Open Access

Melina López Sciarra

• Cytochrome P450 enzymes are a large superfamily of membrane-bound heme-containing monooxygenases. They are essential for the oxidative metabolism of endogenous substrates such as steroids and fatty acids, and biotransformation of xenobiotic substrates such as pollutants and drugs. Although the highest expression of CYPs is found in the liver, their cardiovascular expression is not negligible with CYP450 subfamilies being responsible for the production of vasoactive lipids. Of importance, the enzymatic activity of all microsomal CYP450 isoenzymes is dependent on the cytochrome P450 reductase (POR), an electron donor. In the first part of this work, the role of cytochrome P450 monooxygenases on the biotransformation of organic nitrates was investigated. Recombinant SupersomesTM were selected and incubated with NTG and PETN, where nitrite release was measured as a nitric oxide (NO) footprint. The capacity of the recombinant POR/CYP450 system to release nitrite from NO prodrugs was shown to be CYP-specific and dose-dependent. To study the involvement of CYP450 enzymes in the vascular biotransformation of organic nitrates in vivo, a smooth muscle-cell specific, inducible knockout model of POR (smcPOR-/-) was generated. Organ chamber experiments revealed that the vascular POR/CYP450 system had no impact on the dilator response of NTG and PETN. In line with previous publications, inhibition of ALDH2, known as the main enzyme responsible for the activation of NTG and PETN, and/or abolishment of the endogenous NO production did not reveal a contribution of the POR/CYP450 system to the dilator response of NTG and PETN. To better understand these results, we looked at the expression of the hepatic and vascular expression of the POR/CYP450 system where the hepatic was increased by 10- to 40-fold as shown by Western blot analysis. We concluded that due to insufficient vascular expression of CYP450 enzymes their contribution to the bioactivation of NTG and PETN is only minor. The second part of this work focused on the cardiac relevance of endothelial isoenzymes. For that purpose, an endothelial cell-specific, tamoxifen-inducible knockout model of POR was generated and characterized in the present study. RNA-sequencing of the heart of healthy mice revealed that the CYP450 expression is cell-specific with cardiac endothelial cells (ECs) exhibiting an enrichment in the expression of the Cyp4 family (ω-oxidation of fatty acids) and of the Cyp2 family (production of EETs). Under non-stredded conditions (i.e. 30 days after inducing the knockout by tamoxifen feeding), endothelial deletion of POR was associated with cardiac remodelling as observed by an increase in the ratio of heart weight to body weight and an increase in the cardiomyocyte area. RNA-sequencing of cardiac ECs suggested that loss of POR might alter ribosomal biogenesis and protein synthesis, which could potentially affect the cardiac contractility in ecPOR-/- mice. Metabolomics from cardiac tissue of CTL and ecPOR-/- mice were not indicative for an important metabolic function of the endothelial POR/CYP450 system in the heart. The combination of transverse aortic constriction (TAC) with endothelial deletion of POR accelerates the development of heart failure in mice as detected by a reduction in cardiac output and stroke volume. These effects were mediated most likely by a reduction in vascular EETs production, which increases vascular stiffness, resulting in cardiac remodeling.
• Die Cytochrom-P450-Enzyme sind eine große Superfamilie von membrangebundenen Häm-Monooxygenasen. CYP450-Enzyme sind für den oxidativen Stoffwechsel endogener Substrate wie Steroide und Fettsäuren sowie für die Biotransformation xenobiotischer Substrate wie Schadstoffe und Arzneimittel von wesentlicher Bedeutung. Die Leber weißt dabei die höchste CYP450-Expression auf, von denen alleine 15 Isoenzyme an der Phase I des Arzneimittelstoffwechsels beteiligt sind. Die Expression der CYP450-Familie im kardiovaskulären System ist jedoch nicht zu vernachlässigen, da Epoxygenasen (CYP2C- und CYP2J-Sufamilien) und ω-Hydroxylasen (CYP4A- und CYP4F-Subfamilien) für die Produktion vasoaktiver Lipide (epoxyeicosatrienoic acids, EETs) und hydroxyeicosarrienoic acids, HETEs) verantwortlich sind. Wichtig ist dabei, dass die enzymatische Aktivität aller CYP450 Enzyme von der Cytochrom-P450-Reduktase (POR), abhängig ist, welche diese mit Elektronen versorgt. Die vorliegenden Arbeit glieder sich in zwei Abschnitte. Der erste Teil fokussiert auf den Metabolismus organischer Nitrate, der zweite auf CYP450 im Herzen. Über ein Jahrhundert lang wurden Angina pectoris oder ischämische Herzerkrankungen mit Nitroglycerin (NTG) oder Pentaerythrittetranitrat (PETN) behandelt. Bei diesen Verbindungen handelt es sich um Produkte, die enzymatisch bioaktiviert werden, um ihre Nitrovasodilatoren freizusetzen: die Aldehyddehydrogenase (ALDH2) bei therapeutischen Konzentrationen oder CYP450-Enzyme bei suprapharmakologischen Konzentrationen. In zahlreichen Studien wurde ALDH2 als das für die Aktivierung organischer Nitrate in den Gefäßen hauptverantwortliche Enzym bezeichnet. Mehrere Studien zeigten, dass das hepatische POR/CYP450-System an der Bioaktivierung von organischen Nitraten beteiligt ist. Die Rolle des vaskulären POR/CYP450-Systems