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

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 (mtO 2 -) 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) microtubuleassociated 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 mtO 2 -, whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO 2 -, 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.

The arterial microenvironment: the where and why of atherosclerosis

“…In contrast, laminar flow reduces endothelial cell metabolism through Klf2-dependent repression of endothelial glycolytic enzymes [76] and promotes endothelial cell autophagy to protect endothelial cells from oxidant-induced cell injury [77, 78]. In contrast, oscillatory flow stimulates a JNK-dependent increase in mitochondrial superoxide production [79] but limits autophagy, allowing the accumulation of oxidant injury and markers of DNA damage (8-hydroxydeoxyguanosine) at sites of disturbed flow [78, 80]. Enhanced oxidant stress at sites of disturbed flow correlates with increased endothelial cell apoptosis [81], and disturbed flow promotes SUMOylation-dependent nuclear export of the apoptosis-regulating transcription factor p53, resulting in Bcl2 binding and induction of endothelial apoptosis [82].…”

Autophagy in vascular endothelial cells

“…The reason(s) for these discrepant observations is not clear, but may be related to different experimental settings. Interestingly, it has been noted that in the aortic arch, which is continuously exposed to turbulent or oscillatory shear stress, the level of the autophagy receptor protein p62 is upregulated, indicating an impaired autophagic flux . In cultured endothelial cells, although oscillatory shear stress significantly increases the abundance of LC3‐II and the number of autophagosome, these effects appear to be primarily due to blockade of the end stage autophagosome clearance, but not an enhanced induction of the autophagic response.…”

“…In cultured endothelial cells, although oscillatory shear stress significantly increases the abundance of LC3‐II and the number of autophagosome, these effects appear to be primarily due to blockade of the end stage autophagosome clearance, but not an enhanced induction of the autophagic response. In this case, the increased autophagosome accumulation to oscillatory shear stress is associated with increased oxidative stress, while further induction of autophagy by starvation or rapamycin normalises the disturbed redox homeostasis …”

Mitochondrial Ca 2+ transport in the endothelium: regulation by ions, redox signalling and mechanical forces

“…Dysfunctional autophagy occurs when autophagy induction (autophagosome formation) becomes highly activated and, as a result, the autophagic flux (rate of autophagosome-lysosome fusion and degradation of autophagosomal cargo) slows down ('impaired' flux) leading to inadequate removal of dysfunctional mitochondria and a tonic increase in oxidative stress. Li et al [204] showed that OS exposure increases the induction of autophagy, but decreases the autophagic flux in cultured HAECs (whereas PS maintains autophagy at levels comparable to static controls); both the increased induction and decreased flux under OS were mediated by JNK activation and elevated mROS. The impaired EC autophagic flux was verified in OS-exposed regions of rabbit aortas [204].…”

“…Li et al [204] showed that OS exposure increases the induction of autophagy, but decreases the autophagic flux in cultured HAECs (whereas PS maintains autophagy at levels comparable to static controls); both the increased induction and decreased flux under OS were mediated by JNK activation and elevated mROS. The impaired EC autophagic flux was verified in OS-exposed regions of rabbit aortas [204]. Graded inhibition of MCU activation in OS-exposed regions will reduce mROS, which, by lowering autophagy induction, should allow for, at least, partial normalization of the impaired flux, in agreement with a recent review [205].…”