Resolving fibrosis by stimulating HSC-dependent extracellular matrix degradation

Abstract

Tissue fibrosis arises from a critical imbalance between the production and breakdown of extracellular matrix (ECM) components. Whereas current strategies predominantly focus on curbing ECM production, the possibility of promoting ECM degradation to resolve fibrosis remains largely untapped. The role of hepatic stellate cells (HSCs) in ECM degradation is an intriguing area for investigation. We previously demonstrated that inhibiting acid ceramidase (aCDase) increases ceramide in HSCs to ameliorate hepatic fibrosis. Here, we uncover a key signaling pathway that promotes ECM degradation in primary human HSCs, which is dependent upon the activation of protein kinase Cα (PKCα) and the induction of matrix metalloproteinase 1 (MMP-1) through extracellular signal-regulated kinase 1/2 (ERK1/2). Genetic reduction and pharmacological inhibition with a small molecule reduced aCDase activity, leading to increased collagen degradation and hepatic fibrosis resolution in the carbon tetrachloride (CCl₄) and fructose, palmitate, cholesterol, and trans-fat (FPC) mouse models. Consistently, ceramide signaling correlated with ECM remodeling and degradation in patients with metabolic dysfunction-associated steatotic liver disease. The findings show that ceramide regulates ECM degradation and establish aCDase as a target for therapeutic regression of fibrosis.

Fig. 1.. Ceramide promotes expression of ECM-degrading enzymes.

(A) Volcano plot of proteins profiled by proteomics in human HSCs treated with ceramide-C6 (25 μM, Cer) compared with ethanol vehicle (Veh). The x axis displays the log₂-transformed fold changes in all proteins in the ceramide-treated samples relative to those in the vehicle control, and the y axis shows the −log₁₀-transformed P values derived from two-sided t tests performed to test the significance of the difference in fold changes. Highlighted in red and blue are ECM-related proteins that are up-regulated or down-regulated, respectively, by ceramide. (B) DAVID functional GO analysis of BP_Direct (top) and KW_Biological Process (bottom) protein enrichment. KW, keyword. Proteins with at least a fourfold difference between ceramide and vehicle conditions and P values of 0.001 or less were included for analysis. Shown on the x axis are the −log₁₀-transformed P values. (C) Heatmap of genes profiled by RNA sequencing (RNA-seq) induced (yellow) or repressed (blue) at ≥1.5-log fold change (FDR < 0.05) in HSCs treated with 25 μM ceramide-C6 compared with ethanol vehicle. Genes that regulate fibrogenesis and fibrolysis are shown. (D) Reverse transcription quantitative polymerase chain reaction (qRT-PCR) quantified expression of indicated genes after treatment with ethanol vehicle or ceramide-C6 (25 μM) for 6 hours. Samples were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (E) HSCs were nucleofected with a control-GFP plasmid (left) or an MMP-1–GFP promoter plasmid (right). After 24 hours, cells were treated with ethanol vehicle or ceramide-C6 (25 μM) for 6 hours. Relative fluorescence intensity was measured to quantify MMP-1 promoter activity. (F) Expression of pro MMP-1, active MMP-1, MMP-3, and GAPDH quantified by Western blotting after 6 hours of vehicle or ceramide treatment (top). Expression of pro MMP-1, active MMP-1, and GAPDH quantified by Western blotting after 48 hours of vehicle or ceramide treatment (bottom). Cropped gel images are shown. The antibody 10371–2-AP (Proteintech) was used to detect MMP-1. (G) HSCs were treated with vehicle or ceramide-C6 (25 μM) for 48 hours. MMP-1 expression measured from the conditioned medium. (H) Expression of pro collagen I and degraded collagen I quantified by Western blotting after 48 hours of vehicle or ceramide treatment. Cropped gel images are shown. (I) HSCs were transfected with nontargeting siRNA (Ctrl) or siRNA targeting aCDase (encoded by ASAH1) for 48 hours. qRT-PCR quantified expression of the indicated genes, and samples were normalized to GAPDH. (J) LX2 cells with CRISPR deletion of aCDase (ASAH1^KO) were compared with wild-type cells (ASAH1^WT) after plating on plastic for 48 hours. qRT-PCR quantified expression of indicated genes. Samples were normalized to GAPDH. Results are representative of three independent experiments. Data are means ± SEM. Unpaired two-sided Student’s t tests were used for (D), (E), (G), (I), and (J). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.. Ceramide promotes ECM degradation by HSCs.

(A) Schematic of HSC collagen degradation assay. (B) Human HSCs were transfected with nontargeting siRNA (Ctrl) or siRNA targeting MMP-1 (encoded by MMP1) and plated on rat tail collagen I for 48 hours and treated with ethanol vehicle (Veh) or 25 μM ceramide-C6 (Cer) for 24 hours. IF with F-actin (white), NucSpot (blue), type 1 collagen^3/4 fragment (Col 1^3/4 Frag, green), and SHG (purple). Scale bar, 50 μm. (C) Type 1^3/4 collagen intensity normalized to the overall average of all mean fluorescence intensity (MFI) values. Each dot represents one field with an average of 10 cells per treatment condition imaged at 20×. (D) Total clearance area measured using Imaris. Each dot represents one field imaged at 20×. (E) MMP-1 measured from the conditioned medium. (F) LX2 cells with CRISPR deletion of aCDase (ASAH1^KO) or wild-type cells (ASAH1^WT) were plated on rat tail collagen I for 48 hours. IF with F-actin (white), type 1 collagen^3/4 fragment (green), and SHG (purple). Scale bar, 50 μm. (G) Type 1^3/4 collagen intensity normalized to the overall average of all MFI values. Each dot represents one field with an average of 10 cells per treatment condition imaged at 20×. (H) Total clearance area measured using Imaris software. Each dot represents one field imaged at 20×. (I) Schematic of HSC-derived matrix. HSCs were maintained in ascorbic (5 μg/ml) and TGF-β (5 ng/ml) on glass cover slips for 10 days, treated with ethanol vehicle (Veh) or 25 μM ceramide-C6 (Cer) for 48 hours, and decellularized. (J) IF with type I collagen (collagen I, green) and SHG (purple) images. Scale bar, 50 μm. (K and L) Collagen type I and SHG intensity normalized to the overall average of all MFI values. Each dot represents one field with an average of 10 random fibers per treatment condition imaged at 20×. Results are representative of three independent experiments. Data are means ± SEM. Unpaired two-sided Student’s t tests [(G), (H), (K), and (L)] or one-way ANOVA with Tukey’s method for multiple comparisons was used [(C) to (E)]. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. Ceramide activates PKCα-ERK1/2-AP1 signaling to promote MMP-1 expression.

(A) Phosphoproteomic analyses at 1-, 5-, and 15-min (left to right) of ceramide-C6 (25 μM) treatment time points relative to ethanol vehicle. The significance of log₂-transformed phosphorylation site abundance fold change between the treated sample and the vehicle control was calculated using independent, two-sided t tests. The calculated log₁₀-transformed P values were plotted as a function of fold change (black). Dashed lines indicate a P value of 0.05 on the y axis and a log₂-transformed fold change of 0.5 on the x axis (top). Proteins implicated in collagen degradation are indicated in color (bottom). MARCKS, myristoylated alanine-rich protein kinase C substrate. (B) HSCs were treated with ethanol vehicle (Veh) or 25 μM ceramide-C6 (Cer) for 10 and 30 min. Proteins quantified by Western blotting with GAPDH as loading control. (C) HSCs were nucleofected with an MMP-1–GFP promoter plasmid. After 24 hours, cells were treated with ERK1/2 inhibitor SCH772984 (10 μM) or dimethyl sulfoxide (DMSO) for 1 hour followed by 25 μM ceramide-C6 or ethanol vehicle for 6 hours. Relative fluorescence intensity was measured to quantify MMP-1 promoter activity. (D and E) HSCs were treated with the ERK1/2 inhibitor SCH772984 (10 μM) or DMSO for 1 hour followed by 25 μM ceramide-C6 or ethanol vehicle for 6 hours. qRT-PCR (D) and Western blotting with GAPDH as loading control (E) were performed to quantify MMP-1 expression. (F and G) HSCs were treated with the PKC inhibitor Go6983 (10 μM) or DMSO for 1 hour followed by 25 μM ceramide-C6 or ethanol vehicle for 6 hours. qRT-PCR (F) and Western blotting (G) were performed to quantify MMP-1 expression. (H and I) HSCs were transfected with nontargeting siRNA (siCtrl) or siRNA targeting PKCα (encoded by PRKCA) for 48 hours and treated with 25 μM ceramide-C6 or ethanol vehicle for 10 min (H) or 6 hours (I). Proteins quantified by Western blotting with GAPDH as loading control. Anti-MAB901 antibody (R&D Systems) was used to quantify MMP-1. Results are representative of three independent experiments. Data are means ± SEM. One-way ANOVA with Tukey’s method for multiple comparisons was used for (C), (D), and (F). ***P < 0.001; ****P < 0.0001.

Fig. 4.. Ceramide promotes PKCα-ERK1/2-AP1 signaling to increase collagen degradation by HSCs.

(A) HSCs were treated with the ERK1/2 inhibitor SCH772984 (10 μM) or DMSO for 1 hour followed by 25 μM ceramide-C6 or ethanol vehicle for 24 hours. IF with F-actin (white), type 1 collagen^3/4 fragment (Col 1^3/4 Frag, green), and SHG (purple). Scale bar, 50 μm. (B) Type 1^3/4 collagen intensity normalized to the overall average of all MFI values. Each dot represents one field with an average of 10 cells per treatment condition imaged at 20×. (C) Total clearance area measured using Imaris. Each dot represents one field imaged at 20×. (D) HSCs were treated with the PKC inhibitor Go6983 (10 μM) or DMSO for 1 hour followed by 25 μM ceramide-C6 or ethanol vehicle for 6 hours. IF with F-actin (white), type 1 collagen^3/4 fragment (Col 1^3/4 Frag, green), and SHG (purple). Scale bar, 50 μm. (E) Type 1^3/4 collagen intensity normalized to the overall average of all MFI values. Each dot represents one field with an average of 10 cells per treatment condition imaged at 20×. (F) Total clearance area measured using Imaris. Each dot represents one field imaged at 20×. (G) HSCs were transfected with nontargeting siRNA (siCtrl) or siRNA targeting PKCα (encoded by PRKCA) for 48 hours and treated with 25 μM ceramide-C6 or ethanol vehicle for 24 hours. IF with F-actin (white), type 1 collagen^3/4 fragment (Col 1^3/4 Frag, green), and SHG (purple). Scale bar, 50 μm. (H) Type 1^3/4 collagen intensity normalized to the overall average of all MFI intensity values. Each dot represents one field with an average of 10 cells per treatment condition imaged at 20×. (I) Total clearance area measured using Imaris. Each dot represents one field imaged at 20×. Results are representative of three independent experiments. Data are expressed as means ± SEM. One-way ANOVA with Tukey’s method for multiple comparisons was used for (B), (C), (E), (F), (H), and (I). *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig. 5.. Inducible deletion of acid ceramidase promotes collagen degradation by HSCs in the CCl₄ mouse model.

(A) Asah1^flox/flox; Ai14; Col1a1-EGFP; Col1a2-CreER mice (male, ages 6 to 8 weeks) received a total of 6 weeks of CCl₄ or olive oil by intraperitoneal injection three times per week. During week 5, mice were concomitantly administered olive oil (control mice) or tamoxifen (100 mg/kg) by intraperitoneal injection daily for five doses to induce deletion of aCDase (Asah1^icKO mice). Mice were euthanized 72 hours after the last CCl₄ or olive oil injection. (B) Representative images of liver sections with hematoxylin and eosin (H&E) staining. Scale bar, 100 μm. (C) Representative images of liver sections with Sirius red staining. Scale bar, 100 μm. (D) Morphometric assessment of CPA on Sirius red–stained slides. n = 4, olive oil + Ctrl; n = 4, olive oil + Asah1^icKO; n = 9, CCl₄ + Ctrl; and n = 14, CCl₄ + Asah1^icKO. (E) Hepatic hydroxyproline was measured in mice from each treatment group. n = 4 olive oil + Ctrl; n = 4, olive oil + Asah1^icKO; n = 13, CCl₄ + Ctrl; and n = 12 CCl₄ + Asah1^icKO. (F) Representative IF images of liver tissues for GFP (green), tdTomato (red), and CHP (purple). Scale bar, 50 μm. (G) CHP intensity normalized to the overall average of all MFI values captured. Five liver sections were imaged and averaged per mouse, and each liver section included a minimum of 10 random fibers imaged at 20×. n = 4, olive oil + Ctrl; n = 4, olive oil + Asah1^icKO; n = 5, CCl₄ + Ctrl; and n = 5, CCl₄ + Asah1^icKO. (H) Representative images of decellularized liver sections analyzed by SHG microscopy. Scale bar, 50 μm (I) SHG intensity normalized to the overall average of all MFI values captured. Ten liver sections were imaged and averaged per mouse, and each liver section included a minimum of 10 random fibers imaged at 20×. (J) Quantification of collagen fiber width (micrometers) using CT-FIRE (collagen fiber image reconstruction and extraction). Seven liver sections were imaged and averaged per mouse. n = 3, CCl₄ + Ctrl; n = 3, CCl₄ + Asah1^icKO. (K) Asah1^flox/flox; Ai14; Col1a1-EGFP; Col1a2-CreER mice or Asah1^WT; Ai14; Col1a1-EGFP; Col1a2-CreER mice (male, ages 6 to 8 weeks) received a total of 6 weeks of CCl₄ by intraperitoneal injection three times per week. Beginning at week 5, all mice received tamoxifen (100 mg/kg) by intraperitoneal injection daily for five doses. HSCs were isolated from mice at 72 hours after the last CCl₄ injection. (L) qRT-PCR quantified expression of the indicated genes. Samples were normalized to Gapdh. n = 4, CCl₄+ Asah1^WT, and n = 4, CCl₄ + Asah1^icKO. Data are expressed as means ± SEM. Unpaired two-sided Student’s t tests [(I), (J), and (L)] or one-way ANOVA with Tukey’s method for multiple comparisons was used [(D), (E), and (G)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.. Pharmacologic inhibition of aCDase with compound 5A halts fibrosis progression.

(A) C57BL/6J mice received olive oil or CCl₄ three times a week by intraperitoneal injection for 4 weeks. Mice concomitantly received compound 5A (25 mg/kg) or vehicle by intraperitoneal injection five times a week for 4 weeks. (B) Liver weight–to–body weight ratio (liver proportional weight). (C) Representative images of liver sections with H&E staining. Scale bar, 100 μm. (D and E) Histologic evaluation of lobular inflammation and ballooning performed by blinded pathologist. (F) Representative images of liver sections with Sirius red staining. Scale bar, 100 μm. (G) Morphometric assessment of CPA on Sirius red–stained slides. n = 5, olive oil + Veh; n = 5, olive oil + 5A; n = 10, CCl₄ + Veh; and n = 10, CCl₄ + 5A. (H) Hepatic hydroxyproline was measured in mice from each treatment group. n = 5, olive oil + Ctrl; n = 4, olive oil + 5A; n = 9, CCl₄ + Ctrl; and n = 8, CCl₄ + 5A. (I) Representative IF images of liver tissues for 4′,6-diamidino-2-phenylindole (DAPI; blue) and CHP (purple). Scale bar, 50 μm. (J) CHP intensity normalized to the overall average of all MFI values captured. Five liver sections were imaged and averaged per mouse, and each liver section included a minimum of 10 random fibers imaged at 20×. n = 5, olive oil + Veh; n = 5, olive oil + 5A; n = 9, CCl₄ + Veh; and n = 10, CCl₄ + 5A. Data are expressed as means ± SEM. One-way ANOVA with Tukey’s method for multiple comparisons was used [(B), (D), (E), (G), (H), and (J)]. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7.. Compound 5A promotes fibrosis regression in the FPC model of MASH.

(A) C57BL/6J male mice (ages 6 to 8 weeks) received a normal diet (ND) or the FPC diet for 16 weeks. Beginning at week 13, mice received compound 5A (25 mg/kg) or vehicle by intraperitoneal injection five times a week for 3 weeks. (B) aCDase enzymatic activity measured in hepatic tissues. (C) Liver weight–to–body weight ratio (liver proportional weight). (D) Representative images of liver sections with H&E staining. Scale bar, 100 μm. (E to G) Histologic evaluation of steatosis, lobular inflammation, and ballooning performed by blinded pathologist. (H) Fasting blood glucose measured at week 15. (I and J) Insulin tolerance test (ITT) was performed at 16 weeks, and AUC was determined. (K) Representative images of liver sections with Sirius red staining. Scale bar, 100 μm. (L) Morphometric assessment of CPA on Sirius red–stained slides. (M) Representative images of liver sections imaged with TPEF (red) and SHG (green) microscopy. Scale bar, 50 μm. (N) SHG intensity normalized to the overall average of all MFI values captured. Five liver sections were imaged and averaged per mouse, and each liver section included a minimum of 10 random fibers imaged at 20×. n = 9, FPC + Veh, and n = 9, FPC + 5A. Data are means ± SEM. Unpaired two-sided Student’s t tests (N) or one-way ANOVA with Tukey’s method for multiple comparisons [(B), (C), (E), (F), (G), (H), (J), and (L)] was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8.. Ceramide signaling is associated with ECM remodeling in patients with MASLD.

(A) GSEA of MASLD-associated pathways in response to ceramide. The plot shows the top enriched pathways in the MASLD dataset associated with the CRS from a GSEA analysis using the GO BP gene sets. The top 20 path-ways both positively and negatively associated based on the NES for the pathways are shown. Bars represent individual pathways, with the length indicating the magnitude of the NES. The color gradient from blue to red represents the −log₁₀(P value), with darker red indicating higher statistical significance. Positive NES values (right side) indicate up-regulation, and negative values (left side) indicate down-regulation. Collagen-related pathways are highlighted in bold. UV, ultraviolet; cAMP, cyclic adenosine 3′,5′-monophosphate. (B) IPA of top pathways associated with MASLD ceramide responsiveness. The plot displays pathways in the MASLD dataset associated with the CRS in an IPA pathway analysis ranked by their activation z-score, predicting the activation state of the pathway (activated or inhibited). Bars represent individual pathways, with the length corresponding to the absolute z-score. Color gradient from blue to red indicates the −log₁₀(P value) of the enrichment, with darker red signifying higher statistical significance. Collagen-related pathways are highlighted in bold. GTPases, guanosine triphosphatases; MHC, major histocompatibility complex; L1CAM, L1 cell adhesion molecule; ER, endoplasmic reticulum; NAP1L1, nucleosome assembly protein 1-like 1; PKR, RNA-activated protein kinase; cGAS-STING, cyclic GMP-AMP synthase–stimulator of interferon genes; HER-2, human epidermal growth factor receptor 2; REM, Rad and Gem-related GTP-binding protein; RHOGDI, rho guanine nucleotide dissociation inhibitor; CREB, cAMP response element–binding protein; AMPK, adenosine monophosphate-activated protein kinase; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase; CDX, caudal type homeobox; TOB, transducer of ERBB2.