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. 2015 Nov 20;23(15):1207-19.
doi: 10.1089/ars.2014.5896. Epub 2015 Jun 29.

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis

Rongsong Li  1 , Nelson Jen  2 , Lan Wu  1 , Juhyun Lee  2 , Karen Fang  1 , Katherine Quigley  1 , Katherine Lee  1 , Sky Wang  1 , Bill Zhou  1 , Laurent Vergnes  3 , Yun-Ru Chen  4 , Zhaoping Li  5 , Karen Reue  3 , David K Ann  4 , Tzung K Hsiai  1   2   5
Affiliations

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis

Rongsong Li et al. Antioxid Redox Signal. .

Abstract

Aim: Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO2(•-)) production.

Results: In the disturbed flow or oscillatory shear stress (OSS)-exposed aortic arch, we observed prominent staining of p62, a reverse marker of autophagic flux, whereas in the pulsatile shear stress (PSS)-exposed descending aorta, p62 was attenuated. OSS significantly increased (i) microtubule-associated protein light chain 3 (LC3) II to I ratios in human aortic endothelial cells, (ii) autophagosome formation as quantified by green fluorescent protein (GFP)-LC3 dots per cell, and (iii) p62 protein levels, whereas manganese superoxide dismutase (MnSOD) overexpression by recombinant adenovirus, N-acetyl cysteine treatment, or c-Jun N-terminal kinase (JNK) inhibition reduced OSS-mediated LC3-II/LC3-I ratios and mitochondrial DNA damage. Introducing bafilomycin to Earle's balanced salt solution or to OSS condition incrementally increased both LC3-II/LC3-I ratios and p62 levels, implicating impaired autophagic flux. In the OSS-exposed aortic arch, both anti-phospho-JNK and anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) staining for DNA damage were prominent, whereas in the PSS-exposed descending aorta, the staining was nearly absent. Knockdown of ATG5 with siRNA increased OSS-mediated mtO2(•-), whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO2(•-), mitochondrial respiration, and complex II activity.

Innovation: Disturbed flow-mediated oxidative stress and JNK activation induce autophagy.

Conclusion: OSS impairs autophagic flux to interfere with mitochondrial homeostasis. Antioxid. Redox Signal. 23, 1207-1219.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Spatial variations in shear stress differentially increased p62 accumulation in rabbit aorta. (A) Cross section in rabbit aorta highlights instantaneous spatial variations in WSS in the aortic arch versus descending aorta during systole. The instantaneous WSS is low in the lesser curvature of aortic arch, but is circumferentially high in the descending aorta. Immunohistochemistry reveals more prominent p62 staining in the lesser curvature of aortic arch (B) where ECs were exposed to disturbed flow, including OSS, when compared with the descending aorta (C) where ECs were exposed to PSS. ECs, endothelial cells; OSS, oscillatory shear stress; PSS, pulsatile shear stress; WSS, wall shear stress. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
Temporal variations in shear stress (OSS vs. PSS) differentially induced autophagy in HAECs. (A) HAECs were exposed to OSS versus PSS for 4 h. Autophagy was assessed by normalizing the ratios of LC3-II to LC3-I with the static condition and comparing p62 levels by Western blotting. OSS significantly increased the LC3-II/LC3-I ratios (p<0.001, n=5) when compared with static condition and PSS, whereas PSS minimally increased LC3 ratios (p<0.05, n=5) in comparison with static condition. OSS also increased protein levels of p62 against static condition and PSS (p<0.01, n=4), whereas there was no significant change between static condition and PSS. (B) HAECs were infected with recombinant LC3-GFP adenovirus (1:20) overnight, and then were exposed to OSS versus PSS for 4 h. LC3-GFP puncta or dots/cell (arrows) indicated that OSS significantly increased the number of autophagosomes in comparison with static control (p<0.01, n=3) similarly to EBSS-induced autophagy (p<0.01, n=3). PSS only minimally increased LC3-GFP puncta versus static condition (p<0.05, n=3). (C) HAECs were treated with either OSS or EBSS in the presence or absence of 1 nM of bafilomycin (Baf). While EBSS increased LC3 ratios (p<0.05, n=6) and decreased p62 levels (p<0.05, n=6) over static condition, bafilomycin treatment significantly increased LC3 ratios and p62 to a lesser extent in OSS-exposed cells (LC3: p<0.05, n=6; p62: p<0.01, n=6) than EBSS-treated cells (LC3: p<0.001, n=6; p62: p<0.001, n=6). EBSS, Earle's balanced salt solution; GFP, green fluorescent protein; HAEC, human aortic endothelial cell; LC3, microtubule-associated protein light chain 3. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
OSS induced oxidative stress and JNK activation to activate autophagy. (A) HAECs were infected with control or recombinant MnSOD adenovirus (MOI 1:100) overnight, and then were exposed to static condition or OSS for 4 h. Overexpression of MnSOD significantly attenuated OSS-induced LC3-II/LC3-I ratios (OSS: p<0.05, n=4; MnSOD: p<0.05, n=4). (B) HAECs were exposed to static condition or OSS for 4 h in the presence or absence of NAC at 5 mM or (C) a JNK inhibitor, SP600125 (SP), at 2 μM. Both antioxidant NAC and JNK inhibitor significantly attenuated OSS-induced LC3-II/LC3-I ratios (NAC: p<0.01; SP: p<0.05, n=4). JNK, c-Jun N2-terminal kinase; MnSOD, manganese superoxide dismutase; MOI, multiplicity of infection; NAC, N-acetyl cysteine.
<b>FIG. 4.</b>
FIG. 4.
EBSS-activated autophagy mitigated OSS-induced mitochondrial O2•− production. (A) HAECs were exposed to OSS in DMEM/1% FBS (control) or in EBSS for 4 h. MtO2•− production was measured by the MitoSox Red fluorescent intensity. EBSS attenuated OSS-induced MitoSox Red intensity (p<0.05, n=4). (B) HAECs were transfected with scrambled siRNA or ATG5 siRNA for 48 h, and then were exposed to static condition or OSS. Knockdown of ATG5 augmented OSS-induced MitoSox Red intensity (p=0.059, n=4). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 5.</b>
FIG. 5.
EBSS-activated autophagy restored OSS-mediated reduction in mitochondrial respiration. HAECs were exposed to OSS in DMEM/1% FBS (control) or in EBSS for 4 h. (A) Mitochondrial respiration was assessed by Seahorse Flux Analyzer. EBSS restored OSS-mediated reduction in mitochondrial respiration (OSS: p<0.01, n=4; EBSS: p<0.01, n=4). (B) EBSS restored OSS-mediated reduction in mitochondrial complex II activity (p<0.05, n=4).
<b>FIG. 6.</b>
FIG. 6.
OSS induced mtDNA damage. (A) Immunofluorescent staining revealed mtDNA damage. HAECs exposed to OSS (4 h) developed a prominent intensity for anti 8-OHdG (green) as an indicator for DNA damage, which colocalized with anti-cytochrome C (red) for mitochondria. DAPI (blue) stained for nuclei. (B) HAECs were exposed to OSS versus PSS for 4 h, and mtDNA damage was measured by quantitative PCR. OSS induced a 4.5-fold increase in mtDNA damage (p<0.001, n=4), and PSS induced an insignificant increase (p>0.05, n=3). (C) OSS induces negligible nuclear DNA damage. Tail moment analysis of Comet assay (CometScore software) revealed an insignificant difference in nuclear DNA damage between static and OSS conditions (p=0.9, n=3). 8-OHdG, 8-hydroxy-2′-deoxyguanosine; mtDNA, mitochondrial DNA. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 7.</b>
FIG. 7.
Antioxidants and JNK inhibitor reversed OSS-induced mtDNA damage. (A) HAECs were infected with control or recombinant MnSOD adenovirus (MOI 1:100) overnight, and then were exposed to OSS for 4 h. MnSOD overexpression significantly reversed OSS-induced mtDNA damage (p<0.05, n=4). (B) HAECs were exposed to static condition or OSS for 4 h in the presence or absence of 5 mM of antioxidant (NAC) or (C) 10 μM of JNK inhibitor, SP600125 (SP). Both NAC and SP significantly reversed OSS-induced mtDNA damage (NAC: p<0.05; SP: p<0.01, n=4).
<b>FIG. 8.</b>
FIG. 8.
Spatial variations in shear stress differentially promoted DNA damage and JNK activation in New Zealand White rabbits. Cross sections of the aortic arch versus descending aorta were stained with anti-8-OHdG for DNA damage and anti-phospho-JNK for JNK activation. Prominent anti-8-OHdG staining was observed in the aortic arch, but nearly absent in the descending aorta. Phospho-JNK staining was prominent in the aortic arch, but nearly absent in the descending aorta. The red squares denote enlarged areas (100× magnification). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 9.</b>
FIG. 9.
Scheme of OSS modulation of autophagy. OSS-induced oxidative stress activates JNK, which plays a dual role in inducing mitochondrial dysfunction and initiating autophagy. However, sustained OSS impairs autophagic flux, leading to accumulation of p62, increased mtO2•− production, and mtDNA damage. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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