Ethanol‐Induced Lymphatic Endothelial Cell Permeability: Role of Prox‐1 and Vascular Endothelial Growth Factors

Acute and chronic alcohol (Ethanol, EtOH) administration to rodents promote mesenteric lymphatic vessel permeability leading to immune cell leakage into perilymphatic adipose tissue. We speculate this contributes to impaired immune dialog between the gut and adjacent lymphoid tissue. Mechanistic in vitro studies show that EtOH-induced lymphatic endothelial cell (LEC) permeability is not associated with alterations in tight and adherens junction expression but is partially rescued by p38 MAP kinase inhibition. Transcription factor Prox-1 and Vascular endothelial growth factor receptor-3 (VEGFR-3) play essential roles in maintaining the integrity of mesenteric lymphatic vessels and of lymphatic vascular functions. The lack of Prox-1 and VEGFR-3 expression has been shown to contribute to excessive lymph and vascular leakage, respectively, promoting increased lymphatic permeability. Prox-1 and VEGFR-3 are negatively regulated by notch signaling. Notch activation suppresses Prox-1 and notch target genes, Hey 1 and Hey 2, inhibit VEGFR-3. We hypothesize that EtOH-induced LEC permeability is mediated by decreased Prox-1 and VEGFR-3 expression via notch signaling activation. We used an in vitro model of rat LEC to characterize Prox-1 and VEGFR-3 expression in EtOH-induced LEC permeability. Cells were seeded in T-75 flask and cultured with rat endothelial cell medium supplemented with EtOH (0, 25mM, and 50mM) for 30 minutes in an EtOH water bath to limit EtOH evaporation loss from culture media. Gene expression of Prox-1, VEGFR-3, Notch 1, Notch 4, Hey 1, and Hey 2 was measured using Quantitative Polymerase Chain Reaction. EtOH at 50mM concentration for 30 minutes significantly decreased gene expression of Prox-1 and VEGFR-3 and increased gene expression of Notch 1 and Hey 2. No significant changes were observed in LEC exposed to 25mM EtOH. These findings support our hypothesis that decreases in Prox-1 and VEGFR-3 expression involving notch signaling activation may be implicated in EtOH induced LEC permeability leading to mesenteric lymphatic dysfunction.