Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown.

NADPH oxidases (NOX) constitute the main reactive oxygen species (ROS) source in blood vessels. An oxidative stress situation due to ROS overproduction can lead into endothelial dysfunction, a molecular mechanism that precedes cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, and stroke. NOX5 is the last discovered member of the NOX family, studied in a lesser extent due to its absence in the rodent genome. Our objective was to describe the phenotypic alterations produced by an oxidative stress situation derived from NOX5 overexpression in an endothelial in vitro model. The in vitro model consists of the hCMEC/D3 cell line, derived from brain microvascular endothelium, infected with a recombinant NOX5-β adenovirus. After an initial proteomic analysis, three phenotypic alterations detected in silico were studied: cell proliferation and apoptosis, general and mitochondrial metabolism, and migration capacity. NOX5 infection of hCMEC/D3 generates a functional protein and an increase in ROS production. This model produced changes in the whole cell proteome. The in silico analysis together with in vitro validations demonstrated that NOX5 overexpression inhibits proliferation and promotes apoptosis, metabolic alterations and cell migration in hCMEC/D3 cells. NOX5 overexpression in endothelial cells leads to phenotypic changes that can lead to endothelial dysfunction, the onset of atherosclerosis, myocardial infarction, and stroke.

Oxidative stress is a main molecular mechanism that underlies cardiovascular diseases. A close relationship between reactive oxygen species (ROS) derived from NADPH oxidase (NOX) activity and the prostaglandin (PG) biosynthesis pathway has been described. However, little information is available about the interaction between NOX5 homolog-derived ROS and the PG pathway in the cardiovascular context. Our main goal was to characterize NOX5-derived ROS effects in PG homeostasis and their potential relevance in cardiovascular pathologies. For that purpose, two experimental systems were employed: an adenoviral NOX5-β overexpression model in immortalized human aortic endothelial cells (TeloHAEC) and a chronic infarction in vivo model developed from a conditional endothelial NOX5 knock-in mouse. NOX5 increased cyclooxygenase-2 isoform (COX-2) expression and prostaglandin E₂ (PGE₂) production through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in TeloHAEC. Protein kinase C (PKC) activation and intracellular calcium level (Ca⁺⁺) mobilization increased ROS production and NOX5 overexpression, which promoted a COX-2/PGE₂ response in vitro. In the chronic infarction model, mice encoding endothelial NOX5 enhanced the cardiac mRNA expression of COX-2 and PGES, suggesting a COX-2/PGE₂ response to NOX5 presence in an ischemic situation. Our data support that NOX5-derived ROS may modulate the COX-2/PGE₂ axis in endothelial cells, which might play a relevant role in the pathophysiology of heart infarction.

Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.

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.

Alzheimer's disease (AD) is the most common form of dementia, characterized by an early olfactory dysfunction, progressive memory loss, and behavioral deterioration. Albeit substantial progress has been made in characterizing AD‐associated molecular and cellular events, there is an unmet clinical need for new therapies. In this study, olfactory tract proteotyping performed in controls and AD subjects (n = 17/group) showed a Braak stage‐dependent proteostatic impairment accompanied by the progressive modulation of amyloid precursor protein and tau functional interactomes. To implement a computational repurposing of drug candidates with the capacity to reverse early AD‐related olfactory omics signatures (OMSs), we generated a consensual OMSs database compiling differential omics datasets obtained by mass‐spectrometry or RNA‐sequencing derived from initial AD across the olfactory axis. Using the Connectivity Map‐based drug repurposing approach, PKC, EGFR, Aurora kinase, Glycogen synthase kinase, and CDK inhibitors were the top pharmacologic classes capable to restore multiple OMSs, whereas compounds with targeted activity to inhibit PI3K, Insulin‐like growth factor 1 (IGF‐1), microtubules, and Polo‐like kinase (PLK) represented a family of drugs with detrimental potential to induce olfactory AD‐associated gene expression changes. To validate the potential therapeutic effects of the proposed drugs, in vitro assays were performed. These validation experiments revealed that pretreatment of human neuron‐like SH‐SY5Y cells with the EGFR inhibitor AG‐1478 showed a neuroprotective effect against hydrogen peroxide‐induced damage while the pretreatment with the Aurora kinase inhibitor Reversine reduced amyloid‐beta (Aβ)‐induced neurotoxicity. Taken together, our data pointed out that OMSs may be useful as substrates for drug repurposing to propose novel neuroprotective treatments against AD.