Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.

NADPH oxidase 4 expression in the normal endometrium and in endometrial cancer.

The aim of this study was to explore the role of NOX4 in the biology of the normal endometrium and endometrial cancer. NOX4 plays a key role in other adenocarcinomas and has been implicated in the pathogenesis of diabetes and obesity, which are important risk factors for endometrial cancer. NOX4 expression was assessed in 239 endometrial cancer and 25 normal endometrium samples by quantitative real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. DNA methylation of the NOX4 promoter was determined by means of MethyLight PCR. Data were correlated with clinicopathological parameters and analyzed in the context of diabetes and body mass index. In the normal endometrium, NOX4 microRNA expression was significantly higher in the secretory transformed compared with proliferative endometrium ( p = 0.008). In endometrial cancer specimens, NOX4 expression did not differ between diabetic and non-diabetic patients, but was the highest in patients with a body mass index ≤ 26 ( p = 0.037). The lowest NOX4 expression was found in carcinosarcomas ( p = 0.007). High NOX4 expression predicted poorer clinical outcome with regard to overall survival, especially in non-diabetic patients and those with a body mass index > 20. Independent prognostic significance of NOX4 transcripts was retained in type I endometrial cancer and was the most meaningful in patients with a body mass index > 20. No prognostic impact was shown for NOX4 promoter methylation in endometrial cancer. For the first time, we demonstrate that NOX4 plays a considerable role in the cycle-dependent changes in the normal endometrium and in the biology of endometrial cancer.

NADPH oxidases and vascular remodeling in cardiovascular diseases.

Reactive oxygen species (ROS) are key signaling molecules that regulate vascular function and structure in physiological conditions. A misbalance between the production and detoxification of ROS increases oxidative stress that is involved in the vascular remodeling associated with cardiovascular diseases such as hypertension by affecting inflammation, hypertrophy, migration, growth/apoptosis and extracellular matrix protein turnover. The major and more specific source of ROS in the cardiovascular system is the NADPH oxidase (NOX) family of enzymes composed of seven members (NOX1-5, DUOX 1/2). Vascular cells express several NOXs being NOX-1 and NOX-4 the most abundant NOXs present in vascular smooth muscle cells. This review focuses on specific aspects of NOX-1 and NOX-4 isoforms including information on regulation, function and their role in vascular remodeling. In order to obtain a more integrated view about the role of the different NOX isoforms in different types of vascular remodeling, we discuss the available literature not only on hypertension but also in atherosclerosis, restenosis and aortic dilation.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

ROS production and mitochondrial dysfunction driven by PU.1-regulated NOX4-p22phox activation in Aß-induced retinal pigment epithelial cell injury.

Rationale: Amyloid ß (Aß) deposition, an essential pathological process in age-related macular degeneration (AMD), causes retinal pigment epithelium (RPE) degeneration driven mostly by oxidative stress. However, despite intense investigations, the extent to which overoxidation contributes to Aß-mediated RPE damage and its potential mechanism has not been fully elucidated. Methods: We performed tandem mass-tagged (TMT) mass spectrometry (MS) and bioinformatic analysis of the RPE-choroid complex in an Aß1-40-induced mouse model of retinal degeneration to obtain a comprehensive proteomic profile. Key regulators in this model were confirmed by reactive oxygen species (ROS) detection, mitochondrial ROS assay, oxygen consumption rate (OCR) measurement, gene knockout experiment, chromatin immunoprecipitation (ChIP), and luciferase assay. Results: A total of 4243 proteins were identified, 1069 of which were significantly affected by Aß1-40 and found to be enriched in oxidation-related pathways by bioinformatic analysis. Moreover, NADPH oxidases were identified as hub proteins in Aß1-40-mediated oxidative stress, as evidenced by mitochondrial dysfunction and reactive oxygen species overproduction. By motif and binding site analyses, we found that the transcription factor PU.1/Spi1 acted as a master regulator of the activation of NADPH oxidases, especially the NOX4-p22phox complex. Also, PU.1 silencing impeded RPE oxidative stress and mitochondrial dysfunction and rescued the retinal structure and function. Conclusion: Our study suggests that PU.1 is a novel therapeutic target for AMD, and the regulation of PU.1 expression represents a potentially novel approach against excessive oxidative stress in Aß-driven RPE injury.