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Based on the concept of oxidative stress, reactive oxygen species (ROS) have been incriminated as the drivers behind almost every cardiovascular pathology. Redox alterations are, however, omnipresent bystanders to changes in cellular activity state. Even when ROS levels are altered, their contribution to pathology is not necessarily causal. Researchers should hesitate to engage in global ROS measurements and rather aim on identifying individual molecular targets of redox regulation.
The NADPH oxidase Nox4 is a hydrogen peroxide (H2O2)-producing enzyme, with the highest expression in the kidney. As the kidney is involved in volume and blood pressure control through sodium handling, we set out to determine the impact of a low sodium diet on these parameters in WT and Nox4-/- mice. Nox4 expression in the murine kidney was restricted to the proximal tubule. Nevertheless, low-sodium-induced weight loss and sodium sparing function was similar in WT and Nox4-/- mice, disputing an important function of renal Nox4 in sodium handling. In contrast, a low sodium diet resulted in a reduction in systolic blood pressure in Nox4-/- as compared to WT mice. This was associated with a selectively lower pressure to heart-rate ratio, as well as heart to body weight ratio. In general, a low sodium diet leads to activation of sympathetic tone and the renin angiotensin system, which subsequently increases peripheral resistance. Our observations suggest that the control by this system is attenuated in Nox4-/- mice, resulting in lower blood pressure in response to low sodium.
Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise.
Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.
Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.
Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.
Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.
Tolerizing CTL by sustained hepatic PD-L1 expression provides a new therapy spproach in mouse sepsis
(2019)
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.
Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.
Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.
Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.