Hic-5 antisense oligonucleotide inhibits advanced hepatic fibrosis and steatosis in vivo

Abstract

Background & aims: Chronic liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH), pose a significant global health burden. Progressive liver fibrosis can lead to severe outcomes; however, there is a lack of effective therapies targeting advanced fibrosis. Hydrogen peroxide-inducible clone-5 (Hic-5), an adaptor protein in focal adhesion, is critical for promoting liver fibrosis in hepatic stellate cells. This study investigated its clinical applicability by examining hepatic Hic-5 expression in human fibrotic tissues, exploring its association with MASH, and assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting Hic-5 in a MASH mouse model.

Methods: Hepatic Hic-5 expression in human fibrotic tissues underwent pathological image analysis and single-cell RNA sequencing. ASOs targeting Hic-5 were developed and tested using in vitro cell models. An in vivo MASH mouse model was used to evaluate the effects of anti-Hic-5 ASOs on advanced fibrosis and steatosis.

Results: Hepatic Hic-5 expression increased with the progression of fibrosis, particularly in advanced stages. Single-cell RNA sequencing revealed Hic-5 expression primarily in hepatic stellate cells. In MASH-associated fibrosis, Hic-5 expression correlated with the expression of fibrotic genes. In the MASH mouse model, hepatic Hic-5 expression increased with disease progression. Anti-Hic-5 ASOs effectively suppressed Hic-5 expression in vitro and attenuated advanced fibrosis and steatosis in vivo, indicating their therapeutic potential.

Conclusions: Hepatic Hic-5 expression is associated with advanced liver fibrosis and MASH. Anti-Hic-5 ASOs are promising therapeutic interventions for MASH accompanied by advanced fibrosis. These findings provide valuable insights into potential clinical treatments for advanced liver fibrosis.

Impact and implications: This study investigated the role of Hic-5 in liver fibrosis and steatohepatitis, highlighting its potential as a therapeutic target. We developed an antisense oligonucleotide (ASO) that was particularly transportable to the liver, and targeted Hic-5. Anti-Hic-5 ASO exhibited therapeutic efficacy for liver fibrosis and steatosis in vivo, indicating its therapeutic potential for liver fibrosis and steatosis. ASOs have already achieved dramatic therapeutic effects as approved nucleic acid drugs. Thus, anti-Hic-5 ASO is expected to lead the direct generation of seed compounds for the clinical development of drugs for liver fibrosis and steatosis.

Keywords: ASO; Extracellular matrix; Hic-5; Liver fibrosis; MASH.

Graphical abstract

Fig. 1

Hic-5 expression increases with human liver fibrosis progression. (A) Representative images of Hic-5 and Masson’s trichrome (MT) staining of human liver (fibrosis stages F0/F1/F2/F3/F4: n = 11/12/13/10/8). Scale bars: 200 μm. (B) Hic-5-positive areas in patient livers with fibrosis. (C) Linear correlation between fibrosis area and Hic-5-positive area (R, Pearson’s coefficient). (D) Representative immunofluorescence images of Hic-5 (magenta). Nuclei were stained with DAPI. Scale bars: 50 μm. (E) Hic-5 fluorescence intensity per unit Hic-5-positive area. (F) Linear correlation between fibrosis area and Hic-5 fluorescence intensity per unit Hic-5-positive area. Values are means ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, Tukey’s multiple comparison test (B, E).

Fig. 2

HIC-5 is associated with MASLD progression. (A) HIC-5 in healthy and cirrhotic human liver cells. Dashed lines indicate mesenchymal cell clusters. (B) HSCs expressing HIC-5, COL1A1, and COL1A2 in healthy and cirrhotic livers (n = 3/group). (C) HIC-5 in livers of healthy individuals and MASLD with fibrosis (N/F0/F1/F2/F3/F4: n = 31/35/30/27/8/12). N: Healthy. (D) Linear correlation between HIC-5 and COL1A1 and between HIC-5 and COL1A2. (E) HIC-5 in livers of patients with MASLD and MASH (MASLD/MASH: n = 47/51). (F) Linear correlation between HIC-5 and COL1A1 and between HIC-5 and COL1A2. Values are mean ± SEM. ∗p <0.05, ∗∗∗p <0.001, two-tailed Student’s t test (B) and Tukey’s multiple comparison test (C, E). HSCs, hepatic stellate cells; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; SMCs, smooth muscle cells.

Fig. 3

Hic-5 expression in a MASH model. (A) Representative images of H&E, SR, and Oil Red O staining in livers of mice fed standard chow for 6 weeks (control) or CDAHFD for 3–15 weeks. (B) Quantitation of hepatic fibrosis. Scale bars: 200 μm (n = 2–3/group). (C) Quantitation of steatosis. (D) Hic-5 expression. (E) Col1a1 expression and analysis of its linear correlation with Hic-5. (F) Col1a2 expression and analysis of its linear correlation with Hic-5. Control: mice fed standard chow for 6 weeks. Values are mean ± SEM. ∗p <0.05, ∗∗p <0.01, and ∗∗∗p <0.001, Dunnett’s multiple comparison test. CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; SR, Sirius Red.

Fig. 4

Hic-5 deletion attenuates fibrosis and steatosis. (A) Representative images of H&E, SR, and Oil Red O staining in livers of mice fed standard chow for 6 weeks (control) or CDAHFD for 3–15 weeks. (B) Quantitation of hepatic fibrosis. Scale bars: 200 μm (n = 4–7/group). (C) Quantitation of steatosis. (D) Col1a1 and Col1a2 expression. (E) Lox, Loxl1, and Loxl2 expression. (F) Representative immunofluorescence images of Hic-5 (magenta) and α-SMA (green). Nuclei were counterstained with DAPI (blue). Scale bars: 10 μm. Values are mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, two-tailed Student’s t test. α-SMA, alpha-smooth muscle actin; CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; SR, Sirius Red.

Fig. 5

In vivo knockdown of Hic-5 decreases fibrosis and steatosis. (A) Timeline of the experimental procedure (n = 8/group). (B, C) Representative images of HE, SR, and Oil Red O staining in mouse livers. ‘Start’ indicates baseline at initiation of siRNA administration. Scale bars: 200 μm (n = 3–8/group). Quantitation of (D) hepatic fibrosis and (E) steatosis. (F) Col1a1 and Col1a2 expression. (G) Lox, Loxl1, and Loxl2 expression. Values are means ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, two-tailed Student’s t test. CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; siRNA, small interfering RNA; SR, Sirius Red.

Fig. 6

Activities of anti-Hic-5 ASOs in vitro. (A) Alignment of Hic-5 mRNA sequences from mammalian genomes. (B, C) Hic-5 mRNA in cells transfected with anti-Hic-5 ASOs. (-) Control. (D) Cell viability of mouse VSMCs and NIH3T3 cells with ASOs. (E) Caspase 3/7 activation induced by ASOs in mouse VSMCs and NIH3T3 cells. (F) Representative double immunofluorescence images of Hic-5 (green) and phalloidin (red) in human HSCs transfected with anti-Hic-5 ASOs. (-) Control. Nuclei were counterstained with DAPI (blue). Original magnification: 400 × . Scale bars: 20 μm. (G) Hic-5 protein levels. (H) Gymnosis experiments. Values are mean ± SEM. ∗p <0.05, ∗∗p <0.01, Dunnett’s multiple comparison test. ASOs, antisense oligonucleotides; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HSCs, hepatic stellate cells; VSMCs, vascular smooth muscle cells.

Fig. 7

Anti-Hic-5 ASO attenuates advanced fibrosis and steatosis. (A) Timeline of the experimental procedure (n = 6/group). (B, C) Representative images of HE, SR, and Oil Red O staining in mouse livers. ‘Start’ indicates baseline at initiation of ASO administration. Scale bars: 200 μm (n = 6/group). (D) Quantitation of hepatic fibrosis. (E) Quantitation of steatosis. (F) Col1a1 and Col1a2 expression. (G) Lox, Loxl1, and Loxl2 expression. Values are mean ± SEM. ∗p <0.05, ∗∗p <0.01, two-tailed Student’s t test. ASOs, antisense oligonucleotides; CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; SR, Sirius Red.