The CXCR3 ligand CXCL9 attenuates neoangiogenesis and liver fibrosis in vivo

Aims: Recently, we could show that CXCR3-/- mice display exaggerated liver fibrosis compared to wild-type littermates after chronic CCl4 treatment (Wasmuth et al. Gastroenterology 2009). Notably, CXCL9, the ligand of CXCR3, has also angiostatic effects. The aims of this project were to quantify angiogenesis in CXCR3-/- and wild-type mice after CCl4 treatment and to study the feasibility of modulating liver fibrosis and neoangiogenesis by CXCL9.

Methods: Liver fibrosis was induced by repeated injections of CCl4 in CXCR3-/- and wild-type mice. In a separate experiment, CXCL9 or vehicle was administered in parallel with CCl4 to wild-type mice for 6 weeks. Neoangiogenesis was assessed by fluorescence staining of endothelial-specific molecules CD31 and vWF and in vivo by VEGFR2 detection using fluorescence molecular tomography. Liver fibrosis was quantified by staging of histology after sirius red staining. In vitro, the VEGF induced tube formation of endothelial cells and its inhibition by CXCL9 was analyzed by Matrigel experiments.

Results: The treatment of wild-type mice with CCl4 led to increased neoangiogenesis. Compared to wild-type mice, CXCR3-/- mice displayed a further significant increase of intrahepatic CD31/vWF positive microvessel density (p<0.01) and VEGFR2 enrichment in vivo. Notably, the systemic administration of CXCL9 resulted in a significant attenuation of neoangiogenesis as well as of liver fibrosis (p<0.05). In vitro, CXCL9 inhibited the VEGF induced tube formation of endothelial cells (p<0.05).

Discussion: Genetic deletion of CXCR3 leads to increased neoangiogenesis. Conversely, systemic administration of CXCL9 attenuates liver fibrosis associated angiogenesis. Therefore, the CXCR3/CXCL9 pathway could be a potential therapeutic target of fibrosis and neovascularisation.