Endothelial-to-mesenchymal transition (EndMT) is a process that encompasses extensive transcriptional reprogramming of activated endothelial cells leading to a shift toward mesenchymal cellular phenotypes and functional responses. Initially observed in the context of embryonic development, in the last few decades EndMT is increasingly recognized as a process that contributes to a variety of pathologies in the adult organism. Within the settings of cardiovascular biology, EndMT plays a role in various diseases, including atherosclerosis, heart valvular disease, cardiac fibrosis, and myocardial infarction. EndMT is also being progressively implicated in development and progression of pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH). This review covers the current knowledge about EndMT in PH and PAH, and provides comprehensive overview of seminal discoveries. Topics covered include evidence linking EndMT to factors associated with PAH development, including hypoxia responses, inflammation, dysregulation of bone-morphogenetic protein receptor 2 (BMPR2), and redox signaling. This review amalgamates these discoveries into potential insights for the identification of underlying mechanisms driving EndMT in PH and PAH, and discusses future directions for EndMT-based therapeutic strategies in disease management.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21^cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21^cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21^cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21^cip. Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.

Objective—: Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress. Methods and Results—: Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O ₂ ^•− ) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O ₂ ^•− in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47 ^phox and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. Conclusion—: Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.

Reactive oxygen species (ROS)-mediated intracellular signalling is well described in the vasculature, yet the precise roles of ROS in paracrine signalling are not known. Studies implicate interstitial ROS hydrogen peroxide (H₂O₂) in vascular disease, and plasma H₂O₂ levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H₂O₂ was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H₂O₂ activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H₂O₂ induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1. Treatment of rat aortic SMCs (rASMC) with exogenous H₂O₂ resulted in a concentration-dependent increase in Nox-derived superoxide (O₂^•–), determined by L-012 chemiluminescence, cytochrome c and electron paramagnetic resonance. Nox1 was verified as the source of O₂^·– by siRNA. Aqp1 siRNA attenuated H₂O₂ cellular entry and H₂O₂-induced O₂^•– production. H₂O₂ treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H₂O₂-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it. Our results demonstrate for the first time that extracellular H₂O₂, at pathophysiological concentrations, stimulates rASMC Nox1-derived O₂^•–, subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H₂O₂ enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.