Ivacaftor attenuates gentamicin-induced ototoxicity through the CFTR-Nrf2-HO1/NQO1 pathway.

OBJECTIVES: Gentamicin is one of the most common ototoxic drugs that can lower patients' quality of life. Oxidative stress is a key factors inducing sensory hair cell death during gentamicin administration. So far, there are no effective drugs to prevent or treat gentamicin- induced hearing loss. A recent study found cystic fibrosis transmembrane conductance regulator (CFTR) as a new target to modulate cellular oxidative balance. The objective of this study was to estimate the effect of the CFTR activator ivacaftor on gentamicin-induced ototoxicity and determine its mechanism. METHODS: The hair cell count was analyzed by Myosin 7a staining. Apoptosis was analyzed by TUNEL Apoptosis Kit. Cellular reactive oxygen species (ROS) level was detected by DCFH-DA probes. The Nrf2 related proteins expression levels were analyzed by western blot. RESULTS: An in vitro cochlear explant model showed that gentamicin caused ROS accumulation in sensory hair cells and induced apoptosis, and this effect was alleviated by pretreatment with ivacaftor. Western blotting showed that ivacaftor administration markedly increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). The protective effect of ivacaftor was abolished by the Nrf2 inhibitor ML385. DISCUSSION: Our results indicate the protective role of the CFTR-Nrf2-HO1/NQO1 pathway in gentamicin-induced ototoxicity. Ivacaftor may be repositioned or repurposed towards aminoglycosides-induced hearing loss.

Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension.

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of H2O2 activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1ß activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1ß in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of Nppa/Nppb mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.

Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease.

Cerebral cavernous malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or can be inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM disease exhibits a range of different phenotypes, including wide interindividual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Mutations of the KRIT1 gene account for over 50% of familial cases. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered homeostasis of intracellular reactive oxygen species (ROS) and abnormal activation of redox-sensitive transcription factors, which collectively result in pro-oxidative, pro-inflammatory, and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease. Consistently, these original discoveries have been confirmed and extended by subsequent findings showing mechanistic relationships between pleiotropic redox-dependent effects of KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis. In this chapter, we describe few basic methods used for qualitative and quantitative analysis of intracellular ROS in cellular models of CCM disease.

Oxidative stress in carotid arteries of patients submitted to carotid endarterectomy. The role of aging process

ABSTRACT PURPOSE: To evaluated the role of oxidative stress on aging process in patients submitted to carotid endarterectomy. METHODS: Twenty patients were divided into two groups: older group (≥ 70 years old); and the younger group (< 70 years old). We evaluated the reactive oxygen species (ROS) concentration, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, superoxide dismutase (SOD) and catalase (CAT) activities as so as nitrite levels in fragments of carotid arteries harvested during carotid endarterectomy for treatment of high grade carotid stenosis. RESULTS: We observed a higher levels of ROS and NADPH oxidase activity in the older group (p<0.05). Furthermore, the nitrite concentration was lower in the older group (14.55 ± 5.61 x 10-3 versus 26.42 ± 8.14 x 10-3 µM; p=0.0123). However, the activities of antioxidant enzymes (CAT and SOD) were similar in both the groups. CONCLUSIONS : Arterial aging is associated with increased concentrations of oxygen species and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity as so as nitrite reduction in human carotid artery specimens. Maybe therapies that block NADPH oxidase activity and enhance nitrite stores would be a good strategy to reduce the effect of oxidative stress in arterial aging.