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Precision Medicine Lab, Biological Resource Center, Department of Transfusion Medicine, Fondazione Ca’ Granda Ospedale Maggiore Policlinico Milano, Milano 20122, Italy
Precision Medicine Lab, Biological Resource Center, Department of Transfusion Medicine, Fondazione Ca’ Granda Ospedale Maggiore Policlinico Milano, Milano 20122, Italy
Division of Gastroenterology and Hepatology, University of Missouri, Columbia, MO 65211, USADepartment of Nutrition and Exercise Physiology, School of Medicine, University of Missouri, Columbia, MO 65211, USA
Division of Gastroenterology and Hepatology, University of Missouri, Columbia, MO 65211, USAResearch Service, Harry S Truman Memorial Veterans Medical Center, Columbia, MO 65201, USADepartment of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65211, USA
Division of Gastroenterology and Hepatology, University of Missouri, Columbia, MO 65211, USADepartment of Nutrition and Exercise Physiology, School of Medicine, University of Missouri, Columbia, MO 65211, USAResearch Service, Harry S Truman Memorial Veterans Medical Center, Columbia, MO 65201, USA
Precision Medicine Lab, Biological Resource Center, Department of Transfusion Medicine, Fondazione Ca’ Granda Ospedale Maggiore Policlinico Milano, Milano 20122, ItalyDepartment of Pathophysiology and Transplantation, Universita`degli Studi di Milano, Milan 20122, Italy
Department of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA
Nonalcoholic steatohepatitis (NASH)-induced liver fibrosis is emerging as the most common cause of liver disease. For evaluation of therapies, there is a pressing need to identify non-invasive, mechanism-based biomarkers. A pro-fibrotic process relevant to human NASH involves a pathway in which a transcriptional regulator called TAZ (WWTR1) in hepatocytes induces the secretion of pro-fibrotic Indian hedgehog (IHH). We therefore reasoned that circulating IHH may be a useful mechanism-based marker to assess changes in NASH fibrosis.
Methods
Circulating IHH was assessed in wild-type and hepatocyte-TAZ-silenced NASH mice and in three separate cohorts of patients with mild-moderate NASH.
Results
Circulating IHH was elevated in mice with diet-induced NASH compared with chow-fed mice or with NASH mice in which hepatocyte-TAZ was silenced, which is an effective means to decrease NASH fibrosis. In patients with fatty liver disease with or without NASH, NASH fibrosis was associated with increased concentrations of circulating IHH.
Conclusion
The results of these analyses support further investigation to determine whether circulating IHH may be useful as a mechanism-based indicator of target engagement in anticipated future clinical trials testing NASH fibrosis therapies that block the IHH pathway.
I.T. and X.W. have has received research funding (unrestricted) from Takeda Pharmaceuticals. L.V. has been an invited speaker for MSD, Gilead, AlfaSigma, and AbbVie; consults for Gilead, Pfizer, Astra Zeneca, Novo Nordisk, Intercept pharmaceuticals, Diatech Pharmacogenetics, IONIS, Viatris, Boehringer and Ingelheim; and has received research funding (unrestricted) from Gilead.
Financial support
This work was supported by NIH grants R01DK116620 and R01DK133694 (to I.T.); an American Liver Foundation Liver Scholar Award (to X.W.); and a grant from the Ines Mandl Research Foundation (to M.P.M.). P.D. and M.M. are supported by the Italian Ministry of Health (Ricerca Corrente 2022 to P.D. and GR-2019-12370172 to M.M.). L.V. is supported by the Italian Ministry of Health (Ministero della Salute), Ricerca Finalizzata RF-2016-02364358 and Rete Cardiologica “CV-PREVITAL”; Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Ricerca corrente; Fondazione IRCCS Ca’ Granda “Liver BIBLE” (PR-0391); Gilead_IN-IT-989-5790; and several European Union (EU) grants: Horizon 2020 research and innovation programme, EFPIA Programme Horizon 2020 under grant agreement No. 777377 for the project LITMUS, and programme “Photonics” under grant agreement “101016726”. NIH grant R01DK113701 supported R.S.R, E.J.P, J.A.I. and also the collection of human samples included in this study (see cohort 3).
Author contributions
M. Moore, X. W, and I.T. were involved in the study conception and experimental design. X.W, M.P.M, and H.S. conducted mouse experiments, liver analyses, and plasma and serum assays. M. Meroni, A.C, L.R, E.J.P, J.A.I, R.S.R, L.V, and P.D. were involved in conducting and analyzing the human studies and providing important intellectual contributions. M. Moore, X.W, and I.T. drafted the manuscript. All authors revised the manuscript and approved the final version.
Introduction
Non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of cirrhosis worldwide
American association of clinical endocrinology clinical practice guideline for the diagnosis and management of nonalcoholic fatty liver disease in primary care and endocrinology clinical settings: co-sponsored by the American Association for the Study of Liver Diseases (AASLD).
Prevalence of nonalcoholic steatohepatitis-associated cirrhosis in the united states: an analysis of national health and nutrition examination survey data.
. Disease progression from simple steatosis to NASH is caused by multiple insults that cause liver inflammation, hepatocellular death, and, most importantly, histological liver fibrosis, which correlates best with clinical outcome in NASH
. As there are no FDA-approved drugs to treat NASH, there is a critical need for novel therapies that can halt or reverse progression to liver fibrosis
. Accordingly, these efforts will require non-invasive approaches to identify patients at increased risk of NASH, assess disease progression, and monitor response to therapy.
A key process in NASH fibrosis is activation of collagen-producing hepatic stellate cells (HSCs)
. One mechanism involves cholesterol-induced upregulation in hepatocytes of the transcriptional regulator TAZ (WWTR1), which induces the synthesis and secretion of the HSC activator, Indian hedgehog (IHH)
. This pathway is supported by showing increased TAZ and IHH in human NASH vs. steatotic liver and by causation studies using primary human and mouse hepatocytes and mouse NASH models
. For example, silencing hepatocyte TAZ or IHH blocks NASH progression, and the improvement in NASH by silencing hepatocyte TAZ is abrogated by genetically restoring hepatocyte IHH
siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.
Given the importance of secretory IHH, we reasoned that circulating IHH may be a useful mechanism-based marker related to histological features of NASH and fibrosis. We show here that circulating IHH increases in NASH in mice in a hepatocyte-TAZ-dependent manner and associates with NAFLD activity score and liver fibrosis in humans with mild-moderate NASH. These findings support further investigation into whether plasma IHH may be useful as an indicator of target engagement in anticipated trials testing NASH fibrosis therapies that block the IHH pathway.
Material and methods
Animal Studies
Male wild-type C57BL/6J mice (10-week-old) were obtained from Jackson Laboratory (#000664; Bar Harbor, ME, USA) and assigned randomly to received chow diet (PicoLab rodent diet 20, #5053) or a NASH-inducing diet rich in fructose, palmitate, and 1.25% cholesterol (FPC; TD.160785; with drinking water containing 23.1 g/L fructose and 18.9 g/L glucose)
genome copies/mouse) at 8 weeks for the 16-week protocol. Plasma was collected and livers were harvested and snap-frozen or formalin fixed as previously described
. Experiments complied with guidelines of the Columbia Animal Care and Use Committee.
Human Samples
Three separate cohorts were included. Patients gave informed consent, and protocols were approved by the Institutional Review Board the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milan (Cohorts 1 and 2) or the University of Missouri (Cohort 3) and conducted according to the World’s Medical Association Declaration. Patient records were pseudo-anonymized and de-identified. See supplemental information for description of tissue and blood collection and analyses.
Results
Plasma IHH is increased in experimental NASH and lowered by silencing hepatocyte-TAZ
We assayed plasma IHH in mice fed the NASH-inducing FPC diet for 16 or 28 weeks and in mice in which hepatocyte TAZ was silenced between weeks 8-16. Metabolic, biochemical, and histologic data for the mice have been previously published
. Plasma IHH levels were very low in chow-fed mice but became markedly increased in mice fed the FPC diet for 16 and 28 weeks (Figure 1A). Further, in 16-week-fed mice, silencing hepatocyte TAZ between weeks 8-16, which lowers liver fibrosis and IHH
, markedly reduced plasma IHH (Figure 1B). Thus, in experimental NASH, plasma IHH is elevated, and it is lowered by silencing hepatocyte-TAZ.
Figure 1Plasma IHH is increased in experimental NASH and lowered by silencing hepatocyte-TAZ. (A) Plasma IHH of mice fed chow or FPC diet for 16 and 28 weeks (n = 4 mice/group; **P<0.01, ***P<0.001, ****P<0.0001). Data were analyzed with a one-way analysis of variance. (B) Plasma IHH of mice fed the FPC diet for 16 weeks, with AAV8-H1-shTaz or control (shCon) vector injected at week 8 (n = 5 mice/group, **P < 0.01). Data were analyzed with a two-tailed Student's t-test.
Plasma IHH is increased in humans with NASH fibrosis
In Cohort 1, which included 96 patients who underwent liver biopsy for suspected NASH, plasma IHH was ∼2-fold higher in subjects with histologically confirmed NASH and mild-moderate fibrosis versus simple steatosis or without non-alcoholic fatty liver disease (NAFLD) (Figure 2A and Supplemental Table 1). Further, plasma IHH was higher in subjects with higher NAFLD activity score (Figure 2B) and in NASH subjects with liver fibrosis versus NASH subjects without liver fibrosis (Figure 2C). Plasma IHH also correlated positively with serum ALT and AST (Figure 2D-E). The finding of increased circulating IHH in NASH versus control subjects was reproduced in Cohort 2, which included patients (n = 22) who were overweight or obese and referred for liver biopsy to diagnosis NASH (Figure 2F and Supplemental Table 2). In addition, there was a positive correlation between IHH-positive area in immunostained liver sections and serum IHH (Figure 2G). In cohort 3, which consisted of patients (n = 48) with morbid obesity undergoing bariatric surgery as described
, serum IHH was higher in NASH subjects with histologically confirmed mild-moderate liver fibrosis versus NASH subjects without fibrosis (Figure 2H and Supplemental Table 3). Although the wedge liver biopsies from these bariatric surgery patients may contain loci of capsular and subcapsular fibrosis, these findings support our findings in cohort 1 and 2 that plasma and serum IHH is elevated in the setting of NASH fibrosis.
Figure 2Plasma IHH is increased in patients with NASH and NASH-associated liver fibrosis. (A) Plasma IHH concentrations in control (n = 29), steatosis (n = 17), and NASH + fibrosis (n = 50) subjects from Cohort 1. (B-C) Data from panel A were stratified based on NAS and fibrosis score. (D-E) Pearson R correlation of the relationship between plasma IHH (LogIHH) and serum ALT (LogALT) or AST (LogAST) in Cohort 1. (F-G) Serum IHH in subjects without NAFLD (control; n = 4) or with NASH (n = 18) and Pearson R correlation of the relationship between serum and liver IHH in subjects from Cohort 2. (H) Serum IHH in obese NASH subjects without or with fibrosis (n = 22 and 25) from Cohort 3. Grouped data were analyzed using a one-way analysis of covariance. For panels A-C, F, and H the data are expressed as mean ± SE relative to the first group in each graph (*P < 0.05, **P < 0.01, ***P < 0.001).
The premise for this study was based on recently published pre-clinical work, backed by studies of human NASH liver and human hepatocytes, suggesting that targeting the TAZ-IHH pathway in hepatocytes NASH may be a therapeutic option to block the progression of NASH fibrosis
. To summarize, TAZ and IHH are upregulated in human and experimental NASH; TAZ-induced secretory IHH is a direct activator of fibrosis-inducing HSCs; and silencing TAZ or IHH in hepatocytes blocks HSC activation and liver fibrosis in experimental NASH
siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.
of this type of therapy in humans. Accordingly, we reasoned that a mechanism-based biomarker of target engagement involving the measurement of circulating IHH, i.e., based on the fact that IHH is a secretory protein, might be useful as investigators begin to consider such clinical trials. The combination of the preclinical and clinical data herein, namely, showing that plasma IHH is markedly decreased by silencing hepatocyte TAZ in NASH mice and that circulating IHH in humans associates with NAS, mild-moderate fibrosis, and liver IHH in humans with mild-moderate NASH, provides preliminary support for our idea and provides the rationale for further work in this area. However, the ultimate value of plasma IHH as a marker of target engagement will not be known until such trials are initiated.
As to whether plasma IHH might be useful as a more general marker of liver fibrosis in NASH cannot be addressed by this study, as we do not have the data to compare plasma IHH with other plasma or imaging markers of NASH. Moreover, the cohorts were relatively small and did not include enough advanced NASH subjects to compare different fibrosis stages, and we did not have certain clinical data that would allow us to correct for all confounding factors, such as medications at the time of biopsy. One interesting direction for a future prospective study would be to determine if plasma IHH, when added to another scoring scheme based on different mechanisms, may be able to improve the sensitivity and/or specificity for predicting the progression of early to advanced NASH.
Conclusion
Circulating IHH is elevated in mice with diet-induced NASH and lowered by silencing hepatocyte TAZ, which blocks IHH-induced liver fibrosis in NASH. In humans, increased concentrations of circulating IHH associates with mild-moderate NASH fibrosis. Although these results should be confirmed in larger cohorts across the full spectrum of NASH and NASH-associated fibrosis, they provide the premise for further investigation into using circulating IHH as a mechanism-based indicator of target engagement in anticipated future trials testing NASH fibrosis-targeting therapies that lower liver IHH.
Declaration of interests
N/A
Data availability statement
Further information and requests for resources, reagents, and data should be directed to, and will be fulfilled by, the lead contact, Ira Tabas ([email protected]).
Acknowledgments
We thank the volunteers and investigators involved in the clinical studies at the University of Milan, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (Sara Margarita) and the University of Missouri Health System. The graphical abstract was created with BioRender.com.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
American association of clinical endocrinology clinical practice guideline for the diagnosis and management of nonalcoholic fatty liver disease in primary care and endocrinology clinical settings: co-sponsored by the American Association for the Study of Liver Diseases (AASLD).
Prevalence of nonalcoholic steatohepatitis-associated cirrhosis in the united states: an analysis of national health and nutrition examination survey data.
siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.
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