Monitoring TGF-β1 effects on liver cells in vivo

Transforming growth factor (TGF-β) plays an important role in the progression of chronic liver diseases by influencing a plethora of cellular processes, such as hepatic stellate cell activation and matrix remodeling, proliferation control or modulating the immunological niche. In order to test direct and chronic effects of TGF-β1, we treated healthy C57BL6/J mice with recombinant (r)TGF-β1 (100µg/kg, iv) for (2 and 24h) and injected the mice intravenously with an AAV8-TGF-β1 construct (10x1010 virus particles). AAV8-YFP (10x1010 virus particle) served as control. rTGF-β1 treated and part of the AAV8 treated mice were used for cell isolation and comparative scRNASeq analyses. Chronically affected mice were additionally used for a thorough morphopathological analysis. Surprisingly, AAV8-TGF-β1 infected mice survived only for 7 days. Plasma of AAV8-TGF-β1 infected mice displays significantly elevated TGF-β1 levels and the liver tissue shows strongly induced Smad phosphorylation, mainly in non-parenchymal cells, since mouse hepatocytes already display intrinsic nuclear pSmad staining. Histopathological investigation of the livers reveals activation of hepatic stellate cells, upregulation of laminin expression and development of a basal membrane in perivenous and sinusoidal compartments, disturbance in zonation with loss of approximately one-third of glutamine synthetase (GLUL) expressing hepatocytes, and gain of additional E-cadherin positive cells. Further, loss of LYVE1 (fenestration marker) and increase in CD34 indicate capillarization. We currently investigating mice exposed to a lower dose of AAV8-TGF-β1 (1x1010 virus particle).We conclude that chronic challenging of liver cells with active TGF-β1 in healthy liver is associated with sinusoid capillarization, HSC activation and perivenous and sinusoidal scarring.