Abstract 12681: Secretory Factor(s) Derived From Human Pulmonary Artery Endothelial Cells Induce Pseudo-hypoxic Activation of HIF-1α Signaling and Metabolic Reprogramming in Human Pulmonary Artery Smooth Muscle Cells

Abstract

Cell-cell communication is required for maintaining physiological functions of the vasculature, and is also involved in the development of vascular disorders, including pulmonary arterial hypertension. Yet, how heterologous cell-cell crosstalk influences cellular metabolism in pulmonary vascular cells is not well understood. Objective: We studied how the interaction of heterologous pulmonary vascular cells regulates vascular metabolism. Methods and Results: Human pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC) were cultured in a transwell coculture system with or without conditioned medium (CM) under ambient oxygen levels. Results from the Seahorse XF24 analyzer showed an increase in extracellular acidification rate (indicative of glycolysis), and a decrease in oxygen consumption rate (indicative of mitochondrial respiration) in PASMC cultured in CM from PAEC, suggesting a metabolic shift toward glycolysis induced by an endothelial product. This metabolic shift correlated with activation of HIF-1α signaling, as demonstrated by normoxic stabilization of HIF-1α protein and upregulation of its target gene expression ( GLUT1 , HK2 , and PDK1 ) in PASMC. Interestingly, these effects were not observed in PAEC cultured with PASMC or in CM from PASMC. We also found that HIF-1α stabilization is independent of its transcription and of the protein levels of prolyl hydroxylase domain protein 2 (PHD2) and pVHL; however, PHD activity decreased in PASMC cultured in CM from PAEC, suggesting that factor(s) secreted by PAEC could suppress PHD activity in PASMC. To identify the potential factor(s), CM from PAEC were pretreated by heat inactivation or with proteinase K and fractionated by filtration column. Results from these experiments suggested that the inhibitor(s) is(are) a small molecule (<10 kDa). We then tested known PHD inhibitors and found that pyruvate, succinate, fumarate, lactate, glutamate, and reactive oxygen species were unlikely to be the mediators. Overall, our study indicates that cellular metabolism of PASMC can be reprogrammed by PAEC through a paracrine mechanism. These results may provide novel insights into mechanisms of vascular cell-cell crosstalk and its consequences for vascular metabolism and disease.