Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Systemic Soluble Receptor for Advanced Glycation Endproducts Is a Biomarker of Emphysema and Associated with AGER Genetic Variants in Patients with Chronic Obstructive Pulmonary Disease

Rationale: Emphysema in chronic obstructive pulmonary disease (COPD) can be characterized by high-resolution chest computed tomography (HRCT); however, the repeated use of HRCT is limited because of concerns regarding radiation exposure and cost.Objectives: To evaluate biomarkers associated with emphysema and COPD-related clinical characteristics, and to assess the relationships of soluble receptor for advanced glycation endproducts (sRAGE), a candidate systemic biomarker identified in this study, with single-nucleotide polymorphisms (SNPs) in the gene coding for RAGE (AGER locus) and with clinical characteristics.Methods: Circulating levels of 111 biomarkers were analyzed for association with clinical characteristics in 410 patients with COPD enrolled in the TESRA study. sRAGE was also measured in the ECLIPSE cohort in 1,847 patients with COPD, 298 smokers and 204 nonsmokers. The association between 21 SNPs in the AGER locus with sRAGE levels and clinical characteristics was also investigated. Measurements and MainResults: sRAGE was identified as a biomarker of diffusing capacity of carbon monoxide and lung density in the TESRA cohort. In the ECLIPSE cohort, lower sRAGE levels were associated with increased emphysema, increased Global Initiative for Chronic Obstructive Lung Disease stage, and COPD disease status. The associations with emphysema in both cohorts remained significant after covariate adjustment (P < 0.0001). One SNP in the AGER locus, rs2070600, was associated with circulating sRAGE levels both in TESRA (P = 0.0014) and ECLIPSE (7.07 x 10(-16)), which exceeded genome-wide significance threshold. Another SNP (rs2071288) was also associated with sRAGE levels (P = 0.01) and diffusing capacity of carbonmonoxide (P = 0.01) in the TESRA study.Conclusions: Lower circulating sRAGE levels are associated with emphysema severity and genetic polymorphisms in the AGER locus are associated with systemic sRAGE level