Hepatic stellate cells (HSC), quiescent and activated - influence of ltbp1-k.o. on activation in vitro

Hepatic stellate cells (HSC) constitute the most important fibrogenic cell type during liver fibrosis. To analyse the different phenotypes of quiescent and activated cells, cells of 5 animals were transdifferentiated in a well established in vitro transdifferentiaition assay and compared to freshly seeded, quiescent HSC. Gene expression profiling of quiescent and in vitro activated HSC revealed well known and new marker genes upregulated upon HSC activation. ltbp1 (latent transforming growth factor beta binding protein 1) is a crucial factor controlling the secretion and bioactivation of TGFß1. Using the data comparing quiescent and activated HSC, we were able to qualify the subtle but reproducible differences in gene expression observed when comparing activated ltbp1-deficient and activted wt HSC. ltbp1-deficient HSC showed a less fibrogenic phenotype after 6 days of in vitro transdifferentiation compared to the wt counterparts. The microarray data was indepently confirmed in vivo using an experimental model for the induction of liver fibrosis (ligation of the common bile duct). ltbp1-/- mice, after 4 weeks of bile obstruction, showed markedly reduced signs of liver fibrosis. Keywords: gene expression profiling, analysyis of a fibrogenic cell type in the liver and the influence of ltbp1-k.o. Five mice per group were sacrificed and HSC were prepared by pronase/collagenase in situ perfusion. Cells wer pooled and seeded on plastic dishes. wt cells were cultured for one day (refered to as quiescent cells) or 6 days to induce transdifferentiation (refered to as activated cells). Cells from ltbp1 deficient animals were also cultured for 6 days to induce transdifferentiation. RNA was prepared thereafter, quantified and checked for integrity. Equal amounts of RNA (out of 5 animals per group) were subjected to linear amplification and labeling and used for hybridization on PIQOR_TM Microarrays. One microgram of RNA was labeled by reverse transcription via Cy3-dCTP (sample 1) and Cy5-dCTP (sample 2) incorporation. Both experiments (A: quiescent_wt_vs_activated_wt and B: activated_wt_vs_activated_ltbp1-/-) were conducted as technical replicates.