Fig. 4. Cortistatin-deficient HSCs show exacerbated fibrotic
responses and excessive differentiation capacity to activated
myofibroblasts. (A ) Nonparenchymal HSC-enriched fractions were
isolated from wild-type (CST+/+ ) and
cortistatin-deficient (CST-/- ) livers and
cultured as described in the diagram. (B ) Analysis of glial
fibrillary acidic protein (GFAP) and α-smooth muscle actin (αSMA)
expression by immunofluorescence of HSC cultures to determine the
percentage of non-activated HSCs
(GFAP+/αSMA-), myofibroblasts
GFAP+/αSMAlow and activated
myofibroblasts αSMAhigh (with stress fibers) at
different times (n=18 for 6d+1d cultures, n=5 for 6d+4d cultures).
Representative images for each cell type used to the analysis are shown
as examples. (C ) Expression of profibrogenic mediators in HSCs
after primary cultures (n=3 cultures by duplicates). (D-G ) Gene
expression profile in CST+/+ andCST-/- HSCs analyzed by next-generation RNA
sequencing. (D ) Volcano plot depicting the differentially
expressed genes (DEGs, with false discovery rate FDR-p<0.05)
between CST-/- andCST+/+ HSCs (description for all DEGs is
included in table S5). (E ) Unsupervised hierarchical clustering
of the 640-top DEGs (with log2 fold-change
>1 or <-1) separated the samples into two major
groups. (F ) Fold-change expression of the top-25 upregulated
DEGs in CST-/- HSCs. Red bars correspond to
muscle-related genes (table S1 shows a detailed description of these
DEGs). (G ) Gene ontology (GO) analysis of the DEGs betweenCST-/- and CST+/+HSCs. The top-10 or top-5 terms are included. Red and blue bar-segments
correspond to numbers of upregulated and downregulated DEGs,
respectively. All DEGs in each GO term are listed in tables S2 to S4.
(H ) Effect of cortistatin addition every other day to primary
cultures of CST-/- HSCs on myofibroblast
activation/differentiation (immunofluorescence at day 4, n=11 cultures)
and expression of critical genes for myofibroblast activation and muscle
differentiation (n=3 cultures by duplicates). Data are the mean±SEM
(unless data in F that are the mean). *p<0.05,
**p<0.01, ***p<0.001, unpaired two-tailed Student’s
t-test.