NADPH Oxidase 5 Induces Changes in the Unfolded Protein Response in Human Aortic Endothelial Cells and in Endothelial-Specific Knock-in Mice

Abstract

Oxidative stress constitutes a key molecular mechanism in the development of cardiovascular diseases. A potential relationship between reactive oxygen species (ROS) driven by the NADPH oxidase family (NOX) and the unfolded protein response (UPR) has been postulated. Nevertheless, there is a lack of information about the crosstalk between NOX5 homologue and the UPR in a cardiovascular context. The main aim was to analyze NOX5-mediated ROS effects in the UPR and its importance in cardiovascular diseases. To this effect, we used an adenoviral NOX5-β overexpression model in human aortic endothelial cells (HAEC) and a conditional endothelial NOX5 knock-in mouse. Using expression arrays, we investigated NOX5-induced genomic changes in HAEC. Compared with the control HAEC, 298 genes were differentially expressed. Gene ontology analysis revealed the activation of numerous cellular routes, the most relevant being the UPR pathway. Using real-time PCR and Western Blot experiments, we confirmed that NOX5 overexpression induced changes in the expression of the UPR components, which were associated with increased apoptosis. Moreover, in endothelial-specific NOX5 knock-in mice, we found changes in the expression of the UPR components genes. In these mice, myocardial infarction was performed by permanent coronary artery ligation; however, NOX5 expression was not associated with differences in the UPR components mRNA levels. In these animals, we found significant associations between the UPR components gene expression and echocardiographic parameters. Our data support the idea that NOX5-derived ROS may modulate the UPR pathway in endothelial cells, which might play a relevant role in cardiac physiology.

NADPH Oxidase 5 (NOX5) Overexpression Promotes Endothelial Dysfunction via Cell Apoptosis, Migration, and Metabolic Alterations in Human Brain Microvascular Endothelial Cells (hCMEC/D3)

“…It works as a transition factor through cell cycle phases, the higher the E2f expression, the lower the proliferative stimuli that cells need to replicate [ 36 ]. In agreement with this, the reanalysis of a transcriptomic study performed by our group on HAEC infected with NOX5 adenovirus [ 19 ], shows that NOX5 promotes the upregulation of different E2f isoforms ( Supplementary Table S5 ). The E2f7 isoform, related with cells apoptosis and DNA damage [ 36 ], is the most overexpressed member.…”

“…NOX5 overexpression enhanced caspase 3/7 levels related with apoptosis, and decreased cell proliferation as it was observed in intact cells by xCELLigence. Our group previously reported that NOX5-β overexpression by this adenovirus in HAECs leads to apoptosis via UPR signaling [ 19 ]. Other groups have also described NOX5 as proapoptotic agent.…”

“…A solution containing the adequate adenovirus quantity was prepared in Vascular Cell Basal Medium (PCS-100-030, ATCC ® , Manassas, VA, USA) supplemented with 2% fetal bovine serum (26140079, Gibco TM ), 0.5% Streptomycin-penicillin (15140148, Gibco TM ), and 0.5% gentamycin (15750060, Gibco TM ). The cell medium was removed and this solution was added for 3 h until it was replaced with fresh medium for 9 h (12 h of infection), 21 h (24 h of infection), or 45 h (48 h of infection) as previously described by our group in transcriptomic array analysis of Human Aortic Endothelial Cells (PCS-100-011, ATCC ® ) [ 19 ].…”

Expression of Endothelial NOX5 Alters the Integrity of the Blood-Brain Barrier and Causes Loss of Memory in Aging Mice

“…NOX5 upregulation and activation is related with many different processes, including the unfolded protein response [ 39 ], the management of lipid accumulation and insulin sensitivity [ 40 ] or the inflammation via COX2 activation and PG pathway induction [ 22 ]. In this context, the COX2 pathway may lead to brain ROS accumulation during aging [ 41 ] or after stroke [ 42 ].…”

Oxidative Stress in Vascular Pathophysiology: Still Much to Learn

“…Cortes et al [ 13 ] study the potential crosstalk between NOX5-derived oxidative stress and the unfolded protein response (UPR) in endothelial cells and its impact on cardiovascular pathophysiology. In a transcriptomic study, gene ontology analysis revealed that NOX5 overexpression activates the UPR pathway.…”