Open Access

Background and Purpose: Organic nitrates such as nitroglycerin (NTG) or pentaerythritol tetranitrate (PETN) have been used for over a century in the treatment of angina or ischaemic heart disease. These compounds are prodrugs which release their nitrovasodilators upon enzymic bioactivation by aldehyde dehydrogenase (ALDH2) or cytochromes P450 (CYP). Whereas ALDH2 is known to directly activate organic nitrates in vessels, the contribution of vascular CYPs is unknown and was studied here. Experimental Approach: As all CYPs depend on cytochrome P450 reductase (POR) as electron donor, we generated a smooth muscle cell-specific, inducible knockout mouse of POR (smcPOR−/−) to investigate the contribution of POR/CYP to vascular biotransformation of organic nitrates. Key Results: Microsomes containing recombinant CYPs expressed in human vascular tissues released nitrite from NTG and PETN with CYP2C9 and CYP2C8 being most efficient. SFK525, a CYP suicide inhibitor, blocked this effect. smcPOR−/− mice exhibited no obvious cardiovascular phenotype (normal cardiac weight and endothelium-dependent relaxation) and plasma and vascular nitrite production was similar to control (CTL) animals. NTG- and PETN-induced relaxation of isolated endothelium-intact or endothelium-denuded vessels were identical between CTL and smcPOR−/−. Likewise, nitrite release from organic nitrates in aortic rings was not affected by deletion of POR in smooth muscle cells (SMCs). In contrast, inhibition of ALDH2 by benomyl (10 μM) inhibited NTG-induced nitrite production and relaxation. Deletion of POR did not modulate this response. Conclusions and Implications: Our data suggest that metabolism by vascular CYPs does not contribute to the pharmacological function of organic nitrates.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR−/−). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR−/− mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.

The free radical theory of aging suggests reactive oxygen species as a main reason for accumulation of damage events eventually leading to aging. Nox4, a member of the family of NADPH oxidases constitutively produces ROS and therefore has the potential to be a main driver of aging. Herein we analyzed the life span of Nox4 deficient mice and found no difference when compared to their wildtype littermates. Accordingly neither Tert expression nor telomere length was different in cells isolated from those animals. In fact, Nox4 mRNA expression in lungs of wildtype mice dropped with age. We conclude that Nox4 has no influence on lifespan of healthy mice.

Endocannabinoids are important lipid-signaling mediators. Both protective and deleterious effects of endocannabinoids in the cardiovascular system have been reported but the mechanistic basis for these contradicting observations is unclear. We set out to identify anti-inflammatory mechanisms of endocannabinoids in the murine aorta and in human vascular smooth muscle cells (hVSMC). In response to combined stimulation with cytokines, IL-1β and TNFα, the murine aorta released several endocannabinoids, with anandamide (AEA) levels being the most significantly increased. AEA pretreatment had profound effects on cytokine-induced gene expression in hVSMC and murine aorta. As revealed by RNA-Seq analysis, the induction of a subset of 21 inflammatory target genes, including the important cytokine CCL2 was blocked by AEA. This effect was not mediated through AEA-dependent interference of the AP-1 or NF-κB pathways but rather through an epigenetic mechanism. In the presence of AEA, ATAC-Seq analysis and chromatin-immunoprecipitations revealed that CCL2 induction was blocked due to increased levels of H3K27me3 and a decrease of H3K27ac leading to compacted chromatin structure in the CCL2 promoter. These effects were mediated by recruitment of HDAC4 and the nuclear corepressor NCoR1 to the CCL2 promoter. This study therefore establishes a novel anti-inflammatory mechanism for the endogenous endocannabinoid AEA in vascular smooth muscle cells. Furthermore, this work provides a link between endogenous endocannabinoid signaling and epigenetic regulation.

Aim: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. Results: TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. Innovation & conclusion: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.