Stem Cells Dev. 2020 May 30. doi: 10.1089/scd.2020.0063. [Epub ahead of print https://pmlegacy.ncbi.nlm.nih.gov/pubmed/32475301
Extracellular vesicles attenuate nitrofen-mediated human pulmonary artery endothelial dysfunction: implications for congenital diaphragmatic hernia.
Zhaorigetu S1, Bair H2, Jin D3, Gupta VS4, Pandit LM5, Bryan RM6, Lally KP7, Olson SD8, Cox CS9, Harting MT10.
Congenital diaphragmatic hernia (CDH) leads to pathophysiologic pulmonary vasoreactivity. Previous studies show that mesenchymal stromal cell-derived extracellular vesicles (MSCEv) inhibit lung inflammation and vascular remodeling. We characterize MSCEv and human pulmonary artery endothelial cell (HPAEC) interaction as well as the pulmonary artery (PA) response to MSCEv treatment. HPAECs were cultured with and without exposure to nitrofen and treated with MSCEv. HPAEC viability, architecture, production of reactive oxygen species (ROS), endothelial dysfunction-associated protein levels (PPARγ, LOX-1, LOX-2, NF-κB, eNOS, ET1), and the nature of MSCEv-cellular interaction were assessed. Newborn rodents with and without CDH (nitrofen model, Sprague-Dawley) were treated with intravascular MSCEv or vehicle control, and their PAs were isolated. Contractility was assessed via wire myography. The contractile (KCL,ET-1) and relaxation (fasudil) responses were evaluated. HPAEC viability correlated inversely with nitrofen dose, while architectural compromise was directly proportional. There was a 2.1x increase in ROS levels in nitrofen HPAECs(p<0.001), and MSCEv treatment attenuated ROS levels by 1.5x versus nitrofen HPAECs(p<0.01). Nitrofen-induced alterations in endothelial dysfunction-associated proteins are shown, and exposure to MSCEv restored more physiologic expression. Nitrofen HPAEC displayed greater MSCEv uptake (80% increase)(p<0.05). Adenosine, a clathrin-mediated endocytosis inhibitor, decreased uptake by 46%(p<0.05). CDH PA contraction was impaired with KCL (108.6±1.4% vs 112.0±1.4%,p=0.092) and ET-1 (121.7±3.0% vs 131.2±1.8%, p<0.01). CDH PA relaxation was impaired with fasudil (32.2±1.9% vs 42.1±2.2%,p<0.001). After MSCEv treatment, CDH PA contraction improved (125.9±3.4% vs 116.4±3.5, p=0.078), and relaxation was unchanged (32.5±3.2% vs 29.4±3.1%,p=0.496). HPAEC exposure to nitrofen led to changes consistent with vasculopathy in CDH, and MSCEv treatment led to a more physiologic cellular response. MSCEv were preferentially taken up by nitrofen-treated cells via clathrin-dependent endocytosis. In vivo, MSCEv exposure improved PA contractile response. These data reveal mechanisms of cellular and signaling alterations that characterize MSCEv-mediated attenuation of pulmonary vascular dysfunction in CDH-associated PH.PMID: 32475301 DOI: 10.1089/scd.2020.0063