Stellate cell-specific adhesion molecule protocadherin 7 regulates sinusoidal contraction

Abstract

Background and aims: Sustained inflammation and hepatocyte injury in chronic liver disease activate HSCs to transdifferentiate into fibrogenic, contractile myofibroblasts. We investigated the role of protocadherin 7 (PCDH7), a cadherin family member not previously characterized in the liver, whose expression is restricted to HSCs.

Approach and results: We created a PCDH7 fl/fl mouse line, which was crossed to lecithin retinol acyltransferase-Cre mice to generate HSC-specific PCDH7 knockout animals. HSC contraction in vivo was tested in response to the HSC-selective vasoconstrictor endothelin-1 using intravital multiphoton microscopy. To establish a PCDH7 null HSC line, cells were isolated from PCDH7 fl/fl mice and infected with adenovirus-expressing Cre. Hepatic expression of PCDH7 was strictly restricted to HSCs. Knockout of PCDH7 in vivo abrogated HSC-mediated sinusoidal contraction in response to endothelin-1. In cultured HSCs, loss of PCDH7 markedly attenuated contractility within collagen gels and led to altered gene expression in pathways governing adhesion and vasoregulation. Loss of contractility in PCDH7 knockout cells was impaired Rho-GTPase signaling, as demonstrated by altered gene expression, reduced assembly of F-actin fibers, and loss of focal adhesions.

Conclusions: The stellate cell-specific cadherin, PCDH7, is a novel regulator of HSC contractility whose loss leads to cytoskeletal remodeling and sinusoidal relaxation.

Figure 1.. PCDH7 is an HSC-specific transcript in liver.

(A) RNAscope in situ hybridization showing the distribution of mRNAs encoding Pcdh7 and the HSC marker Desmin in healthy and fibrotic (4 week CCl₄ injury model) mouse livers. Images are representative of at least 3 experiments. (B) Pcdh7 expression determined by snRNAseq of a murine model of metabolic dysfunction-associated steatohepatitis (MASH) and healthy control liver (n=3 mice / condition).

Figure 2.. Loss of HSC-mediated vasoconstriction in PCDH7^HSC-KO mice.

(A) Illustration of the breeding strategy to generate HSC-specific PCDH7 KO mice. (B) Visualization of hepatic sinusoids by multiphoton microscopy. Representative images of green FITC-dextran fluorescence in the hepatic sinusoids after infusion of PBS (left) and at 4 min after subsequent infusion of ET-1 (middle and right). Right panel shows a magnification of the middle panel. Images are representative of at least 4 experiments. (C) Cross-sectional area occupied by sinusoids in WT and PCDH7HSC-KO groups after sequential PBS and ET-1 infusion. Dose of ET-1 was 2 nmol/kg. Sinusoidal cross sectional area compared by Student's t-test and presented as means ± SE, *, p < 0.05 vs WT areas prior to ET-1 infusion, n = 4 mice/condition.

Figure 3.. PCDH7 knockout impairs HSC contractility.

(A) Western blot verifying PCDH7 knockout in protein lysates from PCDH7 WT and PCDH7 KO HSCs. (B) Representative images of collagen gel discs from the baseline and day 5 timepoints of the collagen contraction assay. White dotted lines added to highlight gel borders. Scale bar 5 mm. (C) Quantification of collagen gel contraction by WT or PCDH7 KO HSCs. Collagen gel area compared by Student's t-test and presented as means ± SD,**** p value < 0.0001. Data are representative of 5 or more experiments. (D) DIC images showing the morphology of WT or KO HSCs when cultured for 24 or 48 hours in collagen.

Figure 4.. Calcium flux is intact in PCDH7 knockout.

(A) Representative images of baseline and peak Fluo-4 dye fluorescence during treatment with 100 μM ATP to stimulate intracellular calcium release. Scale bar is 100 μm. (B) Fluo-4 fluorescence tracings after treatment with 100 μM ATP plotted as F1/F0 (post-treatment fluorescence divided by initial fluorescence). Plots show the average of 10 independent tracings over time. (C) Comparison of peak Fluo-4 fluorescence (F1/F0) between WT and KO cultures. Represents 10 independent tracings. Compared by Student's t-test and presented as means ± SD. Data are representative of 2 or more experiments.

Figure 5.. PCDH7 regulates focal adhesions and actin assembly in HSCs.

(A) Volcano plot visualization of significantly differentially expressed genes in a bulk RNAseq comparison of PCDH7 WT and PCDH7 KO HSCs grown on tissue culture plastic (n=3 biological replicates collected at different passages for each cell line). (B) Gene ontology analysis of differentially enriched pathways in RNAseq of PCDH7 WT versus KO HSCs. (C) Representative images of collagen gel discs at the 48 hour timepoint with or without 25 μM Y-27632 ROCK inhibition. White dotted lines added to highlight gel borders. Scale bar 5 mm. (D) Quantification of collagen gel area in WT vs KO contraction studies +/− ROCK inhibitor. Quantification performed at the 48hr timepoint. Collagen gel area compared by Student's t-test and presented as means ± SD,**** p value < 0.0001. Data are representative of 2 or more experiments. (E) Representative DIC images of HSC morphology during culture in 3D collagen gel matrices +/− Y-27632 ROCK inhibitor. Scale bar 200 μm. (F) Visualization of F-actin by phalloidin staining in HSCs stimulated with TGF-β1. (G) Immunofluorescence imaging of HSCs stained for vinculin and F-actin. Scale bar 50 μm.