Pulmonary arterial hypertension (PAH) is marked by increased proliferation of smooth muscle and endothelial cells, due in part, to altered cellular metabolism. The molecular and biochemical changes driving this metabolic shift likely depend on increased stabilization of the transcriptional regulator HIF through mitochondrial reactive oxygen species (ROS). Using a pre-PAH ovine model of congenital heart disease with increased pulmonary blood flow (PBF, shunt), we have shown that normal function and development of the pulmonary lymphatics is disrupted. We hypothesized that these aberrations are associated with HIF-mediated metabolic remodeling and accelerated proliferation in lymphatic endothelial cells (LECs).
Late gestation fetal lambs underwent in utero placement of an aortopulmonary graft. Four weeks after birth, LECs were isolated and cultured from control and shunt lambs. mRNA and protein expression were quantified. Targeted metabolomic analyses of glycolysis, TCA cycle, and pentose phosphate pathway (PPP) were performed by the University of Michigan Metabolomics Core.
Shunt LECs proliferated faster than control LECs, and had significantly elevated expression of HIF-1α, HIF-2α, and a number of known HIF target genes, p<0.05. Hypoxia promoted control LEC proliferation and increased HIF expression to levels equivalent to shunt LECs at baseline. Targeted metabolomic analyses of shunt and control LECs revealed significant differences in cellular metabolic flux patterns. Glycolytic intermediates were reduced in shunt LECs, with trends towards increased levels of glutamine metabolites; PPP was not changed. Acetyl CoA levels were reduced in shunt LECs, suggesting an inhibition of pyruvate dehydrogenase complex activity. Inhibition of mitochondrial ROS normalized HIF expression and proliferation in shunt LECs.
LECs from lambs exposed to chronically increased PBF are hyperproliferative, have enhanced expression of HIF and its target genes, and demonstrate an altered cell metabolism similar to other highly proliferative cell types. HIF-dependent glutamine metabolism might provide novel therapeutic targets for patients with lymphatic abnormalities.