Pharmacological Inhibition of Lysine-Specific Demethylase 1A Reduces Atherosclerotic Lesion Formation in Apolipoprotein E-Deficient Mice by a Mechanism Involving Decreased Oxidative Stress and Inflammation; Potential Implications in Human Atherosclerosis.

Dysregulated epigenetic mechanisms promote transcriptomic and phenotypic alterations in cardiovascular diseases. The role of histone methylation-related pathways in atherosclerosis is largely unknown. We hypothesize that lysine-specific demethylase 1A (LSD1/KDM1A) regulates key molecular effectors and pathways linked to atherosclerotic plaque formation. Human non-atherosclerotic and atherosclerotic tissue specimens, ApoE-/- mice, and in vitro polarized macrophages (Mac) were examined. Male ApoE-/- mice fed a normal/atherogenic diet were randomized to receive GSK2879552, a highly specific LSD1 inhibitor, or its vehicle, for 4 weeks. The mRNA and protein expression levels of LSD1/KDM1A were significantly elevated in atherosclerotic human carotid arteries, atherosclerotic aortas of ApoE-/- mice, and M1-Mac. Treatment of ApoE-/- mice with GSK2879552 significantly reduced the extent of atherosclerotic lesions and the aortic expression of NADPH oxidase subunits (Nox1/2/4, p22phox) and 4-hydroxynonenal-protein adducts. Concomitantly, the markers of immune cell infiltration and vascular inflammation were significantly decreased. LSD1 blockade down-regulated the expression of genes associated with Mac pro-inflammatory phenotype. Nox subunit transcript levels were significantly elevated in HEK293 reporter cells overexpressing LSD1. In experimental atherosclerosis, LSD1 mediates the up-regulation of molecular effectors connected to oxidative stress and inflammation. Together, these data indicate that LSD1-pharmacological interventions are novel targets for supportive therapeutic strategies in atherosclerosis.

Activated Histone Acetyltransferase p300/CBP-Related Signalling Pathways Mediate Up-Regulation of NADPH Oxidase, Inflammation, and Fibrosis in Diabetic Kidney.

Accumulating evidence implicates the histone acetylation-based epigenetic mechanisms in the pathoetiology of diabetes-associated micro-/macrovascular complications. Diabetic kidney disease (DKD) is a progressive chronic inflammatory microvascular disorder ultimately leading to glomerulosclerosis and kidney failure. We hypothesized that histone acetyltransferase p300/CBP may be involved in mediating diabetes-accelerated renal damage. In this study, we aimed at investigating the potential role of p300/CBP in the up-regulation of renal NADPH oxidase (Nox), reactive oxygen species (ROS) production, inflammation, and fibrosis in diabetic mice. Diabetic C57BL/6J mice were randomized to receive 10 mg/kg C646, a selective p300/CBP inhibitor, or its vehicle for 4 weeks. We found that in the kidney of C646-treated diabetic mice, the level of H3K27ac, an epigenetic mark of active gene expression, was significantly reduced. Pharmacological inhibition of p300/CBP significantly down-regulated the diabetes-induced enhanced expression of Nox subtypes, pro-inflammatory, and pro-fibrotic molecules in the kidney of mice, and the glomerular ROS overproduction. Our study provides evidence that the activation of p300/CBP enhances ROS production, potentially generated by up-regulated Nox, inflammation, and the production of extracellular matrix proteins in the diabetic kidney. The data suggest that p300/CBP-pharmacological inhibitors may be attractive tools to modulate diabetes-associated pathological processes to efficiently reduce the burden of DKD.

Detection of Vascular Reactive Oxygen Species in Experimental Atherosclerosis by High-Resolution Near-Infrared Fluorescence Imaging Using VCAM-1-Targeted Liposomes Entrapping a Fluorogenic Redox-Sensitive Probe.

Excessive production of reactive oxygen species (ROS) and the ensuing oxidative stress are instrumental in all phases of atherosclerosis. Despite the major achievements in understanding the regulatory pathways and molecular sources of ROS in the vasculature, the specific detection and quantification of ROS in experimental models of disease remain a challenge. We aimed to develop a reliable and straightforward imaging procedure to interrogate the ROS overproduction in the vasculature and in various organs/tissues in atherosclerosis. To this purpose, the cell-impermeant ROS Brite™ 700 (RB700) probe that produces bright near-infrared fluorescence upon ROS oxidation was encapsulated into VCAM-1-targeted, sterically stabilized liposomes (VLp). Cultured human endothelial cells (EC) and macrophages (Mac) were used for in vitro experiments. C57BL6/J and ApoE-/- mice were randomized to receive normal or high-fat, cholesterol-rich diet for 10 or 32 weeks. The mice received a retroorbital injection with fluorescent tagged VLp incorporating RB700 (VLp-RB700). After two hours, the specific signals of the oxidized RB700 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DSPE), inserted into liposome bilayers, were measured ex vivo in the mouse aorta and various organs by high-resolution fluorescent imaging. VLp-RB700 was efficiently taken up by cultured human EC and Mac, as confirmed by fluorescence microscopy and spectrofluorimetry. After systemic administration in atherosclerotic ApoE-/- mice, VLp-RB700 were efficiently concentrated at the sites of aortic lesions, as indicated by the augmented NBD fluorescence. Significant increases in oxidized RB700 signal were detected in the aorta and in the liver and kidney of atherosclerotic ApoE-/- mice. RB700 encapsulation into sterically stabilized VCAM-1-sensitive Lp could be a novel strategy for the qualitative and quantitative detection of ROS in the vasculature and various organs and tissues in animal models of disease. The accurate and precise detection of ROS in experimental models of disease could ease the translation of the results to human pathologies.

Pharmacological inhibition of histone deacetylase reduces NADPH oxidase expression, oxidative stress and the progression of atherosclerotic lesions in hypercholesterolemic apolipoprotein E-deficient mice; potential implications for human atherosclerosis.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are instrumental in all inflammatory phases of atherosclerosis. Dysregulated histone deacetylase (HDAC)-related epigenetic pathways have been mechanistically linked to alterations in gene expression in experimental models of cardiovascular disorders. Hitherto, the relation between HDAC and Nox in atherosclerosis is not known. We aimed at uncovering whether HDAC plays a role in mediating Nox up-regulation, oxidative stress, inflammation, and atherosclerotic lesion progression. Human non-atherosclerotic and atherosclerotic arterial samples, ApoE-/- mice, and in vitro polarized monocyte-derived M1/M2-macrophages (Mac) were examined. Male ApoE-/- mice, maintained on normal or high-fat, cholesterol-rich diet, were randomized to receive 10 mg/kg suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, or its vehicle, for 4 weeks. In the human/animal studies, real-time PCR, Western blot, lipid staining, lucigenin-enhanced chemiluminescence assay, and enzyme-linked immunosorbent assay were employed. The protein levels of class I, class IIa, class IIb, and class IV HDAC isoenzymes were significantly elevated both in human atherosclerotic tissue samples and in atherosclerotic aorta of ApoE-/- mice. Treatment of ApoE-/- mice with SAHA reduced significantly the extent of atherosclerotic lesions, and the aortic expression of Nox subtypes, NADPH-stimulated ROS production, oxidative stress and pro-inflammatory markers. Significantly up-regulated HDAC and Nox subtypes were detected in inflammatory M1-Mac. In these cells, SAHA reduced the Nox1/2/4 transcript levels. Collectively, HDAC inhibition reduced atherosclerotic lesion progression in ApoE-/- mice, possibly by intertwined mechanisms involving negative regulation of Nox expression and inflammation. The data propose that HDAC-oriented pharmacological interventions could represent an effective therapeutic strategy in atherosclerosis.

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.