Induction of Cyclooxygenase-2 by Overexpression of the Human NADPH Oxidase 5 (NOX5) Gene in Aortic Endothelial Cells.

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-ß 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.

Histone Acetyltransferase-Dependent Pathways Mediate Upregulation of NADPH Oxidase 5 in Human Macrophages under Inflammatory Conditions: A Potential Mechanism of Reactive Oxygen Species Overproduction in Atherosclerosis.

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45+/CD68+ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

NOX5: Molecular biology and pathophysiology.

NEW FINDINGS: What is the topic of this review? This review provides a comprehensive overview of Nox5 from basic biology to human disease and highlights unique features of this Nox isoform What advances does it highlight? Major advances in Nox5 biology relate to crystallization of the molecule and new insights into the pathophysiological role of Nox5. Recent discoveries have unravelled the crystal structure of Nox5, the first Nox isoform to be crystalized. This provides new opportunities to develop drugs or small molecules targeted to Nox5 in an isoform-specific manner, possibly for therapeutic use. Moreover genome wide association studies (GWAS) identified Nox5 as a new blood pressure-associated gene and studies in mice expressing human Nox5 in a cell-specific manner have provided new information about the (patho) physiological role of Nox5 in the cardiovascular system and kidneys. Nox5 seems to be important in the regulation of vascular contraction and kidney function. In cardiovascular disease and diabetic nephropathy, Nox5 activity is increased and this is associated with increased production of reactive oxygen species and oxidative stress implicated in tissue damage. ABSTRACT: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), comprise seven family members (Nox1-Nox5 and dual oxidase 1 and 2) and are major producers of reactive oxygen species in mammalian cells. Reactive oxygen species are crucially involved in cell signalling and function. All Noxs share structural homology comprising six transmembrane domains with two haem-binding regions and an NADPH-binding region on the intracellular C-terminus, whereas their regulatory systems, mechanisms of activation and tissue distribution differ. This explains the diverse function of Noxs. Of the Noxs, NOX5 is unique in that rodents lack the gene, it is regulated by Ca2+ , it does not require NADPH oxidase subunits for its activation, and it is not glycosylated. NOX5 localizes in the perinuclear and endoplasmic reticulum regions of cells and traffics to the cell membrane upon activation. It is tightly regulated through numerous post-translational modifications and is activated by vasoactive agents, growth factors and pro-inflammatory cytokines. The exact pathophysiological significance of NOX5 remains unclear, but it seems to be important in the physiological regulation of sperm motility, vascular contraction and lymphocyte differentiation, and NOX5 hyperactivation has been implicated in cardiovascular disease, kidney injury and cancer. The field of NOX5 biology is still in its infancy, but with new insights into its biochemistry and cellular regulation, discovery of the NOX5 crystal structure and genome-wide association studies implicating NOX5 in disease, the time is now ripe to advance NOX5 research. This review provides a comprehensive overview of our current understanding of NOX5, from basic biology to human disease, and highlights the unique characteristics of this enigmatic Nox isoform.