Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies

Abstract

Hepatic stellate cells (HSC) play a key role in the development of chronic liver disease (CLD). Liraglutide, well-established in type 2 diabetes, showed anti-inflammatory and anti-oxidant properties. We evaluated the effects of liraglutide on HSC phenotype and hepatic microvascular function using diverse pre-clinical models of CLD. Human and rat HSC were in vitro treated with liraglutide, or vehicle, and their phenotype, viability and proliferation were evaluated. In addition, liraglutide or vehicle was administered to rats with CLD. Liver microvascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype were determined. Additionally, the effects of liraglutide on HSC phenotype were analysed in human precision-cut liver slices. Liraglutide markedly improved HSC phenotype and diminished cell proliferation. Cirrhotic rats receiving liraglutide exhibited significantly improved liver microvascular function, as evidenced by lower portal pressure, improved intrahepatic vascular resistance, and marked ameliorations in fibrosis, HSC phenotype and endothelial function. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue and, although requiring further investigation, its underlying molecular mechanisms suggested a GLP1-R-independent and NF-κB-Sox9-dependent one. This study demonstrates for the first time that liraglutide improves the liver sinusoidal milieu in pre-clinical models of cirrhosis, encouraging its clinical evaluation in the treatment of chronic liver disease.

Figure 1

Amelioration of human primary HSC in response to liraglutide. (A) Expression of depicted proteins/genes after in vitro treatment with 50 µM liraglutide or vehicle in: 1-HSC isolated from cirrhotic human livers (left), 2-quiescent human HSC undergoing in vitro activation (middle), and 3-human precision-cut liver slices (PCLS) (right). (B) Effects of liraglutide, or its vehicle, on the contraction of primary human HSC. n = 3 per experimental condition. *p < 0.05 vs. vehicle.

Figure 2

Underlying effects of HSC deactivation due to liraglutide. After 72 h of treatment with 50 µM liraglutide, LX-2 cells were assessed for markers of HSC activation (A), cell viability by double staining with acridine orange (green dense nuclei: apoptosis, indicated by arrowheads) and propidium iodide (red cells: necrosis) (B), HSC proliferation assessed by cell counting and expression of the proliferative marker PDGFRβ (C), and cell contraction (D). n = 3 per experimental condition. *p < 0.05 vs. vehicle.

Figure 3

Analysis of HSC phenotype and liver fibrosis in CLD-rats treated with liraglutide. (A) Expression of HSC activation markers (α-SMA and PDGFRβ) and Desmin in livers from TAA-CLD-rats treated for 15 days with liraglutide or vehicle. (B) Analysis of hepatic fibrosis in rats described in A (collagen I expression and Sirius Red staining). (C) Analysis of the phenotype of HSC freshly isolated from rats described in (A). *p < 0.05 vs. vehicle. n = 8 (A and B) and n = 3 (C) per group. Results are indicated as mean ± s.e.m.

Figure 4

Effects of liraglutide on hepatic endothelial phenotype and microvascular function. (A) Liver sinusoidal fenestrae analysis by means of frequency (no. fenestrae/cell area) and porosity (fenestrae area/cell area) in TAA-CLD-rats treated with liraglutide or vehicle. (B) Hepatic nitric oxide (NO) bioavailability in rats described in A. (C) Hepatic microvascular function, calculated as the decrease in portal pressure in response to increasing doses of the endothelium-dependent vasodilator acetylcholine after vasoconstriction with methoxamine. *p < 0.05 vs. vehicle. n = 3 (A), n = 8 (B) and n = 5 (C) per group.