Increased sinusoidal pressure impairs liver endothelial mechanosensing, uncovering novel biomarkers of portal hypertension

Abstract

Background & aims: Portal hypertension (PH) is a frequent and severe clinical syndrome associated with chronic liver disease. Considering the mechanobiological effects of hydrostatic pressure and shear stress on endothelial cells, we hypothesised that PH might influence the phenotype of liver sinusoidal endothelial cells (LSECs) during disease progression. The aim of this study was to investigate the effects of increased hydrodynamic pressure on LSECs and to identify endothelial-derived biomarkers of PH.

Methods: Primary LSECs were cultured under normal or increased hydrodynamic pressure within a pathophysiological range (1 vs. 12 mmHg) using a microfluidic liver-on-a-chip device. RNA sequencing was used to identify pressure-sensitive genes, which were validated in liver biopsies from two independent cohorts of patients with chronic liver disease with PH (n = 73) and participants without PH (n = 23). Biomarker discovery was performed in two additional independent cohorts of 104 patients with PH and 18 patients without PH.

Results: Transcriptomic analysis revealed marked deleterious effect of pathological pressure in LSECs and identified chromobox 7 (CBX7) as a key transcription factor diminished by pressure. Hepatic CBX7 downregulation was validated in patients with PH and significantly correlated with hepatic venous pressure gradient. MicroRNA 181a-5p was identified as pressure-induced upstream regulator of CBX7. Two downstream targets inhibited by CBX7, namely, E-cadherin (ECAD) and serine protease inhibitor Kazal-type 1 (SPINK1), were found increased in the bloodstream of patients with PH and were highly predictive of PH and clinically significant PH.

Conclusions: We characterise the detrimental effects of increased hydrodynamic pressure on the sinusoidal endothelium, identify CBX7 as a pressure-sensitive transcription factor, and propose the combination of two of its reported products as biomarkers of PH.

Impact and implications: Increased pressure in the portal venous system that typically occurs during chronic liver disease (called portal hypertension) is one of the main drivers of related clinical complications, which are linked to a higher risk of death. In this study, we found that pathological pressure has a harmful effect on liver sinusoidal endothelial cells and identified CBX7 as a key protein involved in this process. CBX7 regulates the expression of E-cadherin and SPINK1, and consequently, measuring these proteins in the blood of patients with chronic liver disease allows the prediction of portal hypertension and clinically significant portal hypertension.

Keywords: Endothelial dysfunction; HCV; HVPG; Hepatic haemodynamic; LSEC; Liver cirrhosis; Liver sinusoidal endothelial cells; Mechanobiology; Mechanotransduction; NASH.

Graphical abstract

Fig. 1

Effects of pathological pressure in LSECs cultured under pathological hydrodynamic pressure. (A) Top upregulated and downregulated canonical pathways and (B) main molecular processes modified owing to pathological hydrodynamic pressure. (C) Venn diagrams comparing differentially expressed genes from LSECs submitted to pathological pressure (n = 3) and LSECs isolated from three cirrhotic portal hypertension preclinical models and LSECs from human patients with cirrhosis (n = 6). Genes were considered significantly deregulated when their fold change was >3 or <-3 and their p value <0.05. cBDL, common bile duct ligation-induced cirrhotic rats; CCl₄, carbon tetrachloride induced-cirrhotic rats; HP, hydrodynamic pressure; hLSEC, LSEC from human patients with cirrhosis; LSEC, liver sinusoidal endothelial cell; TAA, thioacetamide-induced cirrhotic rats.

Fig. 2

Hepatic CBX7 gene expression and correlation with HVPG. Hepatic CBX7 gene expression (top), its correlation with HVPG (middle), and ROC curves (bottom) in the discovery and validation cohorts. (A) The discovery cohort included patients with NP (NP; n = 12) and patients with PH with HCV-associated CLD (PH HCV; n = 13). (B) The validation cohort included healthy individuals with NP (NP; n = 11) and patients with PH (PH; n = 30). The validation cohort is further split in (C) patients with alcohol-associated cirrhosis (PH OH; n = 18) and (D) patients with HCV-related cirrhosis (PH HCV; n = 12). CBX7 gene expression data are presented as the mean ± SD, as the ratios of gene expression relative to beta actin, and expressed as a percentage of the healthy group, set at 1. Data were compared using Student’s t test (∗p <0.05), and correlations were calculated using Pearson’s correlation. CBX7, chromobox 7; CLD, chronic liver disease; HVPG, hepatic venous pressure gradient; NP, normal pressure; PH, portal hypertension; ROC, receiver operating characteristic.

Fig. 3

Expression of miR-181a in LSECs and its effects on CBX7. (A) miR-181a-5p expression in cirrhotic LSECs from rat (Rn) and human (Hs), and miR-181a and CBX7 gene expression in (B and C) LSECs after 48 h of in vitro capillarisation and in (D and E) LSECs transfected with an anti-miR-181a. Gene expression data are presented as the mean ± SD, as the ratios of gene expression relative to RNU6 or Gapdh and expressed in comparison with the 0 h group (B and C) or the vehicle group (D and E), set at 1. All experiments were performed with at least n = 4 independent replicates. Data were compared using Student’s t test (∗p <0.05). AALD, alcohol-associated liver disease; CBX7, chromobox 7; CCl₄, carbon tetrachloride; FC, fold change; GADPH, glyceraldehyde-3-phosphate dehydrogenase; LSEC, liver sinusoidal endothelial cell.

Fig. 4

Analysis of ECAD, SPINK1, and their combination to predict PH. (A) ECAD and (B) SPINK1 in plasma of healthy humans with NP (NP; n = 18) compared with patients with ACLD with PH (PH; n = 47) of three different aetiologies: MASH, OH, or HCV. (C) ROC curve of ECAD, SPINK1, and ES and (D) its performance. Data were compared using Student’s t test (∗p <0.05). Values of AUROC, the 95% CI, the cut-off value with better Sens and Spec, PPV, and NPV. ACLD, advanced chronic liver disease; AUROC, area under the receiver operating characteristics; ECAD, E-cadherin; ES, ECAD + SPINK1; MASH, metabolic-associated steatohepatitis; NP, normal pressure; NPV, negative predictive value; OH, alcohol-associated; PH, portal hypertension; PPV, positive predictive value; ROC, receiver operating characteristic; Sens, sensitivity; Spec, specificity; SPINK, serine protease inhibitor Kazal-type 1.

Fig. 5

Analysis of ECAD, SPINK1, and their combination to predict CSPH. (A) ECAD and (B) SPINK1 in plasma of patients with advanced chronic liver disease with subclinical portal hypertension (HVPG <10; n = 11) or with CSPH (HVPG ≥10; n = 36) of three different aetiologies: MASH, OH, or HCV. (C) ROC curve of ECAD, SPINK1, and ES and (D) its performance. Data were compared using Student’s t test (∗p <0.05). Values of AUROC, the 95% CI, the cut-off value with better Sens and Spec, PPV, and NPV. AUROC, area under the receiver operating characteristics; CSPH, clinically significant portal hypertension; ECAD, E-cadherin; ES, ECAD + SPINK1; HVPG, hepatic venous pressure gradient; MASH, metabolic-associated steatohepatitis; NPV, negative predictive value; OH, alcohol-associated; PPV, positive predictive value; ROC, receiver operating characteristic; Sens, sensitivity; Spec, specificity; SPINK, serine protease inhibitor Kazal-type 1.