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Hedgehog Pathway Activation Parallels Histologic Severity of Injury and Fibrosis in Human Nonalcoholic Fatty Liver Disease
Cynthia D. Guy,1 Ayako Suzuki,2 Marzena Zdanowicz,2 Manal F. Abdelmalek,2 James Burchette,1 Aynur Unalp,3 and Anna Mae Diehl2; for the NASH CRN
The Hedgehog (HH)-signaling pathway mediates several processes that are deregulated in patients with metabolic syndrome (e.g., fat mass regulation, vascular/endothelial remodeling, liver injury and repair, and carcinogenesis). The severity of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome generally correlate. Therefore, we hypothesized that the level of HH-pathway activation would increase in parallel with the severity of liver damage in NAFLD. To assess potential correlations between known histologic and clinical predictors of advanced liver disease and HH-pathway activation, immunohistochemistry was performed on liver biopsies from a large, well-characterized cohort of NAFLD patients (n 5 90) enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database 1 study. Increased HH activity (evidenced by accumulation of HHligand–producing cells and HH-responsive target cells) strongly correlated with portal inflammation, ballooning, and fibrosis stage (each P < 0.0001), supporting a relationship between HH-pathway activation and liver damage. Pathway activity also correlated significantly with markers of liver repair, including numbers of hepatic progenitors and myofibroblastic cells (both P < 0.03). In addition, various clinical parameters that have been linked to histologically advanced NAFLD, including increased patient age (P < 0.005), body mass index (P < 0.002), waist circumference (P < 0.0007), homeostatic model assessment of insulin resistance (P < 0.0001), and hypertension (P < 0.02), correlated with hepatic HH activity. Conclusion: In NAFLD patients, the level of hepatic HH-pathway activity is highly correlated with the severity of liver damage and with metabolic syndrome parameters that are known to be predictive of advanced liver disease. Hence, deregulation of the HH-signaling network may contribute to the pathogenesis and sequelae of liver damage that develops with metabolic syndrome. (HEPATOLOGY 2012;55:1711-1721) onalcoholic fatty liver disease (NAFLD) is strongly associated with obesity. Because of the current obesity epidemic, NAFLD is currently one of the most prevalent liver diseases in the world and a major cause of cirrhosis and liver-related


mortality.1 Fortunately, only some of the many individuals with NAFLD will ever develop progressive liver injury that results in steatohepatitis (SH), cirrhosis, or primary liver cancer. Therefore, efficient, accurate identification of patients who are most likely to

Abbreviations: BMI, body mass index; CI, confidence interval; COR, cumulative odds ratio; DM, diabetes mellitus; ECs, endothelial cells; ER, endoplasmic reticulum; G, histologic grade; GLI2, glioblastoma 2 transcription factor; H&E, hematoxylin and eosin; HH, hedgehog; HOMA-IR, homeostatic model assessment of insulin resistance; HPF, high-power field; HTN, hypertension; IHC, immunohistochemistry; K7, keratin 7; mRNA, messenger RNA; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NASH CRN, NASH Clinical Research Network; NKT, natural killer T cells; S, fibrosis stage; SD, standard deviation; SHH, sonic Hedgehog; SH, steatohepatitis; a-SMA, alpha-smooth muscle actin; VIM, vimentin. From the 1Department of Pathology, Duke University Medical Center, Durham, NC; 2Division of Gastroenterology and Hepatology, Department of Medicine, Duke University Medical Center, Durham, NC; and 3Johns Hopkins Center for Clinical Trials, Baltimore, MD. Received July 25, 2011; accepted December 2, 2011. The Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (grants U01DK061718, U01DK061728, U01DK061731, U01DK061732, U01DK061734, U01DK061737, U01DK061738, U01DK061730, and U01DK061713) and the National Institute of Child Health and Human Development. Several clinical centers use support from General Clinical Research Centers or Clinical and Translational Science Awards in the conduct of NASH CRN studies (grants UL1RR024989, M01RR000750, M01RR00188, UL1RR02413101, M01RR000827, UL1RR02501401, M01RR000065, M01RR020359, and UL1RR025741). M.A. is supported by a NIH/NIDDK K23 Career Development Award (K23-DK062116). The analyses described in this study were supported by an NIH/NIDDK grant (PI: to A.M.D.; R01-DK053792 and R01-DK077794) and discretionary funds from the Duke University Division of Gastroenterology. 1711

3 (bridging fibrosis).D. Fibrosis was staged as 0 (no pathologic fibrosis). inhibit apoptosis of these cell types. 2 (centrilobular and periportal pericellular fibrosis). However. (4) unstained tissue sections available for IHC staining.3 Other work in cultured cells demonstrated that HH ligands stimulate quiescent hepatic stellate cells to become myofibroblastic. representative cohort of NAFLD patients. 1 (centrilobular or periportal pericellular fibrosis). within the past 2 years) or other coexisting causes of chronic liver disease.4. NC 27710. June 2012 develop progressive liver damage is crucial so that such individuals can be targeted for more aggressive surveillance and therapeutic interventions to optimize the outcomes and minimize the costs of the NAFLD epidemic. or 2 (greater than mild). 1.e. as well as the burden of HH-responsive liver subjects.25559 Potential conflict of interest: Nothing to report. We performed a cross-sectional analysis using data and liver sections from a representative subset (n ¼ 90) of all subjects in the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database 1 Study (n ¼ 1. HEPATOLOGY. increased in parallel with fibrosis stage. C 2012 by the American Association for the Study of Liver Diseases. M.044). Case selection was performed by the NASH CRN Data Coordinating Center. 1 (few).edu. .7 Briefly. it is conceivable that interindividual differences in HHpathway activity contribute to the variable outcomes of fatty liver injury in NAFLD patients. Patients and Methods Study Design and Population. Suite 1073. but only 232 of those individuals fulfilled the following criteria: (1) age 18 years. however. The NASH CRN studies were approved by the institutional review boards at each participating center.1002/hep. fax: 919-684-4183.3 The resultant data showed that the hepatic content of HH-ligand–producing cells. This concept was supported by immunohistochemical (IHC) staining of liver-biopsy samples from a small number of NAFLD patients. Research involving experimental animals is often used to delineate key mechanisms and pilot therapies for human diseases with long and/or seemingly idiosyncratic natural histories. Success has been stymied by our relatively poor understanding of the processes that regulate the outcomes of fatty liver injury. Liver biopsies from all of the cases in the present study had been stained with H&E and Masson’s trichrome.. 595 LaSalle Street. hepatocyte Address reprint requests to: Anna Mae Diehl. portal inflammation was graded as 0 (none to minimal). promote proliferation of liver myofibroblasts and progenitors. Copyright V View this article online at wileyonlinelibrary.1712 GUY ET AL.diehl@duke. and scored by the NASH CRN Pathology Committee according to the published NASH CRN scoring system. on average. (2) no significant alcohol consumption (14 drinks/week in men or 7 drinks/week in women. simple steatosis/not definite NASH) with no-to-early fibrosis (stage 0. recent studies in mice demonstrated that the development of SH and fibrosis correlated strongly with the intensity and duration of Hedgehog (HH)-pathway activation that developed during fatty liver injury. Histologic Evaluation of NAFLD. DOI 10. or 4 (cirrhosis). this approach has been hampered by the lack of small animal models of SH and progressive liver fibrosis that also mimic the typical metabolic perturbations of human NAFLD. E-mail: annamae.2 Nonetheless.and Masson-trichrome– stained liver-biopsy slides had already been scored by the NASH CRN Pathology Committee. and (3) NAFL or NASH with advanced fibrosis (stage 3 or 4) (N¼72). Further investigation of this issue is warranted. (2) definite NASH with early fibrosis (N ¼ 73). Synderman Building (GSRB-1). Department of Medicine. and (5) the corresponding hematoxylin and eosin (H&E).5 Therefore. (3) liver biopsy of 15 mm in length performed within 6 months of enrollment into the CRN Database. Division of Gastroenterology and Hepatology. and therefore the aim of the present study was to evaluate the relationship between the level of HH-pathway activity and severity of liver inflammation and fibrosis in a large. Duke University.6 Liver histologic data were available for 864 of these 1. or 2 (many). Hepatocyte ballooning was graded as 0 (none). Additional Supporting Information may be found in the online version of this article. and 2) (N ¼ 87). reviewed. Our study cohort (n ¼ 90) was comprised of the first 30 consecutive cases from each of the following three histologically defined groups: (1) nonalcoholic fatty liver (NAFL) (i. Durham. and up-regulate the production of chemokines for various types of immune cells. the fact that the analysis was performed in only a small number of patients from a single institution raised valid concerns among clinicians who questioned whether the selected cohort was representative of the general NAFLD population. In NAFLD. 1 (mild). In addition to data for portal inflammation.

and liver fibrosis..e. In animal models of NAFLD. n ¼ 14. remaining sections were processed to assess the production of sonic Hedgehog (SHH) ligand or accumulation of HH-responsive cells (demonstrated by nuclear staining for the HH-regulated transcription factor. % Race. Representative sections from each histologic subgroup were used to optimize staining conditions. or GLI2 and vimentin (VIM.. and a-SMA were semiquantified using 10x objective low-power fields (100x magnification) as a percentage of the total surface area and graded into five categories: grade 1 (less than 20%). % (G0:G1:G2:G3) Ballooning grade. with and without adjusting for other factors. % (White:Asian/Pacific islanders:others) BMI (kg/m2) Type II DM. S3. Mean age and BMI of the study population were 48 6 13 years and 35 6 7 kg/m2. and 56% had hyperlipidemia. the Results section below details only the findings that were noted with regard to portal inflammation. SAS institute. Forty-three percent of our cohort had ballooning grade 2. GLI2). remaining sections were stained for alpha-smooth muscle actin (a-SMA). bile ductular cells . % HTN. glioblastoma 2.1 (Free Software Foundation. % (S0:S1:S2:S3:S4) 48 6 13 59 14 81:7:12 35 6 7 30 46 56 4:37:33:26 0:56:38:6 26:31:43 11:62:27 26:25:16:22:11 Statistical Analyses.5. Results Clinical and Histologic Characteristics of the Study Population. 1713 ballooning. grade 3 (40%-59%). MA).. paraffin-embedded liver biopsy were available for IHC.05/numbers of pairs in a comparison. SHH IHC was performed on liver sections from 84 patients with different stages of fibrosis (S0.g. total number of K7þ cells. hepatocyte ballooning. and K7þ/GLI2þ double-positive cells. we performed Wilcoxon’s rank-sum tests or KruskalWallis’ tests. Clinical Information. waist circumference (cm). a myofibroblast marker that correlates with fibrosis severity.0. Because of the restricted number of sections. VIM. Clinical and histologic characteristics of the study population are summarized in Table 1. Clinical characteristics are reported as the mean 6 standard deviation (SD) for continuous variables or as a proportion with a condition for categorical variables. n ¼ 19. SHH positivity was identified in portal tract cells (e. grade 4 (60%-79%). were counted in five 40x objective high-power fields (HPFs. For the GLI2/K7 double stain. n ¼ 9). Positive staining was semiquantified (i. The plots were created using random scatter in R version 2. 30% had DM. bile duct cells. No. and hypertension (HTN) were evaluated. representative subgroups of the larger cohorts were stained solely for GLI2. % Steatosis grade. S4. % Hispanic. graded). kg/m2). S2. SHH Expression Correlates With Severity of Ballooning. Table 1. we performed ordinal logistic regression or linear regression analyses.8 Positive staining for SHH.. and differences were considered to be statistically significant when the P values were less than 0. a liver-progenitor marker). grade 2 (20%39%).3 Therefore. n ¼ 21. 2012 GUY ET AL.13. presence or absence of diabetes mellitus (DM). To assess the associations between IHC scores and H&E and trichrome scores. To assess the associations between the level of HH-pathway activity and known clinical risk factors for advanced fibrosis. Vol. in which a-levels were adjusted by 0. and fibrosis.3. and 33% had advanced fibrosis (S3-S4).HEPATOLOGY. technical constraints (e.05. and Fibrosis Stage in NAFLD. Age. except for the post-hoc comparison. Three unstained slides from each patient’s formalin-fixed. Because no relationships were demonstrated between HH immunostaining and levels of hepatic steatosis or lobular inflammation. Clinical Characteristics of the Study Population Clinical Characteristics (N ¼ 90) Summary Statistics Age (years) Female gender. IHC Evaluations.g. as revealed by immunostaining of banked liver sections from the same patients. All clinical information was collected within 6 months of the liver biopsy. we analyzed the complete histologic data set from this cohort to determine whether any other standard histopathological parameter(s) correlated with evidence of HH-pathway activity. Inc. 55. % (G0:G1:G2:G3) Lobular inflammation grade. 6. 400x magnification) to determine the average number of positively stained cells. JMP statistical software (version 7. secondary antibody cross-reactivity). homeostasis model assessment–insulin resistance (HOMA-IR). Wilcoxon’s rank-sum tests were used. HH-pathway activation has been linked to fibrogenesis. and our desire to characterize different types of cells that were HH responsive. S1. % (G0:G1:G2) Portal inflammation grade. Details of the IHC methods and antibodies have been previously published. as described above. body mass index (BMI. NC) was used for analysis. and grade 5 (80%). Inc. Portal Inflammation. % Hyperlipidemia. a mesenchymal cell marker). Cary. n ¼ 21. Boston. or costained for GLI2 and keratin 7 (K7. For post-hoc comparison. To further clarify correlations between HH-pathway activation and fibrosis stage. Women comprised 59% of the cohort.. 46% had HTN. % (G0:G1:G2) Fibrosis stage.

8 6 1. SHH expression in subjects with grade 1 portal inflammation was higher than in those with grade 0 portal inflammation (P < 0.7 6 0. HEPATOLOGY. and S4 (D) fibrosis show increased numbers of positive cells with increased fibrosis stage (400x magnification). *versus stage 1 and 2. 1E. ballooned hepatocytes (58. and periportal hepatocytes (7.5. In this relatively large cohort of NAFLD patients. 1A-D). 1. Portal inflammation was strongly associated with SHH expression (P < 0.9 we examined the relationship between SHH expression and portal inflammation in these subjects. P < 0. standard deviation. Given that studies in animal models demonstrated that HH ligands stimulate chemokine production by ductular cells and result in the hepatic recruitment of certain types of immune cells. 1. Moreover. including endoplasmic reticulum (ER) stress. P < 0. and endothelial cells. and 2 portal inflammation were 1. Ballooned hepatocytes exhibit features of ER stress. SHH expression correlates with fibrosis stage in NAFLD.0.0% of cases). whereas whiskers with horizontal lines (upper and lower) represent SD.6% of cases). as scored by the NASH CRN Pathology Committee. 1-6).015) and lower than in subjects with grade 2 portal inflammation (P < 0.017).0001. the relationship between the level of SHH expression and fibrosis severity was highly significant (Fig. Open circles represent individual subjects. and 2. the hepatic content of SHH-expressing cells increased with fibrosis stage (Fig.1714 GUY ET AL. June 2012 Fig. and 4.0001 (Kruskal-Wallis’ test). 2. Photomicrographs of SHH IHC in patients with S0 (A).0001) (adjusted a-level ¼ 0. P < 0. 83. and treating mouse hepatocytes with tunicamycin to induce ER stress stimulated them to express SHH messenger RNA (mRNA) and . IHC scoring results were semiquantified into five ranks. S2 (B). The middle line represents the mean value. and the results were plotted according to the fibrosis stage. Inflammatory mediators have been implicated in the pathogenesis of NASH and are known to provoke various types of cellular stress.9.0 6 1. using trichrome-stained liver sections (E).0001): Mean rank and SD of SHH expression in patients with grade 0. 1. respectively. **versus stage 0. S3 (C).3.3% of cases). Supporting Figs.005 (a-level adjusted for 10 post-hoc comparison pairs).

Hepatic accumulation of HH-responsive cells (i. P < 0.0001 (Kruskal-Wallis’ test). we also examined the relationship between hepatic accumulation of GLI2-expressing cells and severity of hepatocyte ballooning. and the level of SHH expression strongly correlated with severity of hepatocyte ballooning (Fig. n ¼ 24. 1715 Fig. P < 0. 2A-C). n ¼ 2. Numbers of GLI2 (1) Cells Increase With Fibrosis Stage. P < 0.9 The same 39 cases exhibited a range of portal inflammation (G0. stromal cells.12 This suggests that injury-related factors might arrest epithelial differentiation of liver .005). 2. n ¼ 9. Portal Inflammation. n ¼ 11. n ¼ 19). SHH expression was plotted relative to the grade of hepatocyte ballooning. we next evaluated the relationship between hepatocyte ballooning and SHH expression. Portal inflammation is a potential consequence of HH-pathway activation5 and has also been linked to fibrogenesis in NAFLD. Ballooned hepatocytes stained strongly for SHH (Fig. GLI2 (þ) cells were identified in portal tracts (e.0001).017 versus grade 1 and 2 (alevel adjusted for three post-hoc comparison pairs). No.02). 3A-D) and was particularly robust in patients with advanced (S3 and S4) fibrosis (Fig. G1. 1 (B). Ballooned hepatocytes produce HH ligands.0001). ductular cells. n ¼ 7) to assess the relationship between SHH production and accumulation of cells with nuclear GLI2 staining. Therefore. G2. n ¼ 5.0001). GLI2. *P < 0. as scored by the NASH CRN Pathology Committee. 6. G2 n ¼ 13). G1. Hepatic progenitors accumulate in parallel with the severity of liver myofibroblast accumulation and liver fibrosis in NAFLD.g. whereas whiskers with horizontal lines (upper and lower) represent SD.. SHH interacts with receptors on the surface of HH-responsive target cells to trigger HH signaling that results in the nuclear localization of the HH-regulated transcription factor. S1. using H&E-stained liver sections (D). and Ballooning in NAFLD.e.10 Hence. Photomicrographs of SHH IHC in patients with grade 0 (A). Hepatic Accumulation of HH-Responsive Liver Progenitors and Myofibroblastic Cells Parallels Fibrosis Stage in NAFLD. small hepatocytic periportal cells. 3E. there was a positive correlation between SHH expression and GLI2 expression in these subjects (P < 0. S2. S3. n ¼ 13. and ECs). and 2 (C) ballooning show increased numbers of positive cells with increased ballooning grade (400x magnification). GLI2-positive cells) increased with fibrosis stage (Fig. and found that the severity of portal inflammation was significantly positively associated with numbers of GLI2 (þ) liver cells in these subjects (Fig. 2012 GUY ET AL. Ballooning and GLI2 expression were found to be significantly associated (P < 0.11 We did staining for GLI2 on 39 liver sections from a representative subset of our NAFLD patients (S0.10 Therefore. P < 0. As expected. n ¼ 7. n ¼ 7. S4. The middle line represents the mean value. protein. 2D. 55. Vol. Open circles represent individual subjects.HEPATOLOGY. we assessed the relationship between the accumulation of GLI2-expressing cells and portal inflammation. Ballooning scores differed widely in these 39 individuals (G0. 3F.. SHH expression correlates with hepatocyte ballooning. although there was considerable overlap in hepatic accumulation of GLI2-expressing cells among subjects with different grades of ballooning. inflammatory cells. and in lobular fibroinflammatory foci adjacent to ballooned hepatocytes.

some K7þ cells coexpressed GLI2 (range. n ¼ 2. Photomicrographs of GLI2 IHC in patients with S0 (A).05).005 (versus stage 0.6 6 14. 1. Because ballooned hepatocytes are a rich source of HH ligands and correlate with fibrosis stage in NAFLD.0% 6 11.8%-72. the proportion of K7 cells expressing GLI2 remained relatively constant at any given level of fibrosis (44. S1.9 6 19. n ¼ 1. raising the possibility that the hepatic content of HH-responsive progenitors might be influenced by the level of portal inflammation. P < 0.4% for S3-S4).4 6 37. The canals of Hering. whereas whiskers with horizontal lines (upper and lower) represent SD.13 Therefore. progenitors while promoting the outgrowth of myofibroblastic populations. epithelial-type progenitors in a relatively undifferentiated state. mean.9%. Regardless of the stage of liver fibrosis. 4E: P < 0.03). n ¼ 3). we correlated numbers of K7/GLI2 double-positive cells with the level of portal inflammation. n ¼ 5. S3 (C). HEPATOLOGY. we did double immunostaining for GLI2 and K7 (which marks a subpopulation of liver epithelial progenitors) in another representative subgroup of liver sections (S0. are thought to provide a niche for liver epithelial progenitors.3% 6 18. Total numbers of K7/GLI2 double-positive cells increased with fibrosis stage (Fig.5 for S0-S2 versus 74.5 To address this issue. Open circles represent individual subjects. S2 (B). Numbers of GLI2þ cells correlate with fibrosis stage and grade of portal inflammation. the most proximal part of the intrahepatic biliary tree.03).1716 GUY ET AL. and .017 versus grade 2 (alevel adjusted for 10 and 3 posthoc comparison pairs for fibrosis stage and portal inflammation grade. S3. GLI2 staining scores were plotted relative to fibrosis stage (E) as well as portal inflammation grade (F).3% for S0-S2 versus 49. HH ligands generally maintain HH-responsive. Significant stage/grade-related differences are shown: *P < 0. respectively). However. 4F. 17. P < 0. S4. June 2012 Fig. The middle line represents the mean value. and 2) (E) and *P < 0. The total number of K7þ cells increased in advanced fibrosis (38.4 for S3-S4. but promote the growth of HH-responsive myofibroblastic cells. S2.9%). 46. 3. and S4 (D) fibrosis show increased numbers of positive cells (nuclear staining) with increased fibrosis stage (400x magnification). Portal inflammatory activity correlated significantly with the hepatic content of HH-responsive epithelial progenitors (Fig. n ¼ 5.3.

Sections with advanced fibrosis (S3-S4) had greater numbers of VIM-positive cells than those with less-advanced fibrosis (S0-S2) (Fig.3 HH-mediated epithelial-to-mesenchymal transition. respectively (KruskalWallis’ test). 4. as well as total K7-positive cells. The middle line represents the mean value.03. 6. 2012 GUY ET AL.03. GLI2positive) cells (Fig. and hepatocyte ballooning in NAFLD. 1717 Fig. Hepatic accumulation of liver progenitor cells increases with fibrosis stage. because expansion of myofibroblastic populations is a hallmark of liver fibrogenesis and tends to parallel the accumulation of immature liver epithelial cells in NAFLD. 0.004 and P < 0. The remaining 27 unstained sections were stained for either VIM (n ¼ 12) or a-SMA (n ¼ 15). and S4 (D) fibrosis show increased numbers of positive cells with increased fibrosis stage (400x magnification). 5C) and demonstrated that . P < 0. K7positive cells and GLI2-positive cells were counted in double-stained liver-biopsy sections. whereas whiskers with horizontal lines (upper and lower) represent SD. 5B. and hepatocyte ballooning (G). P < 0. 4G). Vol.004 for fibrosis. S2 (B). respectively). Open circles represent individual subjects. and ballooning. P < 0. Review of slides that were costained for VIM and GLI2 revealed that the stromal cell populations harbored HH-responsive (i. portal inflammation. progenitor accumulation correlates with fibrosis stage in NAFLD. we also examined the relationship between hepatocyte ballooning and numbers of HH-responsive progenitors.05. 5A.14 and fibrogenic repair. Numbers of K7/GLI2 double-positive cells (Fig. fibrosis severity ranged from S0 to S4 in this representative subgroup of cases. and 0. Finally. 55. portal inflammation (F). No. Similar results were noted when a-SMA-stained sections were examined (Fig. Average cell counts (per 400x HPF) were plotted in relationship to fibrosis (E).HEPATOLOGY.004). were strongly positively associated with the severity of hepatocyte ballooning (P < 0. As was typical of the entire cohort. S3 (C).002).3 we evaluated the relationship between fibrosis stage and hepatic content of stromal cells that expressed myofibroblast markers and GLI2. Photomicrographs of liver sections double stained for K7 (blue) and GLI2 (brown) in patients with S0 (A). portal inflammation..e.

age.9] (P ¼ 0.1 [1. likelihood ratio test).3 [1.002.5] (P < 0.9.9] (P ¼ 0. only HTN was significantly correlated with GLI2 expression. a-SMA grade and VIM grade were higher in livers with advanced fibrosis (P < 0. Similarly.5] (P < 0.9.3] (P ¼ 0.and multivariate analysis to identify clinical correlates of liver fibrosis in our study cohort.0001) 2.08) 3.0001).12) 1.1] (P < 0.1. 1.1 [0. advanced-stage) NAFLD. which (as noted earlier) significantly correlated with hepatic accumulation of GLI2positive cells (P < 0.8.e. (C) The photomicrograph illustrates accumulation of HH-responsive mesenchymal cells in fibrotic (i.6 [1. BMI. and waist circumference were calculated for 5 unit changes.. 6.2. Tissue samples in the NASH CRN repository are linked to relevant clinical information.3] (P < 0. portal inflammation.4. whereas whiskers with horizontal lines (upper and lower) represent SD. 2. Genetic and pharmacologic approaches that modulate HH signaling in experimental animals and liver cell culture models have proven that the HH pathway regulates numbers of GLI2-positive cells and VIM expression were strongly correlated (P < 0.0008) 1.4] (P ¼ 0. ordinal logistic regression. 1.003) 2.0006) 2. even after adjusting for fibrosis stage (cumulative odds ratio [COR] [95% confidence interval (CI)] ¼ 3. and HTN were significantly correlated with GLI2 expression (Table 3).3]. Therefore. Liver biopsies were stained with VIM or a-SMA to identify myofibroblastic mesenchymal cells.9] (P ¼ 0. Liver content of VIM-positive cells (A) or a-SMA-stained cells (B) were quantified as described in Patients and Methods and correlated with fibrosis stage based on NASH CRN Pathology Committee review of trichrome-stained sections.02) 1. then assessed the relationship between these parameters and hepatic HH-pathway activity. waist circumference.004 and P < 0.4 [1.8 [1. After adjusting for SHH expression.0 [0.2. 5.001) 1.1 [0. The middle line represents the mean value. Table 2.e.5] (P < 0.1 [1. 6. 1.005) 1. and HTN (Table 2).3 [1.02) 2.6 [1.5] (P < 0. All of these variables correlated strongly with hepatic expression levels of SHH (Table 2).003.002) 1. we performed uni.1718 GUY ET AL.5. 1. respectively).4] (P < 0.1. Associations of Clinical Variables With Fibrosis Stage and SHH Expression Unadjusted Fibrosis Stage COR and 95% CI Unadjusted SHH Expression COR and 95% CI Adjusted SHH Expression COR and 95% CI Clinical Variables Age. 2.2 [0.5 [1. waist circumference.002) 1. Numbers of myofibroblastic cells increase with fibrosis stage.005) 1.2] (P < 0. Estimates for age. hepatocyte ballooning..2.2] (P ¼ 0.6] (P < 0.002) 2. 1. Open circles represent individual subjects.6 [1. BMI. HEPATOLOGY. Univariate analysis demonstrated significant correlations of fibrosis stage with age.0 [0. 4. suggesting that the presence of HTN was independently associated with higher GLI2 expression at a given SHH ligand level (Table 3).4.8.5] (P < 0.1. 5 units change Waist circumference.96) Unadjusted estimates were calculated using ordinal logistic regression.4.5. 1. whereas adjusted estimates for SHH were calculated in models including fibrosis stage as a covariate.1 [1. 6. Clinical Correlates of Liver Fibrosis Significantly Correlate With Hepatic SHH and/or GLI2 Staining.3 [1.003).1. 2. BMI. 4. 6. 5. 5. log HOMA-IR.0007) 3. . 5 units change Log (HOMA-IR) DM HTN 1. and liver fibrosis) parallels the level of HH-pathway activity in this disease. June 2012 Fig. Log HOMA-IR correlated with SHH expression.75) 1. providing a unique opportunity to assess relationships between clinical parameters and liver histology. 1. log HOMA-IR.25) 3. P < 0.8.2. Discussion This cross-sectional IHC analysis of liver biopsies from a large number of well-characterized patients with NAFLD provides compelling evidence that the severity of liver damage (i. Arrows demonstrate stromal and vascular cells in fibrotic septa double stained for VIM (blue) and GLI2 (brown).7] (P < 0. 6.2 [0.3 [1. 5 units change BMI.5] (P < 0.8.

1719 Table 3. livers removed from patients undergoing liver transplantation for NAFLD-related cirrhosis are dramatically enriched with NKT cells.0 6 17.4. Abbreviations: b.5 (P ¼ 0. Associations of Clinical Variables With GLI2 Expression Clinical Variables Unadjusted b 6 SE Adjusted b 6 SE Age. Although cross-sectional. including the outgrowth of liver progenitor populations. and the HH pathway is an acknowledged regulator of vasculogenesis/angiogenesis. and each of the three clinical variables that have been most consistently linked with advanced liver fibrosis in NAFLD (i.9 Third. beta coefficient computed in linear regression models using GLI2 expression (cell numbers per HPF) as an outcome variable.656) circumference. and cytokine production of lymphocytes in adults.5 6 10. EC dysfunction and vascular remodeling are characteristics of metabolic syndrome.15 but sustained or excessive HH signaling promotes cirrhosis. an HH-inducible gene.0034) Unadjusted estimates were calculated using linear regression. and the diagnosis of insulin resistance/ type 2 diabetes). for example. Though novel. BMI. 2012 GUY ET AL.145) 8. No.024) À21.3 (P ¼ 0.8 6 42.0 6 10. To our knowledge. leptin. NASH-related cirrhosis is prevented by NKT cell depletion. including waist circumference and HTN. metabolic syndrome and mediate damage to the liver and other tissues that occurs in this condition. Vol.9 (P ¼ 0.0 6 20.18 Second.17 Mature fat cells themselves are also capable of producing and releasing HH ligands. tissue localization.3 In animal models of liver injury.011) 25. because in rodents. standard error.2 6 1. evidence that deregulated HH signaling occurs in metabolic syndrome.1 (P ¼ 0.7 6 1. several key aspects of liver repair.597) DM 90.e.04) 0. The aggregate data.145) change BMI.3 Thus.5 (P ¼ 0. NKT cells. and it has been proven that the HH pathway is a major. hepatic accumulation of profibrogenic natural killer T (NKT) cells correlates with the level of HH-pathway activity and tissue expression of the NKT cell chemoattractant. 5 units 55.16 Pathway activation arrests adipogenesis and promotes the accumulation of adipocyte precursors. SE. older age. 5 units 20.13 and fibrogenesis. and the HH pathway is known to have immunomodulatory functions. induces HH ligand production and activates HH signaling. 6.3 6 48. although direct proof that deregulated HH signaling mediates NAFLD progression in humans is lacking.5 (P ¼ 0.7 (P ¼ 0. in turn.0006) 123.16 Moreover. our study is the first to demonstrate an unequivocal relationship between HH-pathway activity at the tissue level and the severity of damage in that tissue in people with metabolic syndrome.169) change Waist 4.8 6 39.9 6 61.9 6 41. Our univariate analysis supports this concept by identifying strong correlations between hepatic levels of SHH ligand production or nuclear accumulation of the HH-regulated transcription factor. obesity is strongly associated with metabolic syndrome. GLI2. our data strongly suggest that interindividual differences in the ability to control HH-pathway activity may contribute to the variable outcomes of fatty liver injury.21 Membranous microparticles released . interaction of a key adipocyte-derived anorexogenic hormone. and ligand generation from adipose depots is increased in obesity. likely play a key role in fibrosis progression.854) HTN 181. directly mediates the effects of leptin in those cells. 5 units change Log (HOMA-IR) 97.20 In rodents and humans with NASH. overweight/obesity.5 generation and accumulation of liver myofibroblasts. and waist circumference were calculated for 5 unit changes. with its receptors on target cells.1 (P ¼ 0. Hepatic production of SHH ligands and/or HH-signaling activity were also demonstrated to correlate with other clinical factors that are associated with liver fibrosis. highly conserved regulator of fat mass.5 (P ¼ 0. CXCL16. metabolic syndrome is a chronic inflammatory state.0 (P ¼ 0. which. and is likely to be directly responsible for related tissue pathology.HEPATOLOGY. Extension of this logic justifies the development of noninvasive tests that quantify HH-pathway activity to identify individuals who are experiencing tissue damage related to metabolic syndrome before irreparable end-organ damage ensues. Estimates for age. 55. whereas adjusted estimates were calculated in models including SHH expression level as a covariate. First. suggest that deregulated HH pathway activity might promote. and/or result from. the results of the present study demonstrate that this is likely to be true and thus identify novel diagnostic and therapeutic targets to improve NAFLD outcomes.006) 15. in turn. Such patients could then be enrolled into prospective clinical trials designed to determine whether decreasing HH-pathway activity restores normal tissue repair and prevents (or reverts) progressive tissue damage.19 HH is required for normal thymic development and regulates the viability.1 6 41. therefore.0 (P ¼ 0. is buttressed by data that were previously reported by our group and others. Conversely.3 (P ¼ 0. suggesting a relationship between overly exuberant HH-pathway activation in the liver and extrahepatic adverse outcomes of metabolic syndrome. transient HHpathway activation is required for liver regeneration..12 hepatic recruitment of inflammatory cells.

D. (20072009). Meryt Hanna. St. Cheryl Saunders. Cleveland.P. M. Mangesh Pagadala. .23 Such findings have prompted speculation that HH signaling is fundamentally involved in the pathogenesis of EC dysfunction. M..D.D. The contents are solely the responsibility of the authors and do not necessarily represent the official view of the NCRR or NIH..D. Yao-Chang Liu. Abrams.N. M. M. Matthew Yeh. M. (2004-2008). Cindy Riazi. R. Andrea Morris. M. Puneet Puri..P. R. Grave. McCullough.. Mark Pabst.D. Mount Sinai Kravis Children’s Hospital. Luketic.N. Chia Wang. Danuta Filipowski.D. Rosemary Hollick (2003-2005). MD. Brunt. Kavita Nair. Elizabeth M. M.D. M. Clark. (2004-2009). James Nelson.D. M..D. Katie Jacques. M. MO.. obesity and metabolic syndrome are known to increase the risk of cancer in various tissues. (2003-2009). M. Frederick L.. VA.22 The latter induces EC activation and alters the production of vasoactive substances. R. June 2012 from cells that produce HH ligands (e. Northwestern University Feinberg School of Medicine/Children’s Memorial Hospital.. M. M. M..28 Thus.S. Oscar W.N.. (2007-2009). Schwimmer. M.. Duke University Medical Center. DC. M. a component of the National Institutes of Health (NIH). Kleiner. M.N. Tessa Steel (2006-2008). Raphael Merriman. M. Jay H. Kathleen Lake. Houston.S. Judy Thompson.H.. Patricia Belt. M. Frederick Suchy. Louis.D. WA.D. Claude Sirlin. Srinivasan Dasarathy. National Institute of Child Health and Human Development. M. R. Heather Patton. Margaret Stager.D.D.D. Duke University Medical Center. Kris V. Girish Subbarao. McCullough. M. (2006-2010). M. Stavra Xanthakos. M. Ph.1720 GUY ET AL.. Carol Hawkins.. R..D..N. M. Laura Wilson. M.N. New York. CA. Manal F. Mark James Tonascia. University of California San Francisco. Sc. Sarah Ackermann. Linda D.. ¨ nalp-Arida. Paul Killenberg. M. M.D. R. Raj Vuppalanchi.. M. Hoofnagle. M. Neuschwander-Tetri.H.. Ph. M. Milana Isaacson. Ryan Colvin.N. CA. M. from Case Western Reserve University Clinical Centers: Arthur J... Wana Kim. The liver is both a target of.H.D.D. Joan Siegner. (2002-2007).. Brancati. Naga Chalasani.D.D. Dawn Piercy.D. Appendix Members of the Nonalcoholic Steatohepatitis Clinical Research Network are: from clinical centers: Stephanie H... Susan Stewart. such as nitric oxide. Ph.. Alison Lydecker. WA. Melanie White.N. Nathan M. (original grant with University of Washington).D... Kiran Bambha. Elizabeth Byam. Patricia Brandt.. Debra King. R. St. M. M. M. M. F.D. R.P.A. Ph.H.D.D. B. Sc.D. B... and NIH Roadmap for Medical Research. Peter F.. M. Johns Hopkins Hospital. Claudia Zein.D.27 which has become one of the main causes of cancer-related death in obese American men. B. Anthony Nguyen.D.. Yi-Ping Pan. Jeanne M.D. NY. Lisa Yerian. R. including hepatocellular carcinoma.D. Cleveland Clinic Foundation. Laura Miriel. MD.. Bradley Aouizerat.D.. Edward C. Nicholette Rogers.D. M.. Jaividhya Dasarathy.....D. (2004-2008). M. Anna Mae Diehl. M. Bass.S... Cummings. MO. Joyce Hoffmann.. Michael Fuchs.P.. Saint Louis University..24 Fourth. (2004-2008).. (2004-2008). MD.D. (2004-2009).D.D. Katherine Yates.H... Melissa J. Whitington. M.. M.D. for creating the graphs presented in this article and acknowledge that the work provided by Miao Yu was supported by Grant Number UL1RR024128 from the National Center for Research Resources (NCRR). Kimberly Pfeifer. Seattle. MD. M. M. Bloomberg School of Public Health (Data Coordinating Center). MetroHealth Medical Center. M. TX. Children’s National Medical Center.N.. Sarah Barlow. R. Sherry Boyett.. metabolic syndrome–related pathophysiology. (2006-2009). MD.. Seattle.D. Cynthia Behling. HEPATOLOGY. Diane Bringman. Additional research is needed to examine this issue and to determine whether plasma levels of SHH identify NAFLD subjects with liver injury who have increased fibrogenesis and/or whether treatments that ‘‘normalize’’ HHpathway activation would improve recovery from NAFLD.. Indianapolis...D. Patricia R.. Virginia Commonwealth University.N. M.. WA. M.H...M... Cincinnati.. Cynthia Guy. Johns Hopkins University. M..M.D.H..D. Steven Rose. Mark H. Arun J. Cincinnati Children’s Hospital Medical Center. B. Ph...P. R.N. Samantha Kwan.D.D.N.H.D. Virginia Mason Medical Center1. Richmond. Pamela Mann.N.. Baltimore.D.P. M. Cleveland. Ph. M. Center for Human Genetics.C. R... M.. Aynur U Van Natta.P.. Resource centers: David E. Srinivasan Dasarathy.N. M. Ann Scheimann. Ph. Melissa Smith. M.D.H. IL. M. California Pacific Medical Center.. National Cancer Institute.D. Ann Klipsch. Fishbein..D. National Institute of Diabetes and Digestive and Kidney Diseases.D..D.. WA. Velimir A. Ruth Sargent.. Acknowledgments: The authors thank Miao Yu... M. Washington University. B.g. Philip Rosenthal.D. Melissa Young. M. Indiana University School of Medicine.N.D. M... M. San Francisco.P. M.. OH. NC. M. Anya Morgan.25 HH ligands promote the viability and growth of many types of stem and progenitor cells. Joel E.D..D. M. Seattle.. Seattle Children’s Hospital and Research Institute.. Michele Donithan. M. Alice Sternberg. Melissa Coffey..26 and deregulated HH signaling is well documented in several obesityassociated cancers. Molleston. M. the cumulative evidence strongly supports the concept that deregulated HH signaling is broadly relevant to the pathophysiology of metabolic syndrome. Bethesda. (2002-2007).. Abdelmalek. and a contributor to. M. Nanda Kerkar.N.S.D.D..D. R. Tarek Hassanein. R. Linda Ragozzino. (20062009). Bethesda. Bethesda. M. Doo. Rohit Loomba. Lavine. Kimberly Noble.S.D. Kowdley.M. M..D. Ferrell.D.S. Brent A.D. Ivana Vaughn.. Vy Trinh.S. Michael Torbenson.N. M. OH. (project scientist).D.D. Parvathi Mohan. and the present study suggests that both aspects of the relationship are likely to involve the HH pathway.D.... Marcia Gottfried..H.. Bimalijit Sandhu.N. Jody Mooney.D.D.D.S.S. Janis Durelle. San Francisco. M.. L.S. Carol Sargeant.. M. Durham.P.D. M.H.N. Sreevidya Narayanappa.. M.D.D. Raphael Merriman.. Stephanie Buie. Gilman D.D. Bo Gu (20092010).D.D.. Baltimore. M.-C. Amy Jones. M. PA. University of Washington Medical Center. M.D. M. M. Baylor College of Medicine. Stephanie DeVore.P. Leanel Angeli Fairly. B.N. IN.. Arthur J. apoptotic T cells and liver cells) contain biologically active HH ligands that interact with HH receptors on vascular ECs and initiate HH signaling.H. OH.D. Jeffrey B. Rohit Kohli. Jose Derdoy. Ph. CA.D. Jean P. M.P. Sarah Galdzicka.S. M.S. Louis. Contos. M.D. Chicago. Washington... M. (2006-2008). Ph. M. Ph. University of California San Diego. Ann Quinn.D. San Diego. Sc. (2004-2009). Robuck..D. M. (2008-2009). 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