Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase

Abstract

Objective: Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism.

Approach and results: On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOS^S1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOS^S1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOS^S1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδ^T505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKCδ^T505 is negated by inhibiting autophagy, (2) shear-induced p-eNOS^S1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδ^T505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOS^S1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs.

Conclusions: Targeted reactivation of purinergic signaling and PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

Keywords: autophagy; cell physiological phenomena; endothelial cells; nitric oxide; reactive oxygen species.

Figure 1. Genetic disruption of Atg3 impairs shear-stress induced autophagy, mitophagy, and NO generation

Relative to static conditions, shear-stress increased Atg3 protein expression (A), LC3 II accumulation (B), LC3-GFP puncta formation (C, E), colocalization of TOM20 with LAMP1 (D, F), p-eNOS^S1177 (G), and NO generation (H–J), in ECs transfected with scrambled siRNA (bar 1 vs. 2) but not in ECs transfected with Atg3 siRNA (bar 3 vs. 4), or in ECs after treatment with 3MA (C, bar 5 vs. 6). Images shown in E, F, and J represent mean data shown in C, D, and I, respectively. Fluorescence images in E, F were individually adjusted to maximize clarity. Calibration bar = 50 µm. For A, B (n=30), C, D (n=10), E, F (n=6), G, H (n=30), I, J (n=10). For A, B, G each n=1 × 10 cm petri dish. For C, D each n=1 well of a 24-well plate. For H, each n = 1 well of a 6-well plate. For C, D, E, F each n=10 cells per field × 10 fields per slide. For I, J each n = 3 wells of a 6-well plate. *p<0.05 vs. (−shear)(−Atg3 siRNA); # p<0.05 vs. (+shear)(−Atg3 siRNA).

Figure 2. Genetic disruption of Atg3 impairs EC glycolysis and NO generation

Relative to static conditions, shear-stress increased GLUT1 protein expression (A), glucose uptake (B), ATP production (C, G), p-eNOS^S1177 (H) and NO generation (I) in ECs transfected with scrambled siRNA (bar 1 vs. 2) but not Atg3 siRNA (bar 3 vs. 4). For A–C (n=8–12). For A, each n = 1 × 10 cm petri dish). For B, C, each n = 1 well of a 6-well plate. For A–C *p<0.05 vs. (−shear)(−Atg3 siRNA); #p<0.05 vs. (+shear)(−Atg3 siRNA). Extracellular acidification rate (ECAR) was assessed in ECs exposed to shear stress ± Atg3 siRNA (D). Relative to basal conditions, ECs transfected with scrambled siRNA displayed increased ECAR in response to 5 mM glucose (bar 1 vs. 3) and 1 µM oligomycin (oligo; bar 1 vs. 5), but these responses were not observed in ECs after Atg3 siRNA (bar 2 vs. 4; bar 2 vs. 6, respectively). Relative to basal conditions, ECAR decreased upon treatment with 50 mM 2-DG in ECs transfected with scrambled (bar 1 vs. 7) but not Atg3 siRNA (bar 2 vs. 8). For D, n=3, each n= 1 seahorse plate. For D, *p<0.05 vs. basal condition (+shear)(−Atg3 siRNA i.e., bar 1); # p<0.05 vs. same condition (+shear)(−Atg3 siRNA). Relative to static conditions, shear-stress increased ATP (E) and p-eNOS^S1177 (F) in ECs transfected with scrambled siRNA (bar 1 vs. 2) but not GLUT1 siRNA (bar 3 vs. 4). For E, n=12, each n = 1 well of a 6-well plate. For F, n=6, each n = 1 × 10 cm petri dish. For E, F *p<0.05 vs. (−shear)(−Atg3 siRNA); #p<0.05 vs. (+shear)(−Atg3 siRNA). After co-transfection with a plasmid vector to increase GLUT1 expression in ECs (G–I), the suppression of shear-induced ATP (G), p-eNOS^S1177 (H), and NO generation (I) after autophagy repression (bar 2 vs. 4) was not observed (bar 6 vs. 8). For G–I, n=4, each n = 1 × 10 cm petri dish). For G–I *p<0.05 vs. (−shear)(−Atg3 siRNA); #p<0.05 vs. (+shear)(−Atg3 siRNA).

Figure 3. Genetic disruption of Atg3 limits purinergic-mediated activation of eNOS

Relative to static conditions, shear-stress increased extracellular ATP accumulation (A–C), p-eNOS^S1177 (D–G), and NO generation (H) in ECs transfected with scrambled siRNA (bar 1 vs. 2) but not Atg3 siRNA (A,C,D,E; bar 3 vs. 4) or GLUT1 siRNA (B; bar 3 vs. 4). After co-transfection with a plasmid vector to increase GLUT1 expression in ECs (C), the suppression of shear-induced ATP after autophagy compromise (bar 2 vs. 4) was normalized (bar 6 vs. 8). The ectonucleotidase apyrase (D), the pharmacological P2Y1-R blocker MRS2179 (E), and genetic disruption of P2Y1-R via siRNA (F) prevented shear-induced p-eNOS^S1177 in ECs with intact Atg3 protein (bar 5 vs. 6). Conversely exogenous 2-methylthioadenosine diphosphate (ADP) restored shear-induced p-eNOS^S1177 (G) and NO generation (H) in ECs with suppressed autophagy (bar 7 vs. 8). For A–C, H, n=6, each n = 1 well of a 6-well plate. For D–G, n=5, each n = 1 × 10 cm petri dish. * p<0.05 vs. (−shear)(−Atg3 siRNA); # p<0.05 vs. (+shear)(−Atg3 siRNA).

Figure 4. Defective purinergic-mediated p-PKCδ^T505 activation of eNOS after autophagy compromise is normalized by genetic and pharmacological approaches

Relative to static conditions, shear-stress increased p-PKCδ^T505 (A,E,F,G) and p-eNOS^S1177 (C,D,H) in ECs transfected with scrambled siRNA (bar 1 vs. 2) but not Atg3 siRNA (bar 3 vs. 4). Relative to static conditions, shear-stress increased p-eNOS^S1177 in ECs transfected with scrambled but not PKCδ siRNA (B) (bar 2 vs. 4). Suppressed shear-induced p-eNOS^S1177 after Atg3 siRNA was restored in ECs co-transfected with constitutively active (CA) PKCδ (bar 7 vs. 8; C) but not dominant-negative (DN) PKCδ (D). Suppressed shear-induced p-PKCδ^T505 after Atg3 siRNA (A,E,F,G) could be recapitulated in ECs with intact Atg3 protein that were transfected with P2Y1-R siRNA (bar 5 vs. 6; E) or restored in ECs with autophagy compromise by ADP (bar 7 vs. 8; F). Suppressed shear-induced p-PKCδ^T505, p-eNOS^S1177, and NO generation in ECs transfected with Atg3 siRNA was restored by the PKCδ agonist bryostatin-1 (bry; bar 4 vs. 8, G, H, I). For A–H, n=5, each n = 1 × 10 cm petri dish. For I, n=6, each n = 3 wells of a 6-well plate. *p<0.05 vs. (−shear)(−Atg3 siRNA); # p<0.05 vs. (+shear)(−Atg3 siRNA).

Figure 5. Pharmacological inhibition of autophagy impairs shear-stress induced NO generation in human arterial endothelial cells

Relative to static conditions, shear-stress increased Atg3 (A), LC3 II (B), GLUT1 (D), p-PKCδ^T505 (E) and p-eNOS^S1177 (F) protein expression, NO generation (G), and p62 degradation (C) (bar 1 vs. 2). All responses were prevented by concurrent treatment with 3MA (bar 3 vs. 4). Images shown in the “merge” portion of H represent mean data shown in G. Calibration bar = 100 µm. For A–F, n=3, each n= 2 wells of a 6-well plate. For G and H, n=2, each n = 1 well of a 4 well chamber slide. *p<0.05 vs. (−shear)(− 3MA); # p<0.05 vs. (+shear)(− 3MA).

Figure 6. iecAtg3KO mice exhibit impaired endothelial cell autophagy, GLUT1 protein expression, and p-eNOS protein expression vs. WT mice

To assess mRNA, endothelial cells (ECs) and media + adventitia were isolated from iliac arteries of 5-month old iecAtg3KO and WT mice treated 30-days earlier with tamoxifen. Purity of the EC and media + adventitia fraction was confirmed by PECAM (A) and α-SMA (B) staining, respectively. Atg3/18S mRNA was similar in the media + adventitia component of both groups (C), but was lower in the EC fraction of iecAtg3KO vs. WT mice (D). For A–D, n=4 mice per group. *p<0.05 vs. media + adventitia (A); vs. EC (B); vs. WT (D). EC protein was assessed in a different cohort of 5-month old mice treated 30 days earlier with tamoxifen. Atg3:GAPDH (E), LC3 II:GAPDH (F), GLUT1 : GAPDH (G), and p-eNOS : eNOS (H) was lower in ECs from iecAtg3KO vs. WT mice. For E–H, n=3, each n = ECs obtained from 4 entire aortae. *p<0.05 vs. WT.