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

Shear Stress Increases Autophagy and Mitochondrial Turnover in ECs

Bovine aortic ECs were exposed to no shear (−shear) or ≈20 dyn/cm² for 3 hours (+shear). Shear stress increased Atg3 (Figure 1A) and Atg5 (Figure IA in the online-only Data Supplement) protein expression, LC3 (microtubule-associated protein light chain 3)-II accumulation (Figure 1B) and p62 degradation (Figure IB in the online-only Data Supplement), and LC3:GFP (green fluorescent protein) puncta formation (Figure 1C and 1E).

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Figure 1.

Genetic disruption of Atg3 (autophagocytosis-associated protein 3) impairs shear stress–induced autophagy, mitophagy, and nitric oxide (NO) generation. Relative to static conditions, shear stress increased Atg3 protein expression (A), LC3 (microtubule-associated protein light chain 3) II accumulation (B), LC3-GFP (green fluorescent protein) puncta formation (C and E), colocalization of TOM20 (translocase of outer membrane 20) with LAMP1 (lysosome-associated membrane protein 1; D and F), p-eNOS^S1177(G), and NO generation (H–J) in endothelial cells (ECs) transfected with scrambled small interfering RNA (siRNA; bar 1 vs 2) but not in ECs transfected with Atg3 siRNA (bar 3 vs 4) or in ECs after treatment with 3-methyladenine (3-MA; 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) and (F) were individually adjusted to maximize clarity. Calibration bar, 50 µm. For A and B, n=30; C and D, n=10; E and F, n=6; G and H, n=30; I and J, n=10. For A, B, and G, each n=1×10 cm petri dish. For C and 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, and F, each n=10 cells per field×10 fields per slide. For I and J, each n=3 wells of a 6-well plate. *P<0.05 vs (−shear; −Atg3 siRNA); #P<0.05 vs (+shear; −Atg3 siRNA). DAF indicates diaminofluorescein; eNOS, endothelial nitric oxide synthase; and EPR, electron paramagnetic resonance spectroscopy.

Autophagy is an important regulator of mitochondrial turnover (ie, mitophagy).¹⁰ We observed shear-induced degradation of mitochondrial aconitase and translocase of outer membrane 20 (Figure IC and ID in the online-only Data Supplement), together with colocalization of translocase of outer membrane 20 with lysosome-associated membrane protein 1 (Figure 1D and 1F) and translocase of outer membrane 20 with LC3 (Figure IE and IF in the online-only Data Supplement). These latter findings demonstrate that mitochondrial turnover is elevated in response to shear stress.

Shear-induced LC3-II accumulation was greater, whereas p62 degradation was prevented in the presence versus the absence of bafilomycin A-1 (Figure IG and IH in the online-only Data Supplement), confirming that shear stress increases autophagosome formation rather than decreases autophagosomal degradation by the lysosome.⁷ All indices of shear-induced EC autophagy and mitophagy we measured were prevented by repressing Atg3 (Figure 1A–1F; Figure IB–IF in the online-only Data Supplement) or Atg5 (Figure IA, II, and IJ in the online-only Data Supplement) protein expression. Using an alternative approach, shear-induced LC3:GFP puncta formation was negated by pharmacological inhibition of autophagy using the class III PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine (Figure 1C and 1E). These data indicate that shear stress increases EC autophagy and mitophagy and demonstrate that genetic and pharmacological approaches we used to limit autophagy do not alter cell viability (Figure IIA–IIC in the online-only Data Supplement).

Autophagy Suppression Limits Shear Stress–Induced NO Generation in ECs

Shear stress increased p-eNOS^S1177 and NO generation in ECs transfected with scrambled but not Atg3 (Figure 1G–1J) or Atg5 siRNA (Figure IIIA and IIIB in the online-only Data Supplement). Impairment of autophagy exacerbated shear-induced ROS production (Figure IVA in the online-only Data Supplement) and unleashed markers of inflammation and adhesion (Figure IVB–IVF in the online-only Data Supplement). These findings confirm that vascular autophagy plays a critical role in maintaining NO bioavailability and regulating oxidant/antioxidant balance and inflammatory/anti-inflammatory balance in ECs.

Shear-Induced Phosphatase, Kinase, and ROS Signaling After Autophagy Suppression

Protein phosphatases and kinases, respectively, remove and add phosphate groups from target proteins,^11,12 including eNOS.^13–17 Vascular PP2A (protein phosphatase 2A) activity is increased in pathologies associated with suppressed p-eNOS^S1177 and compromised endothelial function.^16,18–20 We observed no evidence that shear stress altered PP2A activation in the absence or presence of Atg3 siRNA.

To determine whether disrupted kinase signaling to eNOS might compromise NO synthesis secondary to repressed autophagy, we assessed p-Akt^S473, p-Akt^T308, p-ERK^1/2 (phosphorylated extracellular signal regulated kinase 1/2), p-p38MAPK (phosphorylated p38 mitogen-activated protein kinase), and p-AMPK^T172 (phosphorylated 5′ AMP-activated protein kinase) ± Atg3 siRNA ± 180 minutes of shear stress (Figure IVA–IVF in the online-only Data Supplement). Activating phosphorylation was not different in response to shear stress ± autophagy repression, and we concluded that altered signaling from these kinases to eNOS is not responsible for compromised EC NO generation. It was surprising to us that impairment of autophagy per se elevated p-AMPK^T172 (Figure VE in the online-only Data Supplement) and downstream targets (not shown), and this was pursued (see below).

Exaggerated ROS production after Atg3 siRNA (Figure IVA in the online-only Data Supplement) has potential to decrease the synthesis of NO or increase the destruction of NO, and this was examined. Regarding NO synthesis, MAP kinases, such as p38-MAPK and ERK (extracellular signal regulated kinase), are phosphorylated/stimulated by nicotinamide adenine dinucleotide phosphate–generated ROS in vessels from mice with type 2 diabetes mellitus, which concurrently exhibit reduced p-eNOS^S1177 and impaired vasorelaxation.²¹ However, shear-induced p-ERK^1/2 and p-p38MAPK were not altered by autophagy compromise (Figure VC and VD in the online-only Data Supplement). Furthermore, pharmacological inhibition of mitogen-activated protein kinase using PD98059 (Figure VC and VF in the online-only Data Supplement) and ERK^1/2 using FR180204 (not shown) did not restore blunted shear-induced p-eNOS^S1177 (Figure VF in the online-only Data Supplement) after autophagy suppression.

Amplified shear-induced ROS production after Atg3 siRNA has potential to precipitate NO destruction, and this was investigated. After its formation, O₂^•− reacts rapidly with NO to form peroxynitrite (ONOO⁻).²² ONOO⁻ causes tyrosine nitration. Nitrotyrosine (ie, 3-NT) is an established estimate for ONOO⁻ formation.²² 3-NT formation assessed via immunoblot (Figure VIA and VIB in the online-only Data Supplement) or ELISA (not shown) was not greater in ECs exposed to shear stress after autophagy compromise. To strengthen these findings, specific sources of ROS were inhibited in an attempt to restore shear-induced p-eNOS^S1177 and NO generation in ECs after autophagy suppression. Mitochondria were targeted first because mitophagy was repressed after Atg3 siRNA (Figure 1D and 1F; Figure IC and ID in the online-only Data Supplement), and it is not unreasonable to hypothesize that accumulation of these organelles might provide a potent source for ROS production. Even though mito-tempo was efficacious with regard to attenuating shear-induced ROS accumulation in ECs with compromised autophagy (Figure VIC in the online-only Data Supplement), a restoration of p-eNOS^S1177 and NO generation was not observed (Figure VID and VIE in the online-only Data Supplement). Likewise, while the intracellular ROS scavenger N-acetyl cysteine prevented ROS accumulation in the context of autophagy repression (Figure VIF in the online-only Data Supplement), neither p-eNOS^S1177 nor NO generation was preserved in ECs exposed to shear stress (Figure VIG and VIH in the online-only Data Supplement). Neither mito-tempo (Figure VIIA in the online-only Data Supplement) nor N-acetyl cysteine (Figure VIIB in the online-only Data Supplement) altered shear-induced autophagy ± Atg3 siRNA. A similar pattern of results concerning ROS and p-eNOS^S1177 was recapitulated with the intracellular O₂^•− scavenger tiron, the complex IV inhibitor potassium cyanide, and the nicotinamide adenine dinucleotide phosphate oxidase inhibitor N-vanillynonanamide^23,24 (not shown). The ability of 2′,7′-dichlorofluorescin diacetate fluorescence to detect ROS and results concerning cell viability ± the various antioxidant treatments are shown in Figure VIIC and VIID in the online-only Data Supplement, respectively.

Autophagy Suppression Compromises Glycolysis in ECs

Elevated p-AMPK^T172 in ECs after autophagy compromise (Figure VE in the online-only Data Supplement) suggests an energy stress. ECs derive ATP primarily from glycolysis.^25,26 We examined whether autophagy suppression evokes an energetic deficit in ECs. When ECs transfected with scrambled siRNA were exposed to shear stress, they displayed the expected increase in GLUT1 (glucose transporter 1) expression—the main glucose transporter in ECs—^3H-deoxyglucose uptake, and cellular ATP production. Relative to control ECs, each response was prevented in ECs transfected with Atg3 (Figure 2A–2C) or Atg5 siRNA (not shown). Because these data suggested that autophagy compromise impairs EC glycolysis, we measured the extracellular acidification rate (ECAR; a surrogate measure of lactic acid production) using the XF24 Seahorse Bioanalyzer.²⁷ Compared with ECs exposed to no shear, shear stress increased ECAR in ECs transfected with scrambled but not Atg3 siRNA, and results were similar under basal and maximal (ie, +carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone) conditions (Figure VIII in the online-only Data Supplement). These findings motivated us to complete a glycolysis stress test, that is, ECAR was assessed in ECs challenged with shear stress ± autophagy suppression under the following conditions: 0 mmol/L glucose, 5 mmol/L glucose (to stimulate glycolysis, lactate production, and increase ECAR), 1 µmol/L oligomycin (to inhibit mitochondrial ATP production), and 50 mmol/L 2-deoxyglucose (to inhibit glycolysis; Figure 2D). Basal, glucose-stimulated, and oligomycin-stimulated ECAR was suppressed after Atg3 siRNA. As expected, 2-deoxyglucose inhibited ECAR in ECs transfected with scrambled but not Atg3 siRNA. These findings collectively indicate that EC autophagy suppression impairs EC glycolysis.

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Figure 2.

Genetic disruption of Atg3 (autophagocytosis-associated protein 3) impairs endothelial cell (EC) glycolysis and nitric oxide (NO) generation. Relative to static conditions, shear stress increased GLUT1 (glucose transporter 1) protein expression (A), glucose uptake (B), ATP production (C and G), p-eNOS^S1177 (H), and NO generation (I) in ECs transfected with scrambled small interfering RNA (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 and 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 mmol/L glucose (bar 1 vs 3) and 1 µmol/L 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 on treatment with 50 mmol/L 2-DG (2-deoxy D glucose) 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 ie, 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 and F, *P<0.05 vs (−shear; −Atg3 siRNA); #P<0.05 vs (+shear; −Atg3 siRNA). After cotransfection 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). DAF indicates diaminofluorescein; and eNOS, endothelial nitric oxide synthase.

To determine whether impaired EC glycolysis after autophagy compromise might negate shear-induced NO generation, we first used a loss-of-function approach. ECs with ≈65% knockdown of GLUT1 via siRNA (Figure IXA in the online-only Data Supplement) exhibited depressed shear-induced ATP production (Figure 2E) and were refractory to shear-induced p-eNOS^S1177 (Figure 2F). Second, we used a gain-of-function approach. When ECs with suppressed autophagy were transfected with a plasmid vector that increased GLUT1 expression by ≈50% (Figure IXB in the online-only Data Supplement), shear-induced ATP production, p-eNOS^S1177, and NO generation (Figure 2G–2I) were restored. Cell viability and indices of autophagy, respectively, were not altered by GLUT1 siRNA (Figure IXC an IXD in the online-only Data Supplement) or GLUT1 overexpression (Figure IXE and IXF in the online-only Data Supplement).

Autophagy Suppression Impairs Purinergic-Mediated Activation of eNOS

It is established that ECs produce ATP in response to shear stress and that extracellular ATP/ADP signals via purinergic receptors to activate p-eNOS^S1177 and NO generation.^28,29 As would be predicted from cellular ATP results shown in Figure 2C, 2E, and 2G, shear-induced extracellular ATP accumulation was robust in ECs transfected with scrambled but not Atg3 siRNA (Figure 3A), prevented in control ECs transfected with GLUT1 siRNA (Figure 3B), and restored by GLUT1 overexpression in ECs with compromised autophagy (Figure 3C).

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Figure 3.

Genetic disruption of Atg3 (autophagocytosis-associated protein 3) limits purinergic-mediated activation of eNOS (endothelial nitric oxide synthase). Relative to static conditions, shear stress increased extracellular ATP accumulation (A–C), p-eNOS^S1177 (D–G), and nitric oxide (NO) generation (H) in endothelial cells (ECs) transfected with scrambled small interfering RNA (siRNA; bar 1 vs 2) but not Atg3 siRNA (A, C, D, and E; bar 3 vs 4) or GLUT1 (glucose transporter 1) siRNA (B; bar 3 vs 4). After cotransfection 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 (P2Y purinoceptor 1 receptor) blocker MRS2179 (2′-deoxy-N⁶-methyladenosine 3′,5′-bisphosphate tetrasodium salt; 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-methylthio ADP restored shear-induced p-eNOS ^S1177 (G) and NO generation (H) in ECs with suppressed autophagy (bar 7 vs 8). For A–C and 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).

If limited extracellular ATP is responsible for suppressed shear-induced NO generation in ECs with compromised autophagy, then promoting extracellular ATP breakdown in ECs with intact autophagy or blocking the dominant purinergic receptor in ECs with intact autophagy³⁰ should independently recapitulate the phenotype displayed by ECs with impaired autophagy. Supporting this notion, shear stress did not increase p-eNOS^S1177 when ECs transfected with scrambled siRNA were treated concurrently with apyrase, an ectonucleotidase that hydrolyzes ATP to AMP and inorganic phosphate (Figure 3D), or MRS2179 (2′-deoxy-N⁶-methyladenosine 3′,5′-bisphosphate tetrasodium salt), a P2Y1 (P2Y purinoceptor 1) receptor blocker (Figure 3E).³¹ A genetic approach was used to strengthen these findings. When the P2Y1 receptor was silenced by ≈45% via siRNA in ECs with intact autophagy (Figure XA in the online-only Data Supplement), shear stress was incapable of activating eNOS when compared with results from ECs transfected with scrambled siRNA (Figure 3F). Importantly, P2Y1 receptor expression was not different ± shear stress ± Atg3 siRNA (Figure XA in the online-only Data Supplement), and neither apyrase nor P2Y1-receptor siRNA altered LC3-II:GAPDH or cell viability (Figure XB–XD in the online-only Data Supplement).

To validate that limited extracellular ATP contributes importantly to preventing shear-induced NO generation in ECs with compromised autophagy, we provided exogenous ADP to the cellular milieu in an attempt to restore NO production. ADP rescued shear-induced p-eNOS^S1177 (Figure 3G) and NO generation (Figure 3H) in ECs with suppressed autophagy, and neither LC3-II:GAPDH (Figure XE in the online-only Data Supplement) nor cell viability (Figure XD in the online-only Data Supplement) was altered by this treatment.

Autophagy Suppression Impairs Purinergic-Mediated Activation of eNOS via PKCδ^T505

To this point, our data indicate that when autophagy is suppressed in ECs, a glycolytic defect limits shear-induced ATP production to an extent that purinergic-mediated activation of eNOS is compromised. We sought to identify the link between P2Y1 activation and eNOS phosphorylation. An exploration of the literature revealed that extracellular nucleotide-mediated activation of p-eNOS^S1177 in human umbilical vein ECs exposed to shear stress occurs via p-PKCδ^T505 (phosphorylated protein kinase C delta T505).³⁰ Because we did not assess this kinase originally, historical cellular homogenates treated ± shear ± Atg3 siRNA were retrieved, re-examined, and results indicated that shear-induced p-PKCδ^T505 was prevented in ECs with suppressed autophagy (Figure 4A). Confirming the importance of this kinase in the context of the current experimental conditions, eNOS activation was refractory to shear stress in ECs transfected with PKCδ versus scrambled siRNA (Figure 4B; producing ≈40% reduction in PKCδ gene expression, Figure XIA in the online-only Data Supplement) and in ECs with intact autophagy treated with the PKCδ inhibitor rottlerin (not shown).

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Figure 4.

Defective purinergic-mediated p-PKCδ^T505 (phosphorylated protein kinase C delta T505) activation of eNOS (endothelial nitric oxide synthase) after autophagy compromise is normalized by genetic and pharmacological approaches. Relative to static conditions, shear stress increased p-PKCδ^T505 (A, E, F, and G) and p-eNOS^S1177 (C, D, and H) in endothelial cells (ECs) transfected with scrambled small interfering RNA (siRNA; bar 1 vs 2) but not Atg3 (autophagocytosis-associated protein 3) 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 cotransfected 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, and G) could be recapitulated in ECs with intact Atg3 protein that were transfected with P2Y1-R (P2Y purinoceptor 1 receptor) 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, and 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).

Using a gain-of-function approach, when ECs with suppressed autophagy were transfected with constitutively active PKCδ and subsequently challenged with shear stress, p-eNOS^S1177 (Figure 4C) and NO generation (Figure XIB and XIC in the online-only Data Supplement) were restored, and indices of autophagy (Figure XID in the online-only Data Supplement) were not altered. Consistent with these findings, when ECs with suppressed autophagy were transfected with dominant-negative PKCδ and subsequently challenged with shear stress, p-eNOS^S1177 was not restored (Figure 4D), and indices of autophagy (Figure XIE in the online-only Data Supplement) were unaltered. Protein expression was ≈65% greater or ≈50% greater in ECs transfected with constitutively active or dominant-negative PKCδ, respectively (Figure XIF and XIG in the online-only Data Supplement). Representative EPR (electron paramagnetic resonance spectroscopy) traces and cell death for all treatments are shown in Figure XIIA–XIIC in the online-only Data Supplement.

Next, we confirmed that p-PKCδ^T505 is a downstream target of purinergic-mediated signaling in our system. Specifically, phosphorylation of this kinase was refractory to shear stress in ECs with intact autophagy after transfection with P2Y1-receptor versus scrambled siRNA (Figure 4E). Substantiating these findings, shear stress evoked robust p-PKCδ^T505 (and p-eNOS^S1177 and NO generation; Figure 3G and 3H) in ECs with repressed autophagy that were treated concurrently with exogenous ADP (Figure 4F).

Bryostatin-1 is a small molecule that binds with and directly activates PKCδ.^32–34 To determine whether pharmacological activation might preserve NO production in the context of suppressed autophagy, ECs ± Atg3 siRNA were treated ± shear stress ± bryostatin-1. Defective shear-induced PKCδ^T505, p-eNOS^S1177, and NO generation after autophagy compromise were restored by concurrent treatment with bryostatin-1 (Figure 4G–4I; Figure XIIIA and XIIIB in the online-only Data Supplement). Taken together, our findings indicate that impaired EC autophagy represses glycolysis to an extent that extracellular ATP accumulation is limited, and purinergic signaling via the P2Y1-R and PKCδ to eNOS is blunted.

Translation to Human ECs and Intact Mice

We determined whether major findings observed in bovine aortic endothelial cells (BAECs) might be observed in human cells. Human arterial ECs were treated ± 20 dyn/cm² for 3 hours ± 5 mmol/L 3-methyladenine. Shear stress increased Atg3:GAPDH, LC3-II:GAPDH, p62 degradation, GLUT1:GAPDH, p-PKCδ^T505: PKC, p-eNOS^S1177: eNOS, and NO generation in the absence but not the presence of autophagy inhibition (Figure 5A–5H). The 3-methyladenine did not alter cell viability (Figure XIVA–XIVC in the online-only Data Supplement). These data are congruent with results from BAECs and demonstrate strong proof of concept for translational relevance.

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Figure 5.

Pharmacological inhibition of autophagy impairs shear stress–induced nitric oxide (NO) generation in human arterial endothelial cells. Relative to static conditions, shear stress increased Atg3 (autophagocytosis-associated protein 3; A), LC3 (microtubule-associated protein light chain 3) II (B), GLUT1 (glucose transporter 1; D), p-PKCδ^T505 (phosphorylated protein kinase C delta T505; E) and p-eNOS^S1177 (phosphorylated endothelial NO synthase at its positive regulatory site S1117; F) protein expression, NO generation (G), and p62 degradation (C; bar 1 vs 2). All responses were prevented by concurrent treatment with 3-methyladenine (3-MA; 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; −3-MA); #P<0.05 vs (+shear; −3-MA). DAF (diaminofluorescein)-FM indicates 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; and DAPI, 4′,6-diamidino-2-phenylindole.

Next, we examined whether major findings from ECs studied in vitro could be recapitulated in mice with compromised EC autophagy (Figure 6A–6D). Arterial ECs obtained from mice with tamoxifen-inducible deletion of Atg3 specifically in ECs (iecAtg3KO mice) exhibit the anticipated reduction in Atg3 protein expression and LC3-II:GAPDH, concurrent with suppressed GLUT1:GAPDH and p-eNOS^S1177:eNOS, versus results obtained from wild-type controls (Figure 6E–6H). Thus, several key nodes of dysregulation observed in BAECs and human arterial ECs studied in vitro after genetic and pharmacological repression of autophagy are recapitulated in vivo in mice with inducible disruption of EC autophagy. Studies are ongoing to determine the physiological and pathophysiological relevances of these findings.

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Figure 6.

iecAtg3KO mice exhibit impaired endothelial cell autophagy, GLUT1 (glucose transporter 1) protein expression, and p-eNOS (phosphorylated endothelial nitric oxide synthase) protein expression vs wild-type (WT) mice. To assess mRNA, endothelial cells (ECs) and media+adventitia were isolated from iliac arteries of 5-mo-old iecAtg3KO and WT mice treated 30 d earlier with tamoxifen. Purity of the EC and media+adventitia fraction was confirmed by PECAM (platelet endothelial cell adhesion molecule-1; A) and α-SMA (smooth muscle actin; B) staining, respectively. Atg3 (autophagocytosis-associated protein 3)/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-mo-old mice treated 30 d earlier with tamoxifen. Atg3:GAPDH (E), LC3 (microtubule-associated protein light chain 3) 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. KO indicates knockout.