; UU College of Health</jats:p>