New and classical methods to compare oxidative stress levels and parameters of vascular function in rat models of hypertension, diabetes and nitrate tolerance
Authors: Kalinovic Sanela , Oelze Matthias , Kröller-Schön Swenja , Steven Sebastian , Vujacic-Mirski Ksenija , Kvandova Miroslava , Schmal Isabella , Al Zuabi Ahmad , Münzel Thomas , Daiber Andreas 
Published: April 2020

BackgroundOxidative stress was identified as a hallmark of almost all cardiovascular and neurodegenerative diseases. The term oxidative stress describes a condition that is either characterized by increased generation of reactive oxygen and nitrogen species (RONS) and/or dysregulated cellular antioxidant defense mechanisms (e.g. decreased expression of central antioxidant enzymes). Since the transition between oxidative stress and redox signaling is a thin line, the exact knowledge of the identity of formed reactive species, the cellular and subcellular localization of their formation as well as their time duration and concentration are of high clinical and pharmacological importance.Methods and ResultsWith the present studies, we evaluated the specific probe for mitochondrial superoxide formation, mitochondria‐targeted, triphenylphosphonium‐linked hydroethidium (mitoSOX), for its usefulness in the detection of mitochondrial superoxide formation ex vivo/in vivo and compared this HPLC‐based assay with alternative/traditional methods. For this purpose, we used three different animal models with well‐established oxidative stress burden, namely diabetic, hypertensive and nitrate tolerant rats. With our previous work we have stressed that the traditional or old RONS assays are not necessarily inferior as compared to up‐to‐date or even cutting‐edge assays and at least have their specific features and application spectra. Vascular function was assessed by isometric tension methodology and was impaired in the rat models of oxidative stress. Vascular dysfunction correlated with increased mitoSOX oxidation but also classical RONS detection assays as well as typical markers of oxidative stress. Additionally, we successfully applied this method for the mitochondrial superoxide detection in hypertensive (AT‐II induced), aircraft noise exposure mouse model as well as in the combination of these two stressors. The application of this method was also effective in the wild type and Nox2 knockout mice exposed to e‐cigarette vapor over 3 days for 2 h/d.ConclusionIn conclusion, we report that mitoSOX/HPLC indeed represents a suitable method for quantification of mitochondrial superoxide formation in biological samples of animals with cardiometabolic disease, pharmacologically induced cardiovascular complications and cardiovascular diseases induced by novel (environmental/behavioral) stressors. The knowledge on the spatial distribution of RONS formation may be of specific interest for drug development and when it comes to therapy.Support or Funding InformationOur experimental studies were supported by vascular biology research grants from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”.