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

Abstract

Dysfunctional lymphangiogenesis is a component of several diseases, including secondary lymphedema, allergic asthma, and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in secondary complications such as edema, increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Hypoxic microenvironments drive dysfunctional vessel growth by upregulating vascular endothelial growth factor receptors, migratory signaling pathways, and glycolytic metabolism. Despite the potential benefits of normalizing lymphatic vasculature, most treatments for vascular normalization focus on blood vasculature, ignoring the unique properties of lymphatic endothelial cells (LECs). Moreover, previous targets for vascular normalization center around vascular endothelial growth factors, which risk adverse side effects. To address these issues, we approached lymphatic normalization from a metabolic perspective. In this study, we investigated the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing the concentration of glutamine in media results in increased lactate production and the expression of glycolytic genes HK2, GLUT1, and GLUT3 under hypoxia. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a 2D 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.