Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury

Abstract

Background & aims: Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury.

Methods: We analyzed primary isolated LSECs from Atg7^control and Atg7^endo mice as well as rats after CCl₄ induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells.

Results: Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis.

Conclusions: Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression.

Lay summary: Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.

Keywords: Atg7; Autophagy; Endothelial cell; Endothelial dysfunction; LSEC; Liver fibrosis; Nitric oxide; Nrf2; Oxidative stress; eNOS.

Fig. 1.. Autophagy is upregulated during in vitro and in vivo induced capillarization.

Primary LSECs were isolated from untreated SD rats, directly plated (t = 0 h) and grown on plastic tissue culture or onto coverglasses for 24 h (t = 24 h) and 48 h (t = 48 h). Cells were then fixed and processed for immunofluorescent microscopy. For autophagy flux assay by western blot cells were treated with CQ or vehicle during 2 h and collected thereafter. (A) mRNA changes (qPCR analysis) associated with endothelial dysfunction, showing a decrease in Vegfr2 and an increase in Edn1 and (B) LC3B11 immunoblotting with and without addition of CQ showing an increase in autophagy flux at 24 h that decreases at 48 h of culture. (C) Representative immunofluorescent images and quantification of autophagosomes (LC3B, green) with lysosomes (Lamp2, red) colocalization (R value) in LSECs confirming autophagy upregulation during capillarization. Primary LSECs isolated from rats treated with CCl₄ or vehicle for 4 and 6 weeks: (D) mRNA changes (qPCR analysis) associated with endothelial dysfunction, showing a decrease in Vegfr2 and an increase in Edn1 and (E) LC3B11 immunoblotting with and without addition of CQ showing autophagy flux displaying an increase at 4 weeks and incapability of further increase at 6 weeks. Data shows mean value ± SEM of at least 3 experiments. mRNA and protein expressions are expressed as fold-change relative to control (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test or ANOVA). CCl₄, carbon tetrachloride; CQ chloroquine; LSEC, liver sinusoid endothelial cell; qPCR, quantitative real-time PCR; SD, Sprague-Dawley.

Fig. 2.. Generation of Atg7^endo mice.

(A) Schematic view of Atg7^endo mice model generation. (B) mRNA changes (qPCR analysis) and (C) immunoblot of primary LSECs isolated from Atg7^endo mice showing decreased expression of Atg7 and LC3B11/1 and increased of p62 levels. Data shows mean value ± SEM of at least 3 experiments (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). LSEC, liver sinusoid endothelial cell; qPCR, quantitative real-time PCR. (This figure appears in colour on the web.)

Fig. 3.. Loss of LSEC autophagy does not affect liver homeostasis.

(A) Representative images of whole liver H&E staining showing a normal liver architecture. (B) Aminotransferase levels (n = 12) and (C) animal body weight (n = 69) from Atg7^endo and Atg7^control mice under basal conditions. Data shows mean value ± SEM. (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). H&E, hematoxylin and eosin; LSEC, liver sinusoid endothelial cell. (This figure appears in colour on the web.)

Fig. 4.. Loss of LSEC autophagy leads to cellular dysfunction.

Atg7^endo and Atg7^control mice were treated every other day with CC1₄ i.p. for 1 week to induce mild acute liver injury. (A) mRNA changes (qPCR analysis) associated with endothelial dysfunction in primary isolated LSEC, showing a decrease in Vegfr2 and an increase in Edn1. (B) SEM representative graphs of LSEC from Atg7^endo and Atg7^control mice with porosity and number of fenestrae quantification, showing a loss of fenestrae (capillarization) in the Atg7^endo mice. (C) Whole liver sections stained for the endothelial dysfunction marker von Willebrand factor (vWF), displaying an increase value in Atg7^endo mice. Data shows mean value ± SEM (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). LSEC, liver sinusoid endothelial cell; qPCR, quantitative real-time PCR. (This figure appears in colour on the web.)

Fig. 5.. Loss of LSEC autophagy amplifies liver fibrosis without increasing liver injury.

Atg7^endo and Atg7^control mice were treated every other day with CC1₄ i. p. for 1 week to induce mild acute liver injury (n = 24). (A) Whole liver sections stained for Sirius Red and quantification of Sirius Red-positive area (left) and Hydroxyproline stain content (right). (B) Immunoblots for αSMA in isolated HSCs from Atg7^endo and Atg7^control mice and protein quantification. (C) Whole liver sections stained for desmin and αSMA and quantification of positive area. (D) Immunoblots for PDGFRB in whole liver from Atg7^endo and Atg7^control mice and protein quantification. (E) Histological liver analysis for hepatocyte regenerative capacity and liver injury. (F) Aminotransferase levels showing an increase in aspartate aminotransferase with no change in alanine aminotransferase. (G) Representative images of whole liver sections stained for H&E and (H) histological liver analysis for inflammation scoring in Atg7^endo and Atg7^control mice after mild acute liver injury (CCl₄ i.p. for 1 week). Representative images are shown. Data shows mean value ± SEM of at least 3 experiments (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). CCl₄, carbon tetrachloride; H&E, hematoxylin and eosin; HSC, hepatic stellate cell; LSEC, liver sinusoid endothelial cell.

Fig. 6.. Loss of LSEC autophagy increases oxidative stress and decreases NO bioavailability.

Cellular superoxide content measured by dihydroethidium in (A) HUVECs pre-treated with CQfor 12 h before H₂O₂ was added for 15 h, (B) TSECs transduced with empty vector or siAtg7 were also treated with H₂O₂ for 15 h and (C) in whole liver from Atg7^endo and Atg7^control mice after mild acute liver injury (CCl₄ i.p. for 1 week). (D) Quantitative nitrotyrosinated proteins analysis by fluorohistochemistry in whole liver tissue from Atg7^endo and Atg7^control mice after mild acute liver injury (CCl₄ i.p. for 1 week). (E) cGMP levels in liver homogenates from Atg7^endo and Atg7^control mice (CCl₄ i.p. for 1 week) illustrating a significant decrease. (F) Immunoblots for total eNOS and phosphorylated eNOS in whole liver from Atg7^endo and Atg7^control mice and protein quantification. Representative images are shown. Protein is expressed as fold-change relative to control. Data shows mean value ± SEM of at least 3 experiments (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). CQ, chloroquine; HUVEC, human umbilical vein endothelial cell; LSEC, liver sinusoid endothelial cell; qPCR, quantitative real-time PCR; NO, nitric oxide; TSEC, mouse LSEC.

Fig. 7.. Loss of LSEC autophagy is associated with an insufficient antioxidant response.

Atg7^endo and Atg7^control mice were treated every other day with CCl₄ i. p. for 1 week to induce mild acute liver injury and primary LSECs were isolated. (A) mRNA changes (qPCR analysis) showing a downregulation of the classical genes that protect against oxidative stress Gpx1, Gpx4, Sod1, Sod2 and Cat and (B) mRNA changes (qPCR analysis) showing upregulation of the Nrf2-dependent antioxidative stress genes Srxn, Nqol, Gclc, Gclm, and Gstm2. (C) Immunoblots for NQO1 and HMOX1 in isolated LSEC from Atg7^endo and Atg7^control mice confirming an increase at the protein levels. Data shows mean value ± SEM of at least 3 experiments. mRNA expression is expressed as fold-change relative to control (*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test). LSEC, liver sinusoid endothelial cell; qPCR, quantitative real-time PCR