Rho-associated protein kinase 1 inhibition in hepatocytes attenuates nonalcoholic steatohepatitis

Background:

NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis.

Methods and Results:

Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis.

Conclusions:

Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

INTRODUCTION

NAFLD is an umbrella term for a range of conditions characterized by excessive hepatic lipid accumulation (steatosis) in individuals who drink little to no alcohol. Currently, NAFLD affects up to 25% of the world’s population, and the prevalence is also expected to continue to increase, driven in part by the obesity epidemic.1 A subset of NAFLD patients may develop a progressive form of the disease termed NASH. The hallmarks of NASH include hepatocyte lipid–induced injury and cell death (lipotoxicity), liver inflammation associated with the recruitment of monocyte-derived macrophages (MoM), and varying degrees of fibrosis.2 NASH may eventually progress to cirrhosis, end-stage liver disease, or liver cancer. Notably, it is predicted that NASH will become the leading underlying etiology for liver transplantation.3 Moreover, there are currently no FDA-approved drugs for NASH treatment, and the results of recent clinical trials for NASH have been suboptimal.4,5 Therefore, the discovery of critical pathogenic mediators that trigger NASH onset and progression may aid the development of potential mechanism-based therapies.

Rho-associated protein kinase (ROCK) 1 and ROCK2 are ubiquitous serine/threonine–specific protein kinases that regulate cell shape, adhesion, and movement by acting on the actin-myosin cytoskeleton.6 Albeit ROCK1 and ROCK2 have similar functional domains and significant amino acid identity, their cellular functions are often nonredundant.7 For example, ROCK1, but not ROCK2, is a downstream effector of apoptosis, where it mediates plasma membrane blebbing, apoptotic body formation, and cell shrinkage.8,9 We also identified an important role of ROCK1 in mediating the lipotoxicity-induced release of extracellular vesicles (EVs), known mediators of proinflammatory cell-to-cell communication.10–12 Based on animal studies, evolving evidence suggests that ROCK1 signaling also regulates glucose metabolism, insulin signaling, and energy metabolism and, thus, may be involved in the pathogenesis of metabolic syndrome and NAFLD.13 Recently, Huang and colleagues demonstrated the importance of hepatocyte-expressed ROCK1 for the development of obesity and fatty liver in mice on a high-fat diet.14 Specifically, genetic loss of ROCK1 in hepatocytes during high-fat feeding increased energy expenditure and attenuated weight gain and liver steatosis.14 Since this model of high-fat diet feeding does not induce steatohepatitis, it remains unknown whether ROCK1 inhibition can attenuate NASH and consequent fibrosis.

In this study, we investigated the role of ROCK1 in the development of NASH, with a particular focus on ROCK1 activity in hepatocytes and myeloid cells. We found that genetic deletion of ROCK1 in hepatocytes, but not in myeloid cells, attenuated the development of steatohepatitis and fibrosis. Blocking of ROCK1 activity using a novel small-molecule dual ROCK inhibitor (ROCKi) reversed disease severity in mice with established NASH. Therefore, hepatocyte-expressed ROCK1 seems to be a critical contributor to NASH development, and pharmacologic inhibition of ROCK1 may represent a potential therapeutic strategy.

METHODS

Animal studies and experimental design

All animal studies were performed at the Mayo Clinic and in accordance with and approved by the Institutional Animal Care and Use Committee. Male C57Bl/6J mice and LyzM-cre mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). ROCK1^f/f and hepatocyte-specific ROCK1 knockout (ROCK1^∆hep) generated by crossing ^ROCK1f/f mice with albumin-cre mice were described.14 ROCK1^f/f were also crossed with LyzM-cre mice to develop myeloid-specific ROCK1 knockout (ROCK1^∆mye) mice. All mice used had C57Bl/6J background. ROCK1^f/f and conditional knockout mice were cohoused in cages using corn cob bedding. Mice were kept at the Mayo Clinic Rochester animal facility with a 12-hour light-dark cycle. All mice were placed on a diet at 10 weeks of age. The chow diet was a standard rodent diet (PicoLab 5053, LabDiet) with tap water. Fat, fructose, and cholesterol (FFC) diet contains high fat (40% calories) and high cholesterol (0.2%) (#AIN-76A Western Diet; TestDiet), with fructose (18.9 g/L) and glucose (23.1 g/L) added to the drinking water (total sugar 42 g/L) as described.15 ROCK1^∆hep and ROCK1^∆mye mice and their littermate controls (ROCK1^f/f) were fed chow or FFC diet for 24 weeks. For the pharmacologic study, male C57Bl/6J mice were on chow or FFC diet for 24 weeks, after which they were randomized to receive ROCKi or vehicle by daily oral gavage for additional 3 weeks while continuing the diets. The ROCKi was resuspended in 0.4% carboxymethyl cellulose in water. Mice received 50 mg/kg of body weight per day and were weighed before every gavage to calculate the accurate dose of the drug. Vehicle-treated mice received equal volumes of 0.4% carboxymethyl cellulose in water. At the end of the feeding period, mice were killed under general anesthesia induced by a combination of xylazine and ketamine. The blood, epididymal fat, and liver were harvested and processed for further examination.

Mouse liver leukocyte isolation and mass cytometry analysis

Liver leukocytes were isolated using the liver dissociation kit (#130-105-807, Miltenyi Biotec) and Percoll (#17-0891-01, GE Healthcare) gradient as described by us.16,17 Cells were submitted to the Immune Monitoring Core at the Mayo Clinic, where mass cytometry time-of-flight (CyTOF) and data analyses were performed as described by us.16,17 Antibodies were conjugated to stable heavy-metal isotopes to detect cellular antigens (Supplemental Table S2, http://links.lww.com/HC9/A309). Data in .fcs files were normalized using CyTOF Software (version 6.7.1014). Cleaned .fcs files were analyzed by the R-based tool Cytofkit version 3.8. The R-phenograph algorithm clustering and dimensionality reduction were used for all 30 markers in the panel. Clusters were visually presented as tSNE maps and heatmaps.

Statistical analyses

Data are shown as violin plots with individual values and a black horizontal line representing the median. Differences among 3 and more groups were compared using a 1-way ANOVA followed by the Tukey post hoc test, in which p <0.05 was the minimum requirement for a statistically significant difference. The 2-tailed unpaired T test was used to detect differences between the two groups. All analyses were performed using GraphPad Prism 9.0 software.

Full details of the methods are included in the Supplemental Material and Methods document (http://links.lww.com/HC9/A308). (Supplemental Table S1, http://links.lww.com/HC9/A309)

RESULTS

Hepatic ROCK1 activity is increased in NASH

To address the hypothesis that ROCK1 contributes to NASH pathogenesis, we first investigated ROCK1 expression and activity in a well-established murine NASH model based on a FFC diet. The FFC diet feeding leads to obesity, insulin resistance, adipose tissue inflammation, and NASH with fibrosis, thus recapitulating the etiology and features of human metabolic syndrome and NASH.18,19 C57Bl/6J were fed chow or NASH-inducing FFC diet for 24 weeks, after which liver samples were harvested (Figure 1A). Hepatic ROCK1 mRNA level displayed no difference between chow and FFC-fed mice, while ROCK1 protein was significantly increased in the liver of FFC-fed mice (Figure 1B, C). Finally, we performed in vitro kinase activity assay using ROCK1 protein purified from liver lysates. Indeed, ROCK1 protein isolated from FFC livers displayed higher kinase activity compared with chow-fed mice (Figure 1D). We then examined hepatic ROCK1 expression in a cohort of patients with fatty liver (representing isolated steatosis) or NASH. ROCK1 was increased in NASH liver compared with the fatty liver at the mRNA level (Figure 1E), following a similar trend at the protein level (Figure 1F). Collectively, these data demonstrate that ROCK1 expression and activity are increased in murine and human NASH livers.

FIGURE 1.

Hepatic ROCK1 kinase activity is increased in murine NASH. (A) C57Bl/6J mice were fed chow or FFC diet for 24 weeks, after which liver samples were collected for further experiments depicted in “B–D.” (B) Hepatic mRNA expression of ROCK1 was measured in chow and FFC livers by qPCR. (C) Whole liver lysates from chow and FFC-fed mice were used for western blot for ROCK1 and HSP90 (a loading control). ROCK1 expression was normalized to HSP90 for quantification. (D) Whole liver lysates from chow and FFC livers were used to purify ROCK1 protein by immunoprecipitation. The kinase activity of purified ROCK1 was assessed by phosphorylation of an established ROCK1 substrate, recombinant MYPT1. (E and F) Liver specimens from patients with fatty liver or NASH were obtained: (E) RNA was isolated, and qPCR was performed for ROCK1; (F) whole liver protein was used for western blot for ROCK1 and GAPDH (a loading control). ROCK1 levels were normalized to GAPDH for quantification. N of samples indicated in the graphs. *p < 0.05, **p < 0.01. Abbreviations: FFC, fat, fructose, and cholesterol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP90, heat shock protein 90; MYPT1, myosin phosphatase targeting protein 1; ns, nonsignificant; ROCK1, rho-associated protein kinase 1.

Hepatocyte-specific ROCK1 deletion decreases hepatic steatosis and cell death in NASH, without affecting obesity, metabolic phenotype, and insulin resistance

Increased hepatic ROCK1 activity in NASH suggests that ROCK1 may be involved in the disease pathogenesis. To examine the role of ROCK1 in hepatocytes, we used ROCK1^∆hep mice. The lack of ROCK1 protein was confirmed in isolated hepatocytes (Supplemental Figure S1A, http://links.lww.com/HC9/A310). ROCK1^∆hep and littermate ROCK1^f/f mice were fed FFC or chow diet for a total of 24 weeks (Figure 2A). After 16 weeks of feeding, fasting glucose and insulin were measured to calculate HOMA-IR, a surrogate measure of insulin resistance. HOMA-IR values were significantly increased by FFC feeding but were not different between FFC-fed ROCK1^f/f and ROCK1^∆hep mice (Figure 2B, Supplemental Figure S1B, http://links.lww.com/HC9/A310). FFC-fed ROCK1^f/f and ROCK1^∆hep mice were also examined using metabolic cages and an Echo-MRI body composition analyzer after 18 weeks of feeding. We observed no difference in fat and lean mass, food intake, energy expenditure, and metabolic rate between ROCK1^∆hep and ROCK1^f/f mice on the FFC diet (Figure 2C, Supplemental Figure S1C–E, http://links.lww.com/HC9/A310). FFC-fed ROCK1^∆hep and ROCK1^f/f mice developed significant obesity with similar body weight gain, and similar liver and epidydimal white adipose tissue weights (Figure 2D, E, Supplemental Figure S1F–G, http://links.lww.com/HC9/A310). In further phenotypic studies, we noted marked hepatic steatosis in all FFC-fed mice (Figure 2F), while biochemical quantification of liver steatosis demonstrated a significant decrease in liver triglycerides in FFC-fed ROCK1^∆hep mice compared with FFC-fed controls (Figure 2G). Plasma alanine aminotransferase (ALT) activity, a marker of hepatocyte injury, was not statistically different between FFC-fed ROCK1^∆hep and control mice (463 vs 383 U/L, p = 0.1, Figure 2H). However, hepatic cell death, assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, was markedly decreased by ROCK1 deficiency (Figure 2I). Altogether, hepatocyte ROCK1 deficiency decreases liver steatosis and cell death, but it does not impact the development of obesity and metabolic derangements associated with insulin resistance in the FFC diet-based model of NASH.

FIGURE 2.

Hepatocyte-specific ROCK1 deletion reduces liver injury and steatosis in NASH without affecting metabolic phenotype. (A) ROCK1^f/f and ROCK1^∆hep mice were fed chow or FFC diet for 24 weeks, after which samples were collected for further analyses. (B) At 16 weeks of feeding, insulin resistance was approximated by HOMA-IR using fasting glucose and fasting insulin values. (C) After 18 weeks of feeding, mice were placed into an automatic CLAMS to measure energy expenditure. (D) Final body weight at the time of killing. (E) Liver weight normalized to body weight. (F) Representative “H and E” images of liver tissue. (G) Liver triglyceride content measured by a biochemical assay. (H) Plasma ALT activity. (I) Representative images and quantification of TUNEL assay for apoptotic cells in liver tissue. Arrowheads point to TUNEL-positive nuclei; scale bar is 50 µm. N of samples indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ALT, alanine aminotransferase; CLAMS, comprehensive laboratory animal monitoring system; FFC, fat, fructose, and cholesterol; HOMA-IR, homeostatic model assessment for insulin resistance; ns, nonsignificant; ROCK1, rho-associated protein kinase 1; ROCK1^∆hep, hepatocyte-specific ROCK1 knockout; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

ROCK1 deletion in hepatocytes reduces influx of monocyte-derived macrophages into the liver

To better understand the inflammatory response in the fatty liver microenvironment, we characterized liver leukocytes by CyTOF, which provides a high-parameter analysis of immune cells at a single-cell level. The liver leukocytes were labeled with antibodies against 30 markers to distinguish various immune cell types (T cells, B cells, dendritic cells, granulocytes, monocytes, and macrophages) and characterize multiple subpopulations. The clustering algorithm identified 29 clusters of phenotypically similar immune cells (Figure 3A). Unsupervised hierarchical clustering was mapped onto tSNE phenographs, showing the relative density of each cluster (Supplemental Figure S2A, http://links.lww.com/HC9/A310), and heatmaps showing marker intensities for each cluster and cluster frequencies for each sample (Figure 3B, C). Of note, FFC-fed ROCK1^∆hep mice clustered away from FFC-fed ROCK1^f/f mice and closer to chow mice. Moreover, we observed significant differences in the abundance of various leukocyte subpopulations between chow and FFC-fed mice, as well as FFC-fed ROCK1^f/f and FFC-fed ROCK1^∆hep (Figure 3C). The FFC diet increased the percentage of CX3CR1⁺ CD11b⁺ F4/80⁺ cells and lymphocyte antigen 6 complex, locus C (Ly6C)^high CD11b⁺ F4/80⁺ cells (Figure 3D), which represents monocyte-derived macrophages (MoM). The influx of these cells was decreased by hepatocyte ROCK1 deletion. In contrast, the percentage of F4/80⁺ cells coexpressing Tim4 or CD206, markers for resident and restorative macrophages, respectively, was significantly decreased by the FFC diet, and this effect was reversed by hepatocyte ROCK1 deficiency (Figure 3E). In addition, the FFC diet significantly elevated frequencies of lymphocyte antigen 6 complex, locus G (Ly6G)^high Ly6C and CD11b⁺ cells, representing neutrophils (Figure 3F). Similarly, the abundance of neutrophils was significantly reduced in FFC-fed ROCK1^∆hep mice. Together, these immunophenotyping results (Figure 3; Supplemental Figure S2B, http://links.lww.com/HC9/A310) illustrate profound changes in the hepatic immune landscape in relation to the NASH-inducing diet and hepatocyte ROCK1 deletion (Supplemental Figure S2B, http://links.lww.com/HC9/A310). Hepatocyte ROCK1 deficiency overall decreases the influx of immune cells with proinflammatory potential into the liver during FFC feeding.

FIGURE 3.

ROCK1 deletion in hepatocytes reduces the influx of MoM into the liver during NASH. Liver leukocytes were isolated from chow-fed ROCK1^f/f (n = 4), FFC-fed ROCK1^f/f (n = 4), and FFC-fed ROCK1^∆hep (n = 4) mice (24 wk feeding) and subjected to mass cytometry CyTOF. (A) Twenty-nine unique clusters were identified by a 30-marker panel using an R-phenograph clustering algorithm and visualized on a tSNE plot. (B) A heatmap demonstrating the distribution and relative intensity of markers across identified clusters. (C) A heatmap showing the relative abundance of clusters in each sample of liver leukocytes. (D–F) Cluster cell frequencies in total liver leukocytes and mean marker intensities in each cluster. N of samples indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CyTOF, cytometry time-of-flight; FFC, fat, fructose, and cholesterol; MoM, monocyte-derived macrophage; ns, nonsignificant; ROCK1, rho-associated protein kinase 1; ROCK1^∆hep, hepatocyte-specific ROCK1 knockout.

The fact that hepatocyte-specific ROCK1 deletion decreased immune cell recruitment into the liver suggests that ROCK1 may mediate cell-to-cell crosstalk, possibly through secreted factors. Since EVs are known to promote monocyte/macrophage migration,12 we assessed the release of EVs from ROCK1-deficient primary mouse hepatocytes under lipotoxicity. While lipotoxic treatment significantly increased EV release in control hepatocytes, this effect was completely abrogated in ROCK1-deficient hepatocytes (Supplemental Figure S3A, http://links.lww.com/HC9/A310). In addition, hepatocyte ROCK1 deletion also affected the protein composition of lipotoxic EVs, as evidenced by decreased enrichment in C-X-C motif chemokine ligand (CXCL10) (Supplemental Figure S3B, http://links.lww.com/HC9/A310), a chemokine known to be induced by lipotoxicity and mediate monocyte/macrophage migration.11,20,21

Hepatocyte-specific ROCK1 deletion attenuates inflammation and fibrosis during NASH

To corroborate immunophenotyping data suggesting attenuated myeloid cell infiltration in FFC-fed ROCK1^∆hep mice, we further investigated markers of inflammation in whole liver tissue samples. The FFC diet increased the abundance of cells positive for galectin-3 (Lgals3), a marker phagocytically active macrophages,22 within the liver parenchyma, which was significantly reduced in FFC-fed ROCK1^∆hep (Figure 4A). Next, we measured several inflammation markers at the mRNA level: markers of monocytes/macrophages (CD68 and CD14), markers of infiltrating monocytes (C-C motif chemokine receptor 2 (CCR2) and Ly6C), cytokines (TNF and IL12b), and chemokines (CXCL1, CXCL2, CXCL10, CCL3, CCL4, and CCL5). In line with the immunophenotyping data, hepatocyte-specific ROCK1 deletion significantly decreased FFC-induced inflammatory markers, suggesting attenuated inflammatory response in FFC-fed ROCK1^∆hep mice (Figure 4B–E). Because chronic liver injury and inflammation in NASH lead to tissue fibrogenesis, we assessed the extent of liver fibrosis in our cohort of mice using multiple methods. Sirius red staining and immunopositivity for alpha smooth muscle actin, a marker of activated HSCs, were significantly increased by FFC feeding and decreased by hepatocyte ROCK1 deficiency (Figure 4F, G). Collagen 1 mRNA and protein expression were significantly elevated in ROCK1^f/f FFC-fed mice compared with chow and decreased in ROCK1^∆hep FFC-fed mice (Figure 4H, I). Other markers of fibrosis, such as collagen 6a1 and TGFβ, were also decreased in ROCK1^∆hep FFC-fed mice compared with FFC-fed controls (Supplemental Figure S4, http://links.lww.com/HC9/A310). Taken together, hepatocyte-specific ROCK1 deletion decreases liver inflammation in FFC-fed mice. In line with reduced inflammation, we observed that hepatocyte ROCK1 deletion protects against liver fibrosis in murine NASH.

FIGURE 4.

Hepatocyte-specific ROCK1 deletion attenuates FFC diet-induced inflammation and fibrosis. ROCK1^f/f and ROCK1^∆hep mice were fed chow or FFC diet for 24 weeks, after which liver samples were collected for further analyses. (A) Liver tissue sections were stained with Lgals3 antibody, a macrophage marker, and staining was quantified as the percentage of Lgals3-positive area in a blinded manner. (B–E) Whole liver RNA was isolated and qPCR was performed for markers of monocytes/macrophages (CD68 and CD14), markers of infiltrating monocytes (CCR2 and Ly6C), cytokines, and chemokines. (F) Liver tissue sections were used for Sirius red staining detecting collagen deposition, and staining was quantified in a blinded manner. (G) Liver tissue sections were probed with αSMA antibody, a marker for activated HSCs, and quantified as the percentage of αSMA-positive area in a blinded manner. (H) Whole liver RNA was isolated, and qPCR was performed for markers of fibrogenesis. (I) Whole liver lysates from chow ROCK1^f/f, chow ROCK1^∆hep, FFC ROCK1^f/f, and FFC ROCK1^∆hep were isolated, and a western blot was performed for collagen 1 and HSP90 (a loading control). Collagen 1 expression was normalized by HSP90 levels; scale bar is 50 µm. N of samples indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: αSMA, alpha smooth muscle actin; CCR2, C-C motif chemokine receptor 2; FFC, fat, fructose, and cholesterol; HSP90, heat shock protein 90; Lgals3, galectin-3; Ly6C, lymphocyte antigen 6 complex, locus C; ns, nonsignificant; ROCK1, rho-associated protein kinase 1; ROCK1^∆hep, hepatocyte-specific ROCK1 knockout.

Myeloid cell-specific ROCK1 deletion did not affect liver injury, inflammation, and liver fibrosis in fat, fructose, and cholesterol diet-induced NASH

Before embarking on pharmacologic studies to inhibit ROCK1 activity in vivo, we aimed to examine the role of ROCK1 in the myeloid cells that are important drivers of NASH pathogenesis. ROCK1^∆mye mice were generated by crossing ROCK1^f/f and LyzM-cre mice, and ROCK1 deficiency was verified in bone marrow-derived macrophages (Supplemental Figure S5A, http://links.lww.com/HC9/A310). ROCK1^∆mye and ROCK1^f/f mice were placed on FFC or chow diet for 24 weeks (Figure 5A). Myeloid-specific ROCK1 deficiency did not affect FFC diet-induced weight gain, insulin resistance, metabolic features assessed in metabolic cages, liver weight, or liver injury (Figure 5B–E, Supplemental Figure S5B–F, http://links.lww.com/HC9/A310). Furthermore, myeloid-specific ROCK1 deletion did not change frequencies of MoM (CX3CR1⁺ or LyC6⁺) and other macrophages (Tim4⁺ or CD206⁺) in FFC-fed mice (Figure 5F, Supplemental Figure S5G, http://links.lww.com/HC9/A310). Liver inflammation (assessed by galectin-3 immunostaining) and fibrosis (quantified by Sirius Red staining) did not differ between FFC-fed ROCK1^∆mye and ROCK1^f/f mice (Figure 5G, H). Altogether, these data demonstrate that myeloid-specific ROCK1 activity plays an insignificant role in liver injury, inflammation, and fibrosis in the murine NASH model.

FIGURE 5.

Myeloid cell-specific ROCK1 deletion has no effect on liver injury and fibrosis in NASH. (A) ROCK1^f/f and ROCK1^∆mye mice were fed chow or FFC diet for 24 weeks, after which samples were collected for further analyses. (B) At 16 weeks of feeding, insulin resistance was approximated by the HOMA-IR using fasting glucose and fasting insulin values. (C) Final body weight at the time of killing. (D) Liver weight normalized to body weight. (E) Plasma ALT activity. (F) Liver leukocytes were isolated from chow-fed ROCK1^f/f, FFC-fed ROCK1^f/f, and FFC-fed ROCK1^∆mye mice and subjected CyTOF. Cluster frequency and marker expression are shown for a cluster that represents MoM, Kupffer cells, and restorative macrophages. (G) Liver tissue sections were stained with Lgals3 antibody, a macrophage marker, and staining was quantified as the percentage of Lgals3-positive area in a blinded manner. (H) Liver tissue sections were stained with Sirius red to visualize collagen deposition. Sirius red-positive area was quantified using polarized light in a blinded manner; scale bar is 50 µm. N of samples indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ALT, alanine aminotransferase; CyTOF, cytometry time-of-flight; FFC, fat, fructose, and cholesterol; HOMA-IR, homeostatic model assessment for insulin resistance; Lgals3, galectin-3; MoM, monocyte-derived macrophages; ns, nonsignificant; ROCK1, rho-associated protein kinase 1; ROCK1^∆mye,myeloid-specific ROCK1 knockout.

Pharmacological inhibition of ROCK activity ameliorates insulin resistance, NASH, and fibrosis

Despite significant efforts, ROCK1-selective inhibitors have not yet been successfully developed. It is likely because of the similarity between ROCK1 and ROCK2 structure and, thus, the lack of distinct structural features that could be exploited to design ROCK1-selective inhibitors. In contrast, a number of dual ROCK1/2 inhibitors exist. Thus, for our pharmacologic study, we searched through a compound library of dual ROCK1/2 inhibitors to identify a potent ROCK1 inhibitor. The selected ROCK1/2 inhibitor, termed ROCKi (Supplemental Figure S6A, http://links.lww.com/HC9/A310), was characterized in detail using multiple in vitro assays by us (Supplemental Figure S6B, http://links.lww.com/HC9/A310) and others.23 In a ROCK1 activity inhibition assay, ROCKi displayed nanomolar potency against ROCK1. To assess metabolic stability, the elimination rate of ROCKi was estimated by intrinsic clearance using mouse and human microsomes. About 84% and 93% of ROCKi were found to be bound to mouse plasma protein and human plasma protein, respectively. ROCKi had a LogD of 2.9 at pH 7.4 and a thermodynamic solubility of 2.4 μM, indicating favorable features for absorption from the gastrointestinal tract. Pharmacokinetic parameters of ROCKi were defined in C57Bl/6 mice after oral administration of a single dose of ROCKi. ROCKi displayed overall favorable pharmacokinetic properties (summarized in Supplemental Figure S6C, http://links.lww.com/HC9/A310). It is noteworthy that the concentration and exposure of ROCKi in the liver were about 5 and 7 times higher than in plasma, respectively, demonstrating that ROCKi favorably accumulates in the liver. Also, ROCKi showed a half-life (t_1/2) of 6.6 h in the liver and a shorter half-life in plasma (t_1/2 = 3.9 h). Our previous study suggested that ROCK1 activity mediates lipotoxic signaling in hepatocytes.10 To test the efficacy of ROCKi during hepatocyte lipotoxicity in vitro, the Huh7 cells were treated with lysophosphatidylcholine in the presence or absence of ROCKi. We observed that ROCKi effectively inhibited ROCK activity in lipotoxic hepatocytes, as evident by the diminished levels of phosphorylated myosin phosphatase targeting protein 1, a well-established ROCK substrate (Supplemental Figure. S6D, http://links.lww.com/HC9/A310). Finally, we tested ROCKi efficacy in a murine NASH model. C57Bl/6J mice were fed chow or FFC diet for 24 weeks and then received ROCKi (50 mg/kg) or vehicle by daily oral gavage for additional 3 weeks while continuing the diets (Figure 6A). Interestingly, ROCK inhibition significantly decreased fasting blood glucose and HOMA-IR in FFC-fed mice compared with vehicle controls (Figure 6B, Supplemental Figure S6F, http://links.lww.com/HC9/A310). Treatment with ROCKi had no effect on food intake, body weight, liver weight, and epidydimal white adipose tissue weight (Figure 6C, D, Supplemental Figure S6E, G, H, http://links.lww.com/HC9/A310). Although hepatocyte ROCK1 deletion attenuated NASH diet-induced steatosis, we observed no changes in liver triglyceride content and steatosis after ROCKi treatment in FFC-fed mice (Figure 6F, G). ROCK pharmacological inhibition protected the liver against FFC-induced injury and hepatocyte death, as evidenced by reduced plasma ALT levels and TUNEL assay (Figure 6E, H). As described, ROCK1 genetic deletion in hepatocytes attenuated liver inflammation in FFC-fed mice. In line with these findings, ROCKi-treated FFC-fed mice displayed decreased accumulation of liver macrophages (Figure 6I). It is also possible that ROCKi has a direct effect on monocyte recruitment. Indeed, monocyte pretreatment with ROCKi inhibited monocyte chemotaxis in vitro (Supplemental Figure S6I, http://links.lww.com/HC9/A310). Consistent with reduced injury and inflammation in ROCKi-treated mice, we also observed that ROCKi decreased activation of HSCs, measured by immunohistochemistry for alpha smooth muscle actin, and collagen deposition, suggesting that ROCKi can reverse already established liver fibrosis (Figure 6J, K). Taken together, treatment with ROCKi, a small molecule ROCK kinase inhibitor, effectively improved insulin resistance, liver injury, inflammation, and fibrosis in a murine model of NASH.

FIGURE 6.

ROCK1 pharmacological inhibition decreases insulin resistance, liver injury, and fibrosis in FFC-fed mice. (A) C57Bl/6J mice were fed chow or FFC diet for 24 weeks. Then, they received ROCKi or vehicle by daily oral gavage for additional 3 weeks while continuing the diets, after which samples were collected for further analyses. (B) On day 12 of the treatment, insulin resistance was approximated by the HOMA-IR using fasting glucose and fasting insulin values. (C) Final body weight at the time of killing. (D) Liver weight normalized to body weight. (E) Plasma ALT activity. (F) Representative H&E staining of the liver tissue. (G) Liver triglyceride content measured by a biochemical assay. (H) Representative images and quantification of TUNEL assay for apoptotic cells in liver tissue. (I) Liver tissue sections were stained with F4/80 antibody, a marker for macrophages, and quantified as the percentage of F4/80-positive area in a blinded manner. (J) Liver tissue sections were stained with Sirius red to visualize collagen deposition. Sirius red-positive area was quantified using polarized light in a blinded manner. (K) Liver tissue sections were stained with αSMA antibody, a marker for activated HSCs, and quantified as the percentage of αSMA-positive area in a blinded manner. Scale bar is 50 µm. N of samples indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ALT, alanine aminotransferase; αSMA, alpha smooth muscle actin; FFC, fat, fructose, and cholesterol; H and E, hematoxylin and eosin; HOMA-IR, homeostatic model assessment for insulin resistance; ns, nonsignificant; ROCK1, rho-associated protein kinase 1; ROCKi, ROCK inhibitor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

DISCUSSION

The current studies provide insights into mechanisms, by which ROCK1 activity in hepatocytes drives the pathogenesis of NASH. We built on our prior observations implicating ROCK1 in the release of proinflammatory EVs under lipotoxicity and metabolic actions in the context of obesity-induced metabolic disorders.10,14 However, the role of hepatocyte ROCK1 in the development of NASH, the more advanced and progressive form of NAFLD characterized by liver injury, inflammation, and fibrosis, has not been explored up until now. Given that liver fibrosis is associated with an increased risk of mortality and liver-related morbidity in patients with NAFLD,24 the effect of ROCK1 on liver fibrogenesis in NASH is of particular interest to the field and of high importance for potential drug development. Therefore, in the present study, we used a well-established model of NASH with fibrosis, which is based on feeding with the FFC diet that includes FFC.18 This murine model closely mirrors human NASH features, such as liver steatosis, lobular inflammation, and perisinusoidal chicken wire-like fibrosis.18,19,25 The principal findings of the present studies indicate that: (1) ROCK1 activity is increased in NASH liver; (2) hepatocyte-specific ROCK1 deletion attenuates FFC diet-induced liver steatosis, inflammation, and fibrosis, without affecting body weight and metabolic phenotype; (3) ROCK1 deletion in myeloid cells does not affect NASH in mice; and (4) pharmacologic inhibition ROCK activity ameliorates insulin resistance, liver injury, inflammation, and fibrosis in a murine model of NASH. These discoveries are discussed in greater detail in the following.

Prior studies have demonstrated that ROCK1 expression and activity increase in the murine and human steatotic liver compared with a healthy liver.14 Moreover, ROCK1 expression in human fatty liver positively correlated with risk factors clustering around NAFLD and insulin resistance. Following these insights, we were interested in ROCK1 biology in NASH, which, in contrast to fatty liver, may progress to more serious sequelae, such as cirrhosis, liver failure, and liver cancer. We observed that hepatic ROCK1 protein and activity were significantly increased during murine NASH. In addition, hepatic ROCK1 expression was higher in patients with NASH compared with patients with fatty liver. These data support the notion that ROCK1 may play a role in NASH development, representing a potential therapeutical target.

ROCK1 activity has been demonstrated to intricately control glucose metabolism, insulin signaling, and energy metabolism, that is, processes important for the development of NAFLD.14,26 Global ROCK1^-/- mice spontaneously develop systemic insulin resistance, likely due to impaired insulin signaling in skeletal muscle.27 In contrast, ROCK1 in adipocytes plays an inhibitory role in controlling insulin sensitivity and insulin signaling, underscoring that regulation of glucose homeostasis by ROCK1 is complex and tissue-dependent.28 Interestingly, ROCK1^∆hep mice displayed attenuated insulin resistance when on a high-fat diet (58% kcal from fat), which was accompanied by increased energy expenditure, lower body weight, and fat mass compared with the controls.14 In contrast, in our study, mice fed with an NASH-inducing diet (40% kcal from fat, 0.2% cholesterol, and fructose in drinking water) had no change in energy expenditure, fat and lean mass, insulin resistance, and body weight gain. These observations highlight that the composition of obesogenic diet may determine the outcomes of the metabolic phenotype in ROCK1^∆hep deficient mice although the underlying mechanisms are currently unclear. Consistent with prior data describing ROCK1 as an upstream regulator of de novo lipogenesis,14 ROCK1^∆hep mice displayed a decrease in liver steatosis when fed an NASH-inducing FFC diet. Given these and prior findings, ROCK1 represents a key regulator of hepatic lipid accumulation under nutrient excess.

A hallmark of NASH is the heightened recruitment of immune cells into the liver and consequent tissue inflammation. To characterize the liver immune landscape during hepatocyte ROCK1 deficiency, we used a mass cytometry (CyTOF)–based single-cell profiling of liver leukocytes isolated from chow-fed ROCK1^f/f, FFC-fed ROCK1^f/f, and FFC-fed ROCK1^∆hep mice. The CyTOF analysis revealed that FFC-diet causes a substantial influx of immune cells into the liver. As anticipated, this immune response was strongly linked to an increase in MoM and elevated expression of monocyte/macrophage-associated proinflammatory cytokines and chemokines. In contrast, Kupffer cells and restorative macrophages nearly disappeared in NASH livers, consistent with the hypothesis that Kupffer cells during NASH are replaced with bone marrow–derived macrophages.29 Interestingly, we found that ROCK1 deletion in hepatocytes attenuated the influx of MoM, restored numbers of resident macrophages, and attenuated inflammatory response as evidenced by decreased expression of inflammatory cytokines and chemokines. The exact link between hepatocyte ROCK1 activity and liver inflammation is presently unclear, but we suspect possible intercellular crosstalk through hepatocyte-derived EVs in this process. Our prior studies demonstrated that lipotoxicity in cultured hepatocytes and hepatocyte cell lines leads to increased release of EVs, which was inhibited by ROCK1 knockdown or kinase inhibition10, which was validated in primary mouse hepatocytes in the current study. Prior mass spectrometry analyses of control and lipotoxic hepatocyte EVs showed that lipotoxic EVs are enriched in various proteins and lipids known to mediate the chemotaxis of monocytes or macrophages.2,11 Indeed, lipotoxic hepatocyte EVs have a potent, CXCL10-dependent chemotactic potential for monocytes/macrophages, and the capacity to promote macrophage proinflammatory activation and cytokine secretion.10,11,30 Thus, it is plausible that loss of ROCK1 in hepatocytes during NASH prevents the release of lipotoxicity–induced proinflammatory EVs, resulting in attenuated monocyte recruitment and immune response. This hypothesis warrants further mechanistic in vivo studies. We also observed that NASH-associated pericellular collagen deposition was attenuated by hepatocyte-specific ROCK1 deletion. As tissue inflammation is a strong driver of liver fibrogenesis, we speculate that reduced liver fibrosis in FFC-fed ROCK1^∆hep mice might have been a consequence of attenuated macrophage-associated hepatic inflammation in these mice. However, we cannot exclude a direct link between hepatocyte ROCK1 and HSCs. For example, it is conceivable that ROCK1 may also contribute to the phenotype of attenuated fibrosis in FFC-fed ROCK1^∆hep mice through the release of lipotoxicity induced EVs. Prior in vitro studies have demonstrated that toxic lipid-treated hepatocytes secrete EVs that promote HSC activation and migration,31 suggesting a direct link between hepatocyte stress and liver fibrogenesis.

Given the beneficial outcomes of hepatocyte ROCK1 deletion in murine NASH and, thus, the potential for therapeutic targeting, we were interested in the role of ROCK1 in other cell types implicated in NASH, such as monocytes and macrophages. Given that ROCK1 in macrophages was shown to regulate macrophage chemotaxis and cholesterol metabolism,32 we hypothesized that ROCK1 in myeloid cells could also be essential for MoM accumulation in NASH livers. Thus, we placed ROCK1^∆mye mice on the FFC diet and performed CyTOF analysis on isolated liver leukocytes. To our surprise, we observed no changes in liver immune cell composition between ROCK1^∆mye mice and control mice on the FFC diet. Likewise, other markers’ NASH-associated liver inflammation and fibrosis were not affected by ROCK1 deficiency in myeloid cells. Thus, ROCK1 specifically in hepatocytes, but not in myeloid cells, is crucial for the hepatic accumulation of MoM and tissue inflammation during NASH.

In the last decade, NASH has become a significant public health concern, further confounded by the lack of FDA-approved drugs for the treatment of the disease. For these reasons, there is a great need to identify novel therapeutic strategies that would promote NASH resolution and fibrosis regression. Our results in ROCK1^∆hep mice indicate that pharmacologic inhibition of ROCK1 may possess a promise for effective NASH therapy. Of note, ROCK1-selective inhibitors have not yet been successfully developed, but there are numerous dual ROCK1/2 inhibitors. Thus, we had to identify a potent ROCK1 inhibitor with some ROCK2 suppressive activity by screening a compound library of dual ROCK1/2 inhibitors. Based on low nanomolar potency against ROCK1, a suitable dual ROCK1/2 inhibitor {3-[4-(1-H-pyrazol-4-yl)-phenyl]-1-(3-methoxybenzyl)-1-methylurea} was chosen and termed ROCKi. The structure and properties of ROCKi were first published by Yin et al.23 who synthesized ROCKi as an ROCK kinase inhibitor based on the structure-activity relationship. ROCKi seems to be quite ROCK1/2-selective as it does not inhibit the activity of MRCKα, JNK3, p38α, and protein kinase A (PKA) in a nanomolar concentration range.23 For example, ROCKi exhibits >400-fold ROCK1 selectivity over PKA. ROCKi was further evaluated by our team in terms of pharmacodynamics and pharmacokinetics, which confirmed a low nanomolar potency for inhibiting ROCK1 and favorable pharmacokinetic properties. Compared with the well-established ROCK1 inhibitor Y-27632, the inhibition constant of ROCKi is about 70-fold smaller than that of Y-27632.33 Administration of ROCKi to mice with already established NASH reversed multiple hallmarks of NASH, including liver injury, macrophage accumulation, and, most importantly, fibrosis. Interestingly, ROCKi treatment also improved insulin resistance in FFC-fed mice, as demonstrated by decreased HOMA-IR. This distinct effect of ROCKi may be due to its potential to inhibit ROCK1/2 in insulin-sensitive tissues. Indeed, adipose-specific deletion of ROCK1 improved insulin sensitivity in mice fed a high-fat diet.28 Also, haplodeficient ROCK2 mice fed a high-fat diet displayed improved insulin sensitivity through enhanced thermogenesis.34 The positive effect of ROCKi on insulin resistance effect would be beneficial, especially for patients with concurrent NASH and type 2 diabetes.

In contrast to ROCK1 genetic deletion in hepatocytes, ROCKi treatment did not affect liver steatosis caused by FFC feeding. This dissimilar effect on steatosis mediated by genetic versus pharmacologic inhibition may be due to the length of ROCK1 inhibition. The genetic inhibition of ROCK1 was present since the initiation of the FFC diet, while pharmacologic inhibition of ROCK1 activity lasted only 3 weeks and was applied to already advanced steatosis. The open question remains whether a prolong treatment with ROCKi or a higher dosage would decrease steatosis in the FFC model. ROCKi treatment also inhibited liver inflammation in NASH mice, which could be a consequence of the decreased hepatocellular injury and EV release, as well as a direct effect of ROCKi on monocytes. In vitro, ROCKi pretreatment inhibited monocyte chemotaxis, which we speculate was due to the inhibition of ROCK2 rather than ROCK1, given that genetic deletion of ROCK1 in myeloid cells did not prevent monocyte recruitment in FFC-fed NASH mice. Indeed, ROCK2 inhibition was shown to decrease monocyte migration.35 Most importantly, liver fibrosis was attenuated by both genetic and pharmacologic inhibition of ROCK1.

In summary, the present studies identified new mechanisms by which hepatocyte ROCK1 activity promotes NASH with fibrosis. Besides the known role of hepatocyte ROCK1 in promoting liver steatosis,14 here, we demonstrate that ROCK1 activity promotes hepatic recruitment of various subsets of myeloid cells, leading to tissue inflammation and fibrosis. In contrast to hepatocyte ROCK1 activity, ROCK1 activity in myeloid cells is dispensable for NASH development. In addition, we show that treatment with a novel ROCKi promotes the resolution of steatohepatitis and the improvement of fibrosis in mice with established NASH. These findings are relevant to potential anti-inflammatory and anti-fibrotic therapeutic strategies for NASH based on ROCK1 kinase inhibition.

Supplementary Material

hc9-7-e0171-s001.pdf ^{(112.2KB, pdf)}

hc9-7-e0171-s002.pdf ^{(123.8KB, pdf)}

hc9-7-e0171-s003.pdf ^{(2.3MB, pdf)}