Specific Endoglin deletion in primary mouse hepatic stellate cells shows a modulation of TGF-b signaling pathways, aggravating liver fibrosis

Background: Hepatic stellate cells (HSCs) are the major source for extracellular matrix (ECM) production in liver fibrosis. TGF-beta and its auxiliary receptor endoglin (ENG, S- and L-Endoglin splicing variants), which is expressed on HSCs, are important cytokines, mediating cellular responses in liver injury. This study analyzes the role of specific Endoglin deletion in primary mice HSCs using Cre-LoxP genetic recombination and the effect of this deletion on stellate cell activation.

Methods: HSCs were isolated from ENGflox/flox and GFAPCre-ENGΔHSC mice in a two step pronase/collagenase digestion. After FACS sorting cells were activated on plastic surface or stimulated with TGF-beta for 1 and 24h. Alternatively ENGflox/flox cells were treated with adenoviral CMVCre-RSVGFP and RSVGFP (control) expressing vector. Cells were suspended for 24h after isolation and infected for 48h with the adenoviral vectors (4000 MOI). Subsequently HSCs as well were stimulated with TGF-beta for 4 and 24h. TGF-beta downstream signaling was analyzed by western blots and qPCR.

Results: Comparison of Alk5-Smad2/3 and Alk1-Smad1/5/8 pathways showed downregulation of Smad1/5/8 in GFAPCre+-ENGΔHSC HSCs after activation on plastic surface or TGF-beta stimulation (> 45%). Moreover GFAPCre+-ENGΔHSC HSCs showed less Smad2/3 expression. However collagen production was upregulated indication a differential ENG modulation of the Alk5-Smad2/3 and Alk1-Smad1/5/8 TGF-beta signaling pathways. Western blot analysis of TGF-beta signaling of adenoviral Cre (AdCre+) treated ENGflox/flox primary HSCs showed an effective downregulation of ENG in AdCre+ treated cells (> 90%). In the absence of ENG activation of Smad1/5/8 as well as Smad2/3 was downregulated. However PAI-1, a downstream target of Alk5-Smad2/3 was increased compared to Id1 (downstream target of Alk1-Smad1/5/8). Since L-ENG is the predominantely expressed ENG isoform (> 90%) signaling mainly through Alk1-Smad1/5/8 and promoting antifibrotic effects, deletion of ENG (S- and L-ENG isoforms) shifts the TGF-beta signaling to the profibrotic Alk5-Smad2/3 pathway leading to higher collagen expression and aggravated fibrosis in vivo.

Conclusion: Endoglin deficiency in hepatic stellate cells has an effect on the modulation of SMADs pathway signaling in HSCs leading to aggravation of liver fibrosis.

Corresponding author: Scholten, David

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