Abstract

Background:

Chronic inflammation due to various etiologies leads to liver fibrosis and subsequently cirrhosis. The liver lymphatic system helps to reduce inflammation. We hypothesized that the liver lymphatic system ameliorates liver fibrosis/cirrhosis.

Methods:

Mice with different stages of liver fibrosis were generated using bile duct ligation (BDL) surgery. Liver lymphatic endothelial cells (LECs) from different stages of liver fibrosis were isolated for RNA sequencing. Liver samples from control and primary sclerotic cholangitis (PSC) patients were analyzed.

Results:

Hepatic lymphatic vessel (LV) numbers were significantly increased until 2 weeks after BDL (fibrosis stage), but decreased by 4 weeks (cirrhosis stage). To determine whether this drop in LV numbers further promotes liver fibrosis, we blocked new LV formation in BDL mice using MAZ51, an inhibitor of VEGFR3 and found aggravated liver fibrosis by increased Sirius Red (1.4-fold, p<0.05), hydroxyproline (1.9-fold, p<0.05), increased MPO-positive neutrophil number (2.8-fold, p<0.05), and decreased lymphatic drainage (2.5-fold, p<0.05). These results suggest that decreased lymphatic drainage promotes liver fibrosis progression. In contrast, administration of VEGF-C, the most potent factor promoting new LV formation, via AAV8-VEGF-C significantly increased LV numbers and inhibited liver fibrosis as indicated by decreased Sirius Red (1.4-fold, p<0.05), hydroxyproline (1.4-fold, p<0.05), and CD68-positive macrophage number (1.7-fold, p<0.05). Transcriptomic analysis of LECs revealed increased TGFβ signaling, which is known to inhibit lymphangiogenesis in cirrhotic mouse livers. Blocking TGFβ signaling via LY364947 significantly increased LV numbers (2.0-fold, p<0.001) and area (1.8-fold, p<0.01) in cirrhotic mouse livers. In livers from PSC patients, we found increased LV number in compensated (3.5-fold, p<0.001, n=7) and decompensated (2.5-fold, p<0.05, n=6) cirrhosis. Importantly, hepatic LV number decreased in decompensated cirrhotic liver (1.4-fold, p<0.05) compared to compensated cirrhotic patients. Histological analysis showed co-localized pSMAD2 expression increased in liver LECs in decompensated liver cirrhosis, indicating TGFβ-driven lymphatic dysfunction may contribute to the decompensation of liver cirrhosis.

Conclusion:

The liver lymphatic system inhibits liver fibrosis and progression to liver cirrhosis. VEGF-C-driven lymphangiogenesis and lymphatic drainage could be a novel therapy for liver fibrosis/cirrhosis.