cells Article Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury James West, Anandharajan Rathinasabapathy , Xinping Chen, Sheila Shay, Shanti Gladson and Megha Talati *   Citation: West, J.; Rathinasabapathy, A.; Chen, X.; Shay, S.; Gladson, S.; Talati, M. Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury. Cells 2021, 10, 2306. https://doi.org/10.3390/ cells10092306 Academic Editors: Alexander E. Kalyuzhny and Rozenn Quarck Received: 2 July 2021 Accepted: 19 August 2021 Published: 3 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Division of Allergy, Pulmonary and Critical Care Medicine, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA; j.west@vumc.org (J.W.); anandharajan.rathinasabapathy@vumc.org (A.R.); pchen@geneticsassociates.com (X.C.); sheila.shay@vumc.org (S.S.); santhi.gladson@vumc.org (S.G.) * Correspondence: Megha.Talati@vumc.org; Tel.: +1-615-322-8095; Fax: +1-615-343-7448 Abstract: Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1 OE ) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1 OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1 OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1 OE mice was similar to controls. In sugen-hypoxia-treated MSX1 OE mice, the number of small (0–25 uM) and medium (25–50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1 OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1 OE , and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1 OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent. Keywords: pulmonary arterial hypertension; Msx1 expression; mouse models; pulmonary vascular endothelial cells; pulmonary vascular smooth muscle cells; BMP pathway; idiopathic PAH; heritable PAH; RNA sequencing 1. Introduction Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disease [1–5]. The prominent pathologic features of PAH are irreversible loss of small peripheral pulmonary arteries with widespread obliterative vasculopathy, resulting in progressive elevation of pulmonary arterial pressure that leads to right ventricular failure and death. In PAH, disorganized growth of pulmonary artery cells, impaired vascular regeneration, and ectopic Cells 2021, 10, 2306. https://doi.org/10.3390/cells10092306 https://www.mdpi.com/journal/cells