The role of ω-3 and ω-6 Polyunsaturated Fatty Acids (PUFAs) in endothelial cell specification, angiogenesis, and lymph-angiogenesis
Authors:   , Ciliberti Giorgia 
Published: January 2025

Polyunsaturated fatty acids (PUFAs) are organic acids, including more than one double bond in their carbon long chains. They can be further classified in ω-3 and ω-6 fatty acids according to the localization of the double bound within the molecule and comprise a wide group of bioactive compounds involved in the regulation of the cardiovascular homeostasis.
PUFAs can be metabolized Cytochrome P450 which can use several metabolic precursors, to synthetize ω-3 and ω-6 epoxides. Epoxides can be further converted to the corresponding diolss via the soluble epoxide-hydrolase (sEH). ω-3/ω-6 epoxides and diols exert multiple functions in the modulation of endothelial physiology and pathophysiology including regulation of vascular tone, angiogenesis and tumor-associated vasculogeneis. However, little is known about how these metabolites can impact on vascular specification and lymph-angiogenesis.
In the present study, we aimed to investigate the role of ω-3/ω-6 epoxides and diols on endothelial cells specification, and subsequently assessed the effects of their metabolic imbalance on angiogenesis and lymph-angiogenesis.
To address these questions, we chose the ω-6 12,13-EpOME and the derived 12,13-DiHOME, while 19,20-EDP and corresponding diol, 19,20 DHDP, were selected from the ω-6 pool. To investigate effect of ω-3/ω-6 epoxides and diols on the establishment of blood (BEC) and lymphatic (LEC) cell identity, endothelial specification was recapitulated in vitro via differentiation of both human induced and mouse pluripotent embryonic cells (hIPSCs and mESCs). Differentiation was performed with or without administration of ω-3/ω-6 epoxides or diols respectively. Subsequently, BEC/LEC differentiation was confirmed via immunohistochemistry and quantitative flow cytometry, while PUFAs-dependent transcriptional changes were assessed in the differentiated cells via single-cells RNA sequencing.
We were able to demonstrate that the ω-6 epoxide and the ω-3 diol were able to mainly promote blood endothelial differentiation of pluripotent cells by favoring expression of vasculogenic mediators. Differently, the ω-6 diol and the ω-3 epoxide enhanced lymphatic specification via up-regulation of lymph-angiogenic mediators such Prox-1, Lyve-1 and Inregrin-α9. Moreover, the same compounds were able to favor an increase of the lymphatic cell portion in murine lung endothelial cells ex vivo.
Using mice harboring a constitutive deletion of the sEH enzyme, we have shown that the subsequent imbalance of the physiologic epoxides/diols ratio, resulted in altered angiogenic and lymph-angiogenic responses both in vivo and vitro. Indeed, whole mount fluorescent-immunohistochemistry and three-dimensional imaging analysis confirmed that sEH-/- mice exhibited an increased blood endothelial vascular angiogenesis in the ear skin as well as enhanced sprouting of aortic explants in culture. Thus, linked to the accumulation of endogenously produced EpOMEs.
On the other hand, the same animals displayed significantly impaired dermal lymph-angiogenesis and decreased lymphatic sprouting responses in vitro. Subsequently, we were able to show that such phenotype could be mainly linked to the lack of DiHOMEs, as exogenous administration was able to rescue the sprouting responses of sEH-/- thoracic duct explants as well as to improve those from the corresponding wild-type controls.
In synthesis, we have shown that, ω-6/ω-3 epoxides diols can differentially contribute to endothelial cell specification as well as angiogenesis and lymph-angiogenesis. Specifically, while
ω-6 epoxides and ω-3 diols mainly favor blood endothelial cell (BECs) specification of pluripotent cells, ω-6 diols and ω-3 epoxides promoted lymphatic differentiation of angioblast cell progenitors.
Moreover, we were able to confirm that EpOME, and on a lower scale DHDP, were able to enhance angiogenesis in vitro and in vivo. On other hand, we have shown that DiHOME, and partially EDP (in vitro), can regulate lymph-angiogenesis and enhance lymph-angiogenic potential of WT and sEH-/- mice in vitro.