Volumetric optoacoustic tomography enables non-invasive in vivo characterization of impaired heart function in hypoxic conditions.

Exposure to chronic hypoxia results in pulmonary hypertension characterized by increased vascular resistance and pulmonary vascular remodeling, changes in functional parameters of the pulmonary vasculature, and right ventricular hypertrophy, which can eventually lead to right heart failure. The underlying mechanisms of hypoxia-induced pulmonary hypertension have still not been fully elucidated while no curative treatment is currently available. Commonly employed pre-clinical analytic methods are largely limited to invasive studies interfering with cardiac tissue or otherwise ex vivo functional studies and histopathology. In this work, we suggest volumetric optoacoustic tomography (VOT) for non-invasive assessment of heart function in response to chronic hypoxia. Mice exposed for 3 consecutive weeks to normoxia or chronic hypoxia were imaged in vivo with heart perfusion tracked by VOT using indocyanide green contrast agent at high temporal (100 Hz) and spatial (200 µm) resolutions in 3D. Unequivocal difference in the pulmonary transit time was revealed between the hypoxic and normoxic conditions concomitant with the presence of pulmonary vascular remodeling within hypoxic models. Furthermore, a beat-to-beat analysis of the volumetric image data enabled identifying and characterizing arrhythmic events in mice exposed to chronic hypoxia. The newly introduced non-invasive methodology for analysis of impaired pulmonary vasculature and heart function under chronic hypoxic exposure provides important inputs into development of early diagnosis and treatment strategies in pulmonary hypertension.

Cross Talk Between p22phox and ATF4 in the Endothelial Unfolded Protein Response.

BACKGROUND: Cardiovascular diseases have been associated with stress in the endoplasmic reticulum (ER) and accumulation of unfolded proteins leading to the unfolded protein response (UPR). Reactive oxygen species (ROS) such as superoxide and H2O2 derived from NADPH oxidases have been implicated in the pathogenesis of cardiovascular diseases. ROS have also been associated with ER stress. The role NADPH oxidases in the UPR is, however, not completely resolved yet. AIM: In this study, we investigated the role of p22phox, an essential component of most NADPH oxidases, in the UPR of endothelial cells. RESULTS: Induction of ER stress increased p22phox expression at the transcriptional level. p22phox was identified as novel target of the UPR transcription factor ATF4 (activator of transcription factor 4) under ER stress conditions by promoter analyses and ChIP. Depletion of ATF4 and p22phox diminished the levels of superoxide and H2O2 under ER stress conditions. On the contrary, p22phox was instrumental in increasing eIF2α phosphorylation and subsequent ATF4 expression on induction of ER stress by chemicals, oxysterols, or severe hypoxia in vitro and in vivo, leading to increased expression of CHOP and activation of effector caspases. INNOVATION: p22phox is a novel target of ATF4 in response to ER stress, which can promote the PERK-ATF4 branch of the UPR in vitro and in vivo. CONCLUSION: p22phox-dependent NADPH oxidases are important mediators of ER stress driving the UPR.

Stabilization of p22phox by Hypoxia Promotes Pulmonary Hypertension.

AIMS: Hypoxia and reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of pulmonary hypertension (PH), a potentially fatal disorder characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right ventricular hypertrophy. However, how they are linked in the context of PH is not completely understood. We, therefore, investigated the role of the NADPH oxidase subunit p22phox in the response to hypoxia both in vitro and in vivo. RESULTS: We found that hypoxia decreased ubiquitinylation and proteasomal degradation of p22phox dependent on prolyl hydroxylases (PHDs) and the E3 ubiquitin ligase protein von Hippel Lindau (pVHL), which resulted in p22phox stabilization and accumulation. p22phox promoted vascular proliferation, migration, and angiogenesis under normoxia and hypoxia. Increased levels of p22phox were also detected in lungs and hearts from mice with hypoxia-induced PH. Mice harboring a point mutation (Y121H) in the p22phox gene, which resulted in decreased p22phox stability and subsequent loss of this protein, were protected against hypoxia-induced PH. Mechanistically, p22phox contributed to ROS generation under normoxia, hypoxia, and hypoxia/reoxygenation. p22phox increased the levels and activity of HIF1α, the major cellular regulator of hypoxia adaptation, under normoxia and hypoxia, possibly by decreasing the levels of the PHD cofactors ascorbate and iron(II), and it contributed to the downregulation of the tumor suppressor miR-140 by hypoxia. INNOVATION: These data identify p22phox as an important regulator of the hypoxia response both in vitro and in vivo. CONCLUSION: p22phox-dependent NADPH oxidases contribute to the pathophysiology of PH induced by hypoxia.