An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Sensory ataxia and cardiac hypertrophy caused by neurovascular oxidative stress in chemogenetic transgenic mouse lines.

Oxidative stress is associated with cardiovascular and neurodegenerative diseases. Here we report studies of neurovascular oxidative stress in chemogenetic transgenic mouse lines expressing yeast D-amino acid oxidase (DAAO) in neurons and vascular endothelium. When these transgenic mice are fed D-amino acids, DAAO generates hydrogen peroxide in target tissues. DAAO-TGCdh5 transgenic mice express DAAO under control of the putatively endothelial-specific Cdh5 promoter. When we provide these mice with D-alanine, they rapidly develop sensory ataxia caused by oxidative stress and mitochondrial dysfunction in neurons within dorsal root ganglia and nodose ganglia innervating the heart. DAAO-TGCdh5 mice also develop cardiac hypertrophy after chronic chemogenetic oxidative stress. This combination of ataxia, mitochondrial dysfunction, and cardiac hypertrophy is similar to findings in patients with Friedreich's ataxia. Our observations indicate that neurovascular oxidative stress is sufficient to cause sensory ataxia and cardiac hypertrophy. Studies of DAAO-TGCdh5 mice could provide mechanistic insights into Friedreich's ataxia.

GPx3 deficiency exacerbates maladaptive right ventricular remodeling in experimental pulmonary artery banding.

The stressed right ventricle (RV) is particularly susceptible to producing and accumulating reactive oxygen species, leading to extracellular matrix deposition and secretion of natriuretic peptides. The role of specific enzymes with antioxidative capacity, like glutathione peroxidase 3 (GPx3), in RV pathogenesis is currently unknown. Here, we use a murine model of pulmonary artery banding (PAB) to study the role of GPx3 in isolated RV pathology. Compared with wild-type (WT) mice undergoing PAB surgery, GPx3-deficient PAB mice presented with higher RV systolic pressure and higher LV eccentricity indices. PAB-induced changes in Fulton's Index, RV free wall thickness, and RV fractional area change were more pronounced in GPx3-deficient mice compared with WT controls. Adverse RV remodeling was enhanced in GPx3-deficient PAB animals, evidenced by increased RV expression levels of connective tissue growth factor (CTGF), transforming growth factor-ß (TGF-ß), and atrial natriuretic peptide (ANP). In summary, GPx3 deficiency exacerbates maladaptive RV remodeling and causes signs of RV dysfunction.

Novel Approaches to Imaging the Pulmonary Vasculature and Right Heart.

There is an increased appreciation for the importance of the right heart and pulmonary circulation in several disease states across the spectrum of pulmonary hypertension and left heart failure. However, assessment of the structure and function of the right heart and pulmonary circulation can be challenging, due to the complex geometry of the right ventricle, comorbid pulmonary airways and parenchymal disease, and the overlap of hemodynamic abnormalities with left heart failure. Several new and evolving imaging modalities interrogate the right heart and pulmonary circulation with greater diagnostic precision. Echocardiographic approaches such as speckle-tracking and 3-dimensional imaging provide detailed assessments of regional systolic and diastolic function and volumetric assessments. Magnetic resonance approaches can provide high-resolution views of cardiac structure/function, tissue characterization, and perfusion through the pulmonary vasculature. Molecular imaging with positron emission tomography allows an assessment of specific pathobiologically relevant targets in the right heart and pulmonary circulation. Machine learning analysis of high-resolution computed tomographic lung scans permits quantitative morphometry of the lung circulation without intravenous contrast. Inhaled magnetic resonance imaging probes, such as hyperpolarized 129Xe magnetic resonance imaging, report on pulmonary gas exchange and pulmonary capillary hemodynamics. These approaches provide important information on right ventricular structure and function along with perfusion through the pulmonary circulation. At this time, the majority of these developing technologies have yet to be clinically validated, with few studies demonstrating the utility of these imaging biomarkers for diagnosis or monitoring disease. These technologies hold promise for earlier diagnosis and noninvasive monitoring of right heart failure and pulmonary hypertension that will aid in preclinical studies, enhance patient selection and provide surrogate end points in clinical trials, and ultimately improve bedside care.

A self-amplifying loop of YAP and SHH drives formation and expansion of heterotopic ossification.

Heterotopic ossification (HO) occurs as a common complication after injury or in genetic disorders. The mechanisms underlying HO remain incompletely understood, and there are no approved prophylactic or secondary treatments available. Here, we identify a self-amplifying, self-propagating loop of Yes-associated protein (YAP)-Sonic hedgehog (SHH) as a core molecular mechanism underlying diverse forms of HO. In mouse models of progressive osseous heteroplasia (POH), a disease caused by null mutations in GNAS, we found that Gnas-/- mesenchymal cells secreted SHH, which induced osteoblast differentiation of the surrounding wild-type cells. We further showed that loss of Gnas led to activation of YAP transcription activity, which directly drove Shh expression. Secreted SHH further induced YAP activation, Shh expression, and osteoblast differentiation in surrounding wild-type cells. This self-propagating positive feedback loop was both necessary and sufficient for HO expansion and could act independently of Gnas in fibrodysplasia ossificans progressiva (FOP), another genetic HO, and nonhereditary HO mouse models. Genetic or pharmacological inhibition of YAP or SHH abolished HO in POH and FOP and acquired HO mouse models without affecting normal bone homeostasis, providing a previously unrecognized therapeutic rationale to prevent, reduce, and shrink HO.