Conjugated Bile Acids activate Reactive Oxygen Species‐p90RSK‐Vascular Endothelial Growth Factor Receptor 3 signaling axis to promote lymphangiogenesis

Conjugated bile acids (BA), as taurocholic acid (TCA) levels, are significantly elevated in several liver pathologies. The liver is the largest lymph producing organ and has a high density of lymphatic vessels but role of hepatic lymphatics is poorly understood. BAs accumulate in metastatic lymph nodes (LN), and the lymphatics are exposed to high levels of bile in liver disease. However, whether BAs play any role in enhancing pathological lymphangiogenesis that exacerbates liver inflammation remains completely unknown. BAs activate oxidative stress pathways in liver cells and promote injury. However, these mechanisms have not been studied in the context of lymphangiogenesis. We hypothesized that the BAs activate oxidative stress response pathways such as reactive oxygen species (ROS) and p90 ribosomal S6 kinase (p90RSK) pathways and promote lymphangiogenesis. Methods BA levels were evaluated in serum and lymph nodes (LN) of wild type (WT, FVB/NJ) and Mdr2−/− mice (mouse model of primary sclerosing cholangitis, PSC). Lymphangiogenesis was assessed in the liver and LN from these animals. Human lymphatic endothelial cells (HLECs) were treated with TCDCA (100 µM), and TCA (100 µM) for 24hrs in the presence or absence of the specific BA receptor, Takeda G-protein receptor 5 (TGR5) antagonist (SBI-115), and changes in lymphangiogenic genes was assessed by qPCR. Effects on LEC proliferation, invasion, migration, and tube formation were also determined. Changes in LEC cellular bioenergetics was determined by Seahorse assay. Results BA levels were elevated in the LN and serum of Mdr2−/− mice compared to WT. Immunohistochemical staining of liver tissue sections with cholangiocyte marker (CK19) and lymphatic marker (LYVE1) showed increased lymphangiogenesis in the MDR2−/− mice compared to WT. BAs enhanced the expression of BA receptors, farnesoid X receptor (FXR), and TGR5 on HLECs and also increased HLEC tube formation, migration, and growth of lymphatic capillaries in a 3-D collagen matrix that was inhibited by SBI-115, ROS inhibitor (NAC), and p90RSK inhibitor (BI-D1870). A significant increase in maximal glycolytic capacity and ATP production was observed in the HLECs in response to BA treatment. BAs enhanced mitochondrial ROS and phosphorylated the redox-sensitive kinase p90RSK (S380), an essential regulator of endothelial cell dysfunction in a ROS-dependent manner. BAs also induced Prospero homeobox 1 (Prox1) SUMOylation via p90RSK activation and enhanced VEGFR3 expression, which was inhibited by BI-D1870 and NAC. Conclusion Overall, our data suggest that BA-induced p90RSK activation plays a crucial role in the induction Prox1-VEGFR3 axis as the precursor of lymphangiogenesis and activates a feedback loop between cellular ROS and oxidative stress activation pathways that could further promote liver injury and inflammation.