Justin Chong, Biological Sciences, Summer 2021

Figure 1: Prox1-Vegfr3 feedback loop regulating the budding of nascent lymphatic endothelial cells (LECs) from the cardinal vein to a vascular endothelial growth factor C (VEGF-C) gradient at different embryonic times in a mouse.

Image Source: Wikimedia Commons

The lymphatic system is part of the vascular system that plays a critical role in maintaining tissue homeostasis and trafficking immune cells to foreign pathogens (Cueni & Detmar 2013). Cells that make up this crucial system are called lymphatic endothelial cells (LECs). In embryonic development, LECs arise from blood endothelial cells (BECs) in the cardinal vein as BECs differentiate, bud off into lymph sacs, and eventually migrate to form lymphatic vessels (Yang et al. 2012). Two important lymphatic genes, Prox1 and Vegfr3, are upregulated and form a feedback loop that is necessary for the differentiation of BECs into LECs, the maintenance of LEC identity throughout development and post-gestational life, and regulation of the amount of LEC progenitors that bud from the cardinal vein in early development to form LECs.

Researchers have found that cellular metabolism regulates this integral feedback loop, as well as endothelial cell function and lymphangiogenesis. For example, Prox1 can upregulate CPT1a to increase fatty acid oxidation, which increases acetyl-CoA levels necessary for LEC differentiation (Wong et al. 2017). Researchers at Northwestern University sought to shed light on how cellular, and specifically mitochondrial, metabolism plays a role in LEC differentiation and fate specification (Ma et al. 2021). Mitochondrial complex III is one of the downstream complexes of the electron transport chain (ETC) in the inner membrane of the mitochondria, responsible for moving electrons down the ETC to generate energy in the form of ATP. In their in vivo study, the QPC subunit, a critical subunit for the functioning of mitochondrial complex III, was deleted in LECs in mouse embryos.

This deletion was lethal as no mice were born alive, and mouse embryos collected throughout gestation revealed a complete lack of lymphatic vasculature, resulting in severe edema in the embryos since there were no lymphatics systems to drain the built-up interstitial fluid surrounding tissues. Vegfr3 expression levels in QPC null mutants were severely reduced. Enlargement of the jugular lymph sac was observed, likely because loss of Vegfr3 signaling prevents LECs from migrating from lymph sacs to surrounding tissues and forming a lymphatic network. Prox1 is the master control gene for lymphatics. It upregulates LEC genes and downregulates BEC genes to maintain stable LEC identity (Johnson et al. 2008). Since Prox1 is under the control of Vegfr3, downregulation of Vegfr3 in these budding mutant LECs reduces Prox1 expression, so mutant LECs will eventually undergo apoptosis or revert back to their original BEC fate.

To confirm this in vivo study, in vitro assays were performed by treating human LECs with antimycin A, which inhibits mitochondrial complex III. Results indicated decreased LEC proliferation (Diebold et al. 2019). LECs were also transduced with alternative oxidase, which is an enzyme capable of accepting electrons and restoring the electron transport chain activity to normal levels. When mitochondrial complex III was inhibited in these LECs, normal proliferation and Prox1 and Vegfr3 levels were observed, indicating that the loss of mitochondrial respiration following the inhibition of mitochondrial complex III leads to the loss of the Prox1-Vegfr3 feedback loop. The loss of mitochondrial respiration backs up the electron transport chain, leading to changes in levels of metabolites involved in the citric acid cycle (TCA) that donates electrons to the ETC. Alteration of TCA metabolites has been shown to affect histone methylation and acetylation (Anso et al. 2017). Histones are proteins that further regulate DNA transcription, and this study found a reduction in histone modifications near Prox1 and Vegfr3 loci, and an increase in those modifications near loci for Nrp1, and ICAM1 (two BEC genes). This suggests that the impairment of mitochondrial respiration alters metabolite levels in LECs, which affects the histone landscape in a way that downregulates LEC genes and upregulates BEC genes, explaining the loss of Vegfr3 and subsequently, LEC identity (Wong et al. 2018).

In addition to maintaining LEC identity in already budded LECs, mitochondrial respiration is important for controlling the amount of budding LECs and terminating budding. This is important because the physical structure of the cardinal vein can weaken if budding progresses for too long. The cessation of budding is most likely facilitated at embryonic day 14.5 (E14.5), when changes in the tissue extracellular environment of the migrating LECs lead to metabolic changes that epigenetically downregulate Vegfr3 and shut down the Prox1-Vegfr3 autoregulatory feedback loop (Srinivasan et al. 2014). This leads to either cell death or transition of the migrating LEC back to a BEC, ensuring that budding eventually concludes. However, the cues that lead to changes in the tissue and organ microenvironment at E14.5 that gradually stops LEC budding are still unclear and need to be explored further.

While more research needs to be done on the specific metabolic pathways that the mitochondria uses to epigenetically regulate the Prox1-Vegfr3 feedback loop, the study revealed a key player in mammalian developmental lymphatics and has clinical implications as the Vegfr3/Vegf-C signaling pathway is critical in lymphangiogenesis and lymphatic diseases (Srinivasan et al. 2014). Focusing on mitochondrial respiration could unlock new innovative therapeutics for patients with lymphatic related diseases and other diseases worsened by subclinical lymphatic dysregulation.

References

Anso, E. et al. (2017). The mitochondrial respiratory chain is essential for haematopoietic stem cell function. Nature Cell Biology 19: 614-625. https://doi.org/10.1038/ncb3529.

Cueni, L. N., & Detmar M. (2013). The lymphatic system in health and disease. Lymphatic Research and Biology 6(3-4): 109-122. https://doi.org/10.1089/lrb.2008.1008.

Diebold, L. P. et al. (2019). Mitochondrial complex III is necessary for endothelial cell proliferation during angiogenesis. Nature Metabolism 1: 158-171. https://doi.org/10.1038/s42255-018-0011-x.

Johnson, N. C. et al. (2008). Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity. Genes and Development 22: 3282-3291. https://doi.org/10.1101/gad.1727208.

Ma, W. et al. (2021). Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance. Science Advances 7(18): eabe7359. https://doi.org/10.1126/sciadv.abe7359.

Srinivasan, R. S. et al. (2014). The Prox1-Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors. Genes and Development 28: 2175-2187. https://doi.org/10.1101/gad.216226.113.

Wong, B. W. et al. (2017). The role of fatty acid β-oxidation in lymphangiogenesis. Nature 542: 49-54. https://doi.org/10.1038/nature21028.

Wong, B. W. et al. (2018). Emerging concepts in organ-specific lymphatic vessels and metabolic regulation of lymphatic development. Developmental Cell 45(3): 289-301. https://doi.org/10.1016/j.devcel.2018.03.021.

Yang, Y. et al. (2012). Lymphatic endothelial progenitors bud from the cardinal vein and intersomitic vessels in mammalian embryos. Blood 120(11): 2340-2348. https://doi.org/10.1089/lrb.2008.1008.