Role of Oxidative Stress and Autophagy in Thoracic Aortic Aneurysms

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SUMMARY
Thoracic aortic aneurysms (TAA) pathogenesis and progression include many mechanisms. The authors investigated the role of autophagy, oxidative stress, and endothelial dysfunction in 36 TAA patients and 23 control patients. Univariable and multivariable analyses were performed. TAA patients displayed higher oxidative stress and endothelial dysfunction then control patients. Autophagy in the TAA group was reduced. The association of oxidative stress and autophagy with aortic disease supports the role of these processes in TAA. The authors demonstrate a putative role of Nox2 and autophagy dysregulation in human TAA. These findings could pinpoint novel treatment targets to prevent or limit TAA progression. A ortic aneurysm is a segmental, fullthickness dilatation of the aorta, exceeding the normal vessel diameter by 50%, which represents the second most frequent aortic disease after atherosclerosis. Aortic aneurysm is often considered a "silent killer" due to its natural predisposition to dissection and/or sudden rupture (1). Formation of aortic aneurysmal disease is multifactorial and involves inflammation (2); infiltration of aortic wall by lymphocytes and macrophages (3); destruction of elastin and collagen in the media and adventitia by proteases, including matrix metalloproteinases (4)(5)(6); loss of smooth muscle cells with weakening of the media (7); and neovascularization (8). More recently, endothelial derangement, which is characterized by endothelial dysfunction and oxidative stress, has emerged as key contributor to thoracic aortic aneurysms (TAA). Nox2, which is the main isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and its role in the development of TAA still remains to be fully clarified.
Moreover, emerging data have recently suggested a possible role of autophagy in development and progression of aortic aneurysm. Autophagy is an intracellular process that is in charge for the degradation of senescent or dysfunctional cellular components through their sequestration by vesicles called autophagosomes and their subsequent degradation in lysosomes. Autophagy machinery is a complicated network of autophagy-related (ATG) proteins, which are involved in the induction and formation of autophagosomes. Autophagy is implicated in the regulation of endothelial homeostasis and function, also by limiting oxidative stress (9). Few studies have investigated the possible association of autophagy with aortic aneurysm, with none specifically focused to date on TAA (10)(11)(12).
Autophagy-related genes were found to be deregulated in human abdominal aneurysmatic tissues, but it remains unclear whether the autophagic process is inhibited or activated, particularly in TAA (13). Activation of autophagy is beneficial for cells in response to stress, including endothelial cells. Autophagy was found to preserve endothelial function through enhanced mitochondrial quality control, attenuation of endoplasmic reticulum stress, reactive oxygen species (ROS) production and inflammatory signaling, and increased nitric oxide (NO) bioavailability.
However, the interplay among autophagy, Nox2, oxidative stress, and TAA still remains to be elucidated. We hypothesized that oxidative-redox balance and autophagy are deranged in patients with TAA and are associated with TAA formation and progression.

METHODS
STUDY DESIGN. We conducted our study using a case-control design. Specifically, we included 36 patients with TAA and an indication for elective surgical treatment. Twenty-three control patients were also included, using as the selection criterion aortic stenosis/regurgitation with indication for elective surgery in the absence of TAA. All performed procedures were in accordance with international guidelines. Aortic tissue was immediately collected after surgical excision and stored at À80 C. Subsequently, the tissue was homogenized, and supernatant collected for biochemical analysis.

This study was approved by the Medical Ethics
SERUM AND TISSUE sNox2-dp RELEASE. Nox2 activation was measured as soluble Nox2-derived peptide (sNox2-dp) with an enzyme-linked immunosorbent assay method as previously reported (14). Briefly, the peptide is recognized by binding to a specific monoclonal antibody against the amino acid sequence (224-268) of the external portion of Nox2, which was released following platelet activation. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm. Values were expressed as picograms per milliliter; intra-assay and interassay coefficients of variation were 8.95% and 9.01%, respectively.    Table 3, Figures 1C and 1D).  Table 3, Figure 1E).  Table 3, Figure 2A). To examine whether the solubility of p62 was altered in TAA group, cellular lysates were fractionated into Triton X-100-soluble and -insoluble fractions. The   (Figures 2B and 2C). Moreover, we quantified the gene expression of p62 in tissue obtained from TAA patients and control patients. The results showed no significant differences in p62 mRNA expression in TAA tissue as compared with control ( Figure 2D).  Table 3, Figures 2E   and 2F).
To further explore the autophagic flux, we examined other ATG proteins. Results showed in the same TAA group a significant reduction of ATG5 plasma levels (P ¼ 0.003) (Supplemental Table 3, Figure 3A).  Table 3, Figure 4).    Overall, these findings indicate that increased Nox2 activity and oxidative stress are associated with autophagy inhibition.
MULTIVARIABLE ANALYSIS. Multivariable analysis was performed to explore for potential moderators of oxidation and autophagy features, confirming the significant and independent association between group (TAA vs control) assignment and sNox2, tissue Nox2, hydrogen peroxide, serum NO, tissue NO, as well as LC3/Actin, even adjusting for clinical, laboratory, and imaging features (Supplemental Table 5).

DISCUSSION
The present study provides the first evidence to our knowledge that patients with TAA have decreased autophagy. This molecular derangement was associated with increased oxidative stress and activation of Nox2, a major source of oxidative stress in the vasculature.
Many vascular diseases are characterized by increased production of ROS in the vessel wall, which leads to reduction NO bioavailability and endothelial dysfunction (17). Moreover, Nox2 activation was also found to be involved in endothelial dysfunction and vascular damage in response to hypertension, diabetes, and aging (18)(19)(20)