Blocking glutamine transport normalizes lymphatic vessels in hypoxic environments by attenuating glycolysis

Dysfunctional lymphangiogenesis is a component of several diseases with hypoxic microenvironments, including secondary lymphedema and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in complications such as increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Current treatments to normalize vasculature have negative effects on healthy vessels and do not specifically target lymphatic endothelial cells (LECs). As hypoxia is known to change endothelial cell metabolism, exploiting LEC-specific metabolic pathways may provide a focused approach to restoring lymphatic function in patients. However, outside of glycolysis, changes to LEC metabolism in hypoxic conditions are understudied. To address this gap in knowledge, we examined the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing glutamine availability results in increased lactate production as well as a hypoxia-specific increase in glycolytic genes HK2, GLUT1, and GLUT3. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a vessel formation assay, we found that glutamine increased vessel formation in normoxic conditions, but lowered vessel connectivity in hypoxic conditions, reflecting the dysfunction seen in hypoxic diseases. However, attenuating glycolysis by blocking glutamine transport caused LECs to form longer, interconnected vascular networks. This study reveals that glutamine availability can modulate LEC glycolysis, and therefore lymphangiogenesis, in a hypoxia-dependent manner. Collectively, our study identifies glutamine availability as a potential target for lymphatic vessel normalization in chronic and hypoxic diseases.