Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries

Authors: LESLIE, ERIC - University Of New Mexico; LOPEZ, VANESSA - Loma Linda University; ANTI, NANA - Loma Linda University; ALVAREZ, RAFAEL - Loma Linda University; KAFEERO, ISAAC - Loma Linda University; WELSH, DONALD - Robarts Research Institute; ROMERO, MONICA - Loma Linda University; KAUSHAL, SHAWN - Loma Linda University; JOHNSON, CATHERINE - California Polytechnic State University; BOSVIEL, REMY - University Of California, Davis; BLAZENOVIC, IVANA - University Of California, Davis; SONG, RUI - Loma Linda University; BRITO, ALEX - First Moscow State Medical University; LA FRANO, MICHAEL - Loma Linda University; ZHANG, LUBO - Loma Linda University; Newman, John; FIEHN, OLIVER - University Of California, Davis; WILSON, SEAN - Loma Linda University

Submitted to: American Journal of Physiology - Lung Cellular and Molecular Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2021
Publication Date: 4/28/2021
Citation: Leslie, E., Lopez, V., Anti, N., Alvarez, R., Kafeero, I., Welsh, D.G., Romero, M., Kaushal, S., Johnson, C.M., Bosviel, R., Blazenovic, I., Song, R., Brito, A., La Frano, M.R., Zhang, L., Newman, J.W., Fiehn, O., Wilson, S. 2021. Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries. American Journal of Physiology - Lung Cellular and Molecular Physiology. 320(5):L770-L784. https://doi.org/10.1152/ajplung.00469.2020.
DOI: https://doi.org/10.1152/ajplung.00469.2020

Interpretive Summary: Gestational high-altitude hypoxia (i.e. low blood oxygen levels) increases the risk of diseases in human infants including pulmonary hypertension of newborns. Fetal sheep lungs are susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including thickening of the pulmonary arterial wall and dysregulation of cardiac functions culminating in a reduction in the amount of blood ejected from the heart with each beat. To explore the mechanisms associated with these hypoxia-induced aberrations in the fetal sheep lung, we investigated changes in small molecule profiles reflecting tissue metabolism, along with functional changes resulting from long-term hypoxia in the womb. In near-term fetuses exposed to low- or high-altitude hypoxia, we used sensitive tissue imaging techniques, probed changes in the machinery that regulate protein synthesis within cells, visualized how pulmonary artery smooth muscle regulated calcium movements when stimulated to contract, and measured a broad array of metabolites involved in cellular basic functions including energetics and inflammatory responses. We found swelling of the calcium storage vesicles in the muscle and an increased separation of these vesicles from the cell surface with hypoxia. Hypoxic animals presented negative impacts on the protein synthesis machinery, and suppressed calcium storage and stimulated release. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and anti-inflammatory regulatory metabolites derived from them. Our results show evidence that prolonged low blood oxygen-induced functional changes in the arteries of the fetal lung include metabolic alterations that contribute to the development of pulmonary hypertension.

Technical Abstract: Gestational high-altitude hypoxia increases the risk of diseases in human infants including pulmonary hypertension of newborns. Fetal sheep lungs are susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity culminating in reduced right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, western immunoblotting, calcium imaging, and metabolomic analyses on sheep pulmonary arteries isolated from near-term fetuses exposed to low- or high-altitude hypoxia (3801 m) for the latter 110+ days of pregnancy. Our results demonstrate sarcoplasmic reticular swelling with higher luminal width and distances to the plasma membrane (p<0.001) in the hypoxic versus normoxic groups. Hypoxic animals presented higher endoplasmic reticulum stress [higher (p<0.05) phospho-PERK and phospho-eIF2a] and suppressed calcium storage [lower (p<0.001) peak fluorescence response, area under the curve and duration of calcium release]. Metabolically, hypoxia was associated with lower levels (p<0.05) of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g. eicosapentanoic acid, docosahexaenoic acid, alpha-linolenic acid, 5-HEPE, 12-HEPE, 15-HEPE, and PGE3,19(20)-EpDPE), and higher levels of some omega-6 metabolites (p<0.02) including 15-Keto PGE2 and linoleoylglycerol. Our results show evidence for hypoxia-induced dysfunction and phenotypic transformations including metabolomic alterations in the pulmonary arteries of fetal sheep that contribute to the development of pulmonary hypertension.