No abstract

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1␣ (but not HIF-2␣) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxiainducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1␣ mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1␣ in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1␣ mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1␣ stabilization and activity. Based on these data and a mathematical model of HIF-1␣ suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1␣ activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1␣-dependent transcription.Tissue hypoxia is a common feature in a range of physiologic and pathophysiologic states, including exercise, development, cancer, and chronic inflammation. The hypoxia-inducible factor (HIF) is a ubiquitous hypoxia-responsive transcription factor that regulates the expression of a range of genes that promote adaptation to hypoxia (32, 57). The mechanism by which HIF is stabilized in hypoxia is well understood and is due to reduced activity of a family of oxygen-dependent HIFhydroxylases that target HIF␣ subunits for degradation and block transactivation in normoxia (5). Several studies (including the present one) have shown that the upregulation of HIF-1␣ that occurs in response to hypoxia is transient and involves a resolution phase even while the cells are maintained in hypoxia (23,26,59). However, the mechanism(s) underpinning the resolution of HIF-1␣ during prolonged hypoxia remains incompletely understood. Negative-feedback mechanisms involving HIF-dependent upregulation of PHD2 and PHD3 have been identified (5,26,47,59). In the present study, we aimed to expand our understanding of how the HIF response is resolved in prolonged hypoxia by investigating a possible role for hypoxiainduced microRNAs (miRNAs). miRNAs are endogenous small RNA molecules of approximately 22 nucleotides that regulate gene expression by destabilizing mRNA or repressing translation (4, 25). Approximately one-third of all genes in mammals have been predicted to be regulated by miRNAs (43,71), and the development of

Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid β-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1A mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1A mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1A mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.