Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis

Abstract

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl(4)) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl(4)- and alcohol-induced liver fibrosis using Cre-LoxP-based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.

Fig. 1.

Regression of liver fibrosis is accompanied by loss of myofibroblasts. (A) A comparison of the livers of Col-GFP mice that were untreated, CCl₄-treated (2 mo), or recovered from CCl₄ (1 and 4 mo) with respect to GFP expression, Sirius Red staining, and α-SMA immunohistochemistry. Representative bright-field and fluorescent micrographs are shown using 10× and 20× objectives. (B) Quantification of the same four groups in A with respect to hydroxyproline content, Sirius Red staining, α-SMA immunofluorescence, GFP expression, collagen-α1(I) mRNA level, and α-SMA mRNA level. *P < 0.01, **P < 0.05. (C) HSCs (vitamin A⁺) constitute >90% of myofibroblasts (vitamin A⁺GFP⁺), as detected by flow cytometry of the nonparenchymal cell fraction from CCl₄-treated (2 mo) Col-GFP mice (n = 3). (D) Working hypothesis: CCl₄ induces activation of qHSCs into aHSCs/myofibroblasts. Cre-loxP–based genetic labeling marks the fate of collagen type I-expressing aHSCs/myofibroblasts (SI Appendix, Fig. S1). During recovery from CCl₄-liver fibrosis, aHSCs may (i) apoptose (no genetically labeled YFP⁺ HSCs will remain in the liver) or (ii) inactivate (all YFP⁺ cells survive) or (iii) some will apoptose and some will inactivate (YFP⁺ iHSCs will number <100% of aHSCs).

Fig. 2.

Genetically labeled aHSCs persist after 1 mo recovery. (A) Livers from collagen-α2(I)^Cre-YFP mice (no injury: n = 4; CCl₄-treated: n = 8; recovered: 1 mo n = 10) were costained for YFP, GFAP, Desmin, or α-SMA. Genetically labeled HSCs were identified after 1 mo recovery by YFP⁺ expression in Desmin⁺ or GFAP⁺ cells. The number of YFP⁺ HSCs is calculated relative to total HSCs (100%, merge 1, P < 0.05 comparing CCl₄ and recovery groups). Nuclei are shown (DAPI, merge 2). (B) HSCs (vitamin A⁺) from collagen-α2(I)^Cre-YFP mice (no injury: n = 4; CCl₄-treated: n = 6; recovered: 1 mo n = 6) were analyzed by flow cytometry. Genetically labeled aHSCs and iHSCs were identified by simultaneous vitamin A⁺ and YFP⁺ expression. Dot plots are shown; P < 0.01 (comparing YFP⁺ aHSCs and YFP⁺ iHSCs). (C) Genetically labeled GFP⁺ HSCs persist in the livers of tamoxifen-inducible Col-α2(1)^ER-Cre-GFP after 1 mo of recovery from CCl₄. To avoid genetic labeling of HSCs during development, tamoxifen-inducible Col-α2(1)^ER-Cre-GFP mice were generated by crossing Col-α2(1)^ER-Cre mice × Rosa26^{flox-Stop-mTRed-flox-mGFP} mice (here labeled as Rosa26^f/f-mTRed mice) and treated with CCl₄ (2 mo), and genetic pulse-labeling of aHSCs was induced by daily tamoxifen administration during the last week of CCl₄ treatment. After this treatment, mice recuperated for 1 mo to reverse liver fibrosis. Genetically labeled HSCs were visualized by immunostaining for membrane-tagged GFP⁺ (and simultaneous loss of mTRed expression: merge 1), DAPI-stained nuclei (merge 2) were taken with a 20× objective. The number of genetically labeled GFP⁺ HSCs was calculated as percentage of Desmin⁺ HSCs (100%, merge 3). Genetic labeling of 35 ± 6% aHSCs was achieved in response to CCl₄. GFP⁺ iHSCs (14 ± 4%) persisted in the liver after 1 mo recovery (P < 0.05; CCl₄ and recovery groups are compared), confirming that CCl₄-activated HSCs (and their progeny) remain in the liver after regression of fibrosis.

Fig. 3.

HSCs (1 mo recovery) acquire a new phenotype distinct from qHSCs. (A) HSCs from Col-α1(1)^Cre-YFP mice, uninjured or after 1 mo recovery, were cultured for 48 h ± TGF-β1 (2 ng/mL for 6 h), and analyzed by RT-PCR for expression of fibrogenic and neural genes; *P < 0.01, **P < 0.05. (B) CCl₄-treated Col-GFP mice (2 × CCl₄; n = 4) recuperated for 6 mo and were then subjected to recurrent CCl₄ injury. Development of liver fibrosis in these mice was compared with littermates treated with CCl₄ only the second time (1 × CCl₄; n = 4) by Sirius Red. The number of aHSCs was estimated by fluorescent microscopy for Desmin and α-SMA (P < 0.05, using 20× objective). Total collagen deposition was measured by hydroxyproline assay; *P < 0.01. (C) HSCs were isolated from collagen-α1(I)-GFP/β-actin-RFP mice, uninjured or after recovery (7 d or 1 mo) from CCl₄ injury, and transferred intrahepatically (2.2 × 10⁵ cells) into 1-d-old Rag2^−/−γc^−/− pups. Following CCl₄ injury, the number of RFP⁺GFP⁺ engrafted qHSCs and HSCs after 7 d and 1 mo was calculated relative to the number of total HSCs (detected by Desmin).

Fig. 4.

Genetically labeled HSCs obtain a new “inactivated” phenotype after 1 mo of recovery. (A) Microarray analysis. Vitamin A⁺ HSCs were sort-purified from Col-α2(I)^Cre-YFP mice that were untreated (n = 6), fibrotic (n = 6), after 7 d of recovery (n = 3), and after 1 mo of recovery (n = 6). YFP⁺ and YFP⁻ HSCs were then subjected to the Whole Mouse Genome Microarray. Representative cell number is shown for each HSC group. (B) YFP⁺ iHSCs (1 mo recovery) down-regulate mRNAs of fibrogenic genes and up-regulate PPARγ and Bambi but not other qHSC genes (Adfp, Adipor1, GFAP). The results show the relative mRNA level (average of normalized values/multiple probes/gene) obtained using the Agilant microarray; *P < 0.01, **P < 0.001. (C) Gene expression profile clustering analysis identifies similarity between the different HSC phenotypes. The correlation coefficient was used to compare the qHSC (1.00) gene expression pattern with YFP⁺ iHSCs (0.76) and aHSCs (0.63) expression patterns. (D) Expression of signature genes was determined for YFP⁺ iHSCs (1 mo) and YFP⁺ HSCs (7 d recovery, 7 d), and fold induction (compared with YFP⁺ aHSCs) is shown for each group.

Fig. 5.

Genetically labeled YFP⁺ HSCs up-regulate prosurvival Hsp1a/b genes at 7 d of recovery. (A) Up-regulation of prosurvival Hsp1a/b genes in YFP⁺ HSCs at 7 d of recovery. The results are expressed as relative mRNA levels (average of normalized values/multiple probes/gene; *P < 0.001) obtained by Agilant microarray. (B) Apoptosis was induced in Hspa1a/b^−/− and wild-type HSCs by glyotoxin (25 nM for 4 h) and TNF-α (20 ng/mL) + actinomycin (0.2 μg/mL) for 18 h. (C) Hspa1a/b^−/− and wild-type (Wt) mice were gradually subjected to CCl₄ injury and recovered for 2 wk, and livers were analyzed by Sirius Red, staining for Desmin and α-SMA (positive areas are indicated). Regression of fibrosis and disappearance of fibrogenic myofibroblasts during recovery were calculated in comparison with CCl₄ treatment (100%) and are shown as percentage of Sirius Red-, Desmin-, and α-SMA–positive areas; *P < 0.01, **P < 0.05.