Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease

textabstractBackground and aim: Circulating fibroblast activation protein (cFAP) is a constitutively active enzyme expressed by activated fibroblasts that has both dipeptidyl peptidase and endopeptidase activities. We aimed to assess the correlation between cFAP activity and antigen levels and to compare variations in levels. Methods: In plasma of 465 control individuals, 368 patients with coronary heart disease (CHD) and 102 hepatitis C virus (HCV) infected patients with severe liver disease before and after liver transplant, cFAP activity levels were measured with a newly developed cFAP activity assay. In the same samples, cFAP antigen levels were measured using a commercially available cFAP ELISA. Correlation analyses between activity and antigen levels were performed by calculating Pearson's correlation coefficient (ρ). Additionally, normal ranges, determinants and differences between cohorts and between anticoagulants were investigated. Results: cFAP activity and antigen levels significantly correlated in controls (ρ: 0.660, p<0.001) and in CHD patients (ρ: 0.709, p<0.001). cFAP activity and antigen levels in the HCV cohort were significantly lower in the samples taken after liver transplantation (p<0.001) and normalized toward levels of healthy individuals. Furthermore, cFAP activity and antigen levels were higher in men and significantly associated with body mass index. Also, cFAP activity and antigen levels were higher in EDTA plasma as compared to the levels in citrated plasma from the same healthy individuals. Conclusions: For analyzing cFAP levels, either activity levels or antigen levels can be measured to investigate differences between individuals. However, it is of importance that blood samples are collected in the same anticoagulant

Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease

<div><p>Background and aim</p><p>Circulating fibroblast activation protein (cFAP) is a constitutively active enzyme expressed by activated fibroblasts that has both dipeptidyl peptidase and endopeptidase activities. We aimed to assess the correlation between cFAP activity and antigen levels and to compare variations in levels.</p><p>Methods</p><p>In plasma of 465 control individuals, 368 patients with coronary heart disease (CHD) and 102 hepatitis C virus (HCV) infected patients with severe liver disease before and after liver transplant, cFAP activity levels were measured with a newly developed cFAP activity assay. In the same samples, cFAP antigen levels were measured using a commercially available cFAP ELISA. Correlation analyses between activity and antigen levels were performed by calculating Pearson’s correlation coefficient (ρ). Additionally, normal ranges, determinants and differences between cohorts and between anticoagulants were investigated.</p><p>Results</p><p>cFAP activity and antigen levels significantly correlated in controls (ρ: 0.660, p<0.001) and in CHD patients (ρ: 0.709, p<0.001). cFAP activity and antigen levels in the HCV cohort were significantly lower in the samples taken after liver transplantation (p<0.001) and normalized toward levels of healthy individuals. Furthermore, cFAP activity and antigen levels were higher in men and significantly associated with body mass index. Also, cFAP activity and antigen levels were higher in EDTA plasma as compared to the levels in citrated plasma from the same healthy individuals.</p><p>Conclusions</p><p>For analyzing cFAP levels, either activity levels or antigen levels can be measured to investigate differences between individuals. However, it is of importance that blood samples are collected in the same anticoagulant.</p></div

Correlation between sFAP activity levels and cFAP antigen levels.

<p>Control cohort (n = 465): open circles and dashed line (r² = 0.435). CHD cohort (n = 368): closed squares and solid line (r² = 0.503). The axes are on a log scale.</p

cFAP activity and antigen levels in the different cohorts.

<p>cFAP activity and antigen levels in the different cohorts.</p

Changes to transcript expression in two liver progenitor cell lines following GLI inhibition.

<p>Heat map representing changes to gene transcript levels in BMOL1.2 (<i>n</i> = 3) or BMOL-TAT (<i>n</i> = 3) lines following GANT61 (10 μM) 8 h treatment. Log₂ intensity scale shown. Downregulated genes (blue) are shown above the yellow dashed line, with upregulated genes (red) below. Grey indicates no data.</p

Inhibition of GLI signaling reduces the viability of liver progenitor cell lines.

<p>LPC lines BMOL1.2 <b>(A, C, E, G)</b> and BMOL-TAT <b>(B, D, F, H)</b> were grown over 72 h in the presence of signaling inhibitors for GLI (GANT61; <b>A, B</b>), Notch (DAPT; <b>C,D</b>) c-Met (SGX523; <b>E, F</b>), Wnt (XAV939; <b>G, H</b>) signaling and matching [DMSO]. Viability was determined by ALAMAR Blue assays, and an interaction between time and dose was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.001) (Prism, GraphPad). **** <i>p</i><0.001.</p

Gene changes in BMOL1.2 cells following GANT61 treatment.

<p>Gene changes in BMOL1.2 cells following GANT61 treatment.</p

A very minimal population of human vimentin⁺ HSCs/myofibroblasts express a primary cilium, with none detected on CD31⁺ endothelial cells.

<p>Human ALD liver tissue was examined for the expression of primary cilia (α-acetylated tubulin, green; γ-tubulin, red) by vimentin⁺ (grey) HSCs/myofibroblasts <b>(A)</b> or CD31+ (grey) ECs <b>(C)</b>. <b>(A)</b> The majority of vimentin⁺ cells were Pc^-ve in the tissues examined. Representative image shown, displaying absence of Pc on vimentin⁺ cells. To confirm this result, ciliary protein Arl13b (green) was co-stained with vimentin (grey). Rare Arl13b ciliary structures (arrow) co-localised with vimentin⁺ cells. Final panel in A illustrates rare Pc⁺ (α-acetylated tubulin, green; γ-tubulin, red) vimentin⁺ (grey) HSCs/myofibroblasts, at the cirrhotic interface. <b>(B)</b> Number of vimentin⁺ Pc⁺ cells or vimentin⁺ Pc^neg cells per FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample). <b>(C)</b> No Pc were detected on CD31⁺ cells in the tissues examined (ALD <i>n</i> = 3, 8 FOV/sample). Representative image shown. All images obtained using confocal microscopy, 63x objective. DAPI, blue. White arrows illustrate Pc. * Non-specific liver autofluorescence.</p