Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension - PubMed (original) (raw)

. 2019 Jul;157(1):193-209.e9.

doi: 10.1053/j.gastro.2019.03.013. Epub 2019 Mar 11.

Tejasav Sehrawat 1, Juan P Arab 1, Zhutian Zeng 2, Jinhang Gao 1, Mengfei Liu 1, Enis Kostallari 1, Yandong Gao 3, Douglas A Simonetto 1, Usman Yaqoob 1, Sheng Cao 1, Alexander Revzin 4, Arthur Beyder 1, Rong A Wang 5, Patrick S Kamath 1, Paul Kubes 2, Vijay H Shah 6

Affiliations

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension

Moira B Hilscher et al. Gastroenterology. 2019 Jul.

Abstract

Background & aims: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes.

Methods: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice.

Results: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel.

Conclusions: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.

Keywords: Chemokine; Congestive Hepatopathy; Extracellular Matrix; Mouse Model.

Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1.

Figure 1.. Cyclic stretch upregulates CXCL1.

(A) Primary murine LSECs were subjected to cyclic stretch with a Flexcell device Microarray analysis of genes relevant to endothelial cell biology reveals upregulation of a number of genes impacting inflammatory cell chemotaxis, including CXCL1 (fold change 6.92). The top 15 genes are shown (THBD: thrombomodulin, SELE: selectin, endothelial cell, CXCL1: C-X-C motif chemokine ligand 1, CXCL2: C-X-C motif chemokine ligand 2, SELL: selectin, lymphocyte, CCL2: chemokine (C-C motif) ligand 2, PTGS2: prostaglandin-endoperoxide synthase 2, TYMP: thymidine phosphorylase, PLG: plasminogen, PROCR: protein C receptor, endothelial, CCL5: chemokine (C-C motif) ligand 5, SELPLG: selectin, platelet (p-selectin) ligand, TGFB1: transforming growth factor, beta 1, PTGIS: prostaglandin I2 (prostacyclin), IL6: interleukin 6). (B) CXCL1 upregulation by cyclic stretch was demonstrated with quantitative PCR (upper panel), and ELISA (lower panel). (C) Neutrophils plated in a fibronectin-coated microfluidic device migrate toward a CXCL1 chemotactic gradient (n=3–5; *P≤0.05 for all panels).

Figure 2.

Figure 2.. Neutrophils and platelets infiltrate liver sinusoids early after pIVCL.

(A) Livers of mice were subjected to in vivo intravital imaging 24 hours after sham (left panel) and pIVCL (right panel) procedures. Mice were injected with fluorescently-conjugated anti-Ly6G and anti-CD49b antibodies and Sytox Green. Neutrophils are shown in red, platelets in blue, and extracellular DNA with Sytox Green. After the sham procedure, scant neutrophils are seen in the sinusoids. In contrast, IVC ligation induces sinusoidal dilation and accumulation of neutrophils which aggregate with platelets. All images were taken at the same fluoresence intensity. (B) Transmission electron microscopy shows infiltration of neutrophils within liver sinusoids 24 hours after pIVCL which associate with erythrocytes (right panel). In contrast, sinusoids are non-dilated with scant erythrocytes after sham operation (left panel) (EC, endothelial cell; N, neutrophil; E, erythrocyte; scale bars, 10 μm). (C) Transmission electron microscopy reveals a neutrophil during late-stage NETosis and its aggregation with platelets 24 hours after pIVCL (N, neutrophil; P, platelets; scale bars, 10 μm). (D)Images of NETs were acquired 24 hours after sham (upper panels) and pIVCL (lower panels) procedures. Extracellular DNA was stained with Sytox Green, histone with red-conjugated antibody, and NE with blue-conjugated antibody. Images from each channel were overlaid to visualize colocalization of NET components. (E) Expression of citrullinated histone 3, a byproduct of NET formation, is increased in liver lysates of mice six weeks after pIVCL (quantification in the adjacent graph). Samples are shown in triplicate. (F) Immunofluorescent staining of liver sections shows increased deposition of fibrin (red) and MPO (green) after pIVCL. Intensity of fibrin and MPO and their colocalization were quantified by ImageJ and displayed in the panel below the images. (G) Serum from patients with cardiac cirrhosis have significantly increased levels of circulating dsDNA-cit-Histone (left panel) and dsDNA-MPO complexes (right panel) compared with healthy controls (n=4–5; *P≤0.05 for all panels).

Figure 3.

Figure 3.. NE−/− mice have attenuated increase in portal pressure and fibrosis after pIVCL.

(A) NE−/− mice have significantly lower portal pressures 6 weeks after pIVCL compared to WT mice (ANOVA P≤0.05). (B) Quantitative reverse transcription polymerase chain reaction from whole liver mRNA shows lower mRNA levels of α-SMA (ANOVA P≤0.05) and collagen 1 (ANOVA P≤0.05) in NE−/− mice after pIVCL compared to WT controls. (C) Western blot analysis reveals decreased fibronectin (ANOVA P≤0.05) and α-SMA (ANOVA P≤0.05) protein levels in whole liver of NE−/− mice after pIVCL compared to WT controls. Hsc70 is a loading control. Quantification is shown in the adjacent panel. (D) Hydroxyproline assay shows lower collagen content in livers of NE−/− mice after pIVCL compared to WT mice (ANOVAP ≤0.05). (E) Collagen (red, ANOVA P≤0.05) and fibrin (green, ANOVA P≤0.05) immunofluorescence was significantly lower in NE−/− mice after pIVCL compared to WT controls. Quantification was performed with ImageJ and displayed in the adjacent graphs (n=5–7; *P≤0.05 for all panels). (F) Pad4−/− mice have significantly lower portal pressures after pIVCL when compared with WT controls (n=4–6; ANOVA P≤0.05).

Figure 4.

Figure 4.. NE−/− mice have decreased PHTN after BDL.

(A) NE−/− mice have lower portal pressures after BDL when compared with WT mice (ANOVA P≤0.05). (B) Immunofluorescent staining shows decreased fibrin content in NE−/− mice after BDL compared to WT mice. Quantification was performed with ImageJ and displayed in the adjacent graph. (C) Sivelestat was administered subcutaneously three times a week for six weeks following pIVCL. Mice treated with sivelestat had lower portal pressures after pIVCL compared with mice treated with DMSO (ANOVA P≤0.05). (D) Fibrin (red) and myeloperoxidase (green, ANOVA P≤0.05) immunofluorescence was significantly lower after sivelestat treatment compared to DMSO treatment. Quantification was performed with ImageJ and displayed in the adjacent graphs (n=5–7; *P≤0.05 for all panels).

Figure 5.

Figure 5.. Cyclic stretch utilizes integrins to activate the Notch pathway.

(A) RNA was isolated from primary murine LSECs that were subjected to cyclic stretch. RNA-seq revealed significant upregulation of genes related to integrin signaling. (B) RGD-peptide prevents stretch-induced upregulation of CXCL1 (ANOVA P≤0.05), Hes1 (ANOVA P≤0.05) and Hey1 (ANOVA P≤0.05). (C) Protein expression of Hes1 is higher in HUVEC subjected to cyclic stretch compared with unstretched controls. Quantification was performed using ImageJ with fold change represented in the adjacent graph. (D) Notch1 agonist Jagged-1 upregulates CXCL1 mRNA levels. (E) HUVEC transfected with an siRNA against the Notch1 receptor have lower mRNA levels of CXCL1 after cyclic stretch compared with HUVEC transfected with siControl (ANOVA P≤0.05). siRNA knockdown of Notch is shown in adjacent panel. (F) Treatment of HUVEC with the Notch inhibitor DAPT decreases mRNA expression of CXCL1 (ANOVA P≤0.05) (n=3–5; *P≤0.05 for all panels).

Figure 6.

Figure 6.. Notch pathway interacts with piezo1 channels to upregulate CXCL1.

(A) Stimulation of HUVEC with the piezo1 activator Yoda1 increases mRNA levels of Hes1, Hey1, and CXCL1. (B) Inhibition of piezo1 channels with ruthenium red decreases mRNA levels of CXCL1, Hes1, and Hey1 in HUVEC (CXCL1 ANOVA P≤0.05; Hes1 ANOVA P≤0.05; Hey1 ANOVA P≤0.05). (C) Transfection of HUVEC with siRNA pool to piezo1 decreases upregulation of CXCL1 as well as the Notch targets, Hes1 and Hey1, by cyclic stretch (Hey1 ANOVA P≤0.05; Hes1 ANOVA P≤0.05; CXCL1 ANOVA P≤0.05). siRNA knockdown of piezo1 is shown. (D) Transfection of HUVEC with siRNA pool to Notch1 attenuates the upregulation of CXCL1 by Yoda1 (P≤0.05). siRNA knockdown of Notch1 is shown. (E) Primary LSECs were isolated from mice 48 hours after pIVCL and sham procedures. Quantitative reverse transcription PCR showed increased mRNA levels of Hes1, Hey1, and CXCL1in primary LSECs after IVC ligation compared to sham controls (n=3–5, *P≤0.05 for all panels). (F) Mice with LSEC-specific deletion of Notch1 (Notch1iΔEC) have lower portal pressures when compared with Notchfl/fl mice 4 weeks after IVC ligation (n=7–11; ANOVA P≤0.05).

Figure 7.

Figure 7.. Proposed model of mechanocrine signaling and PHTN.

LSECs sense cyclic stretch through integrins. The insert box shows proposed molecular interactions between integrins, piezo1 channels, and the Notch1 receptor. Integrin activated piezo channels bind to the Notch1 receptor which leads to production of downstream transcription factors, Hes1 and Hey1 to promote CXCL1 generation. Integrins are thought to transmit mechanical forces to piezo channels through myosin54, 55. CXCL1 attracts neutrophils which induce sinusoidal thromboses through formation of NETs. Sinusoidal thromboses are pivotal mediators of PHTN through volume-pressure effects within the sinusoidal lumen.

Comment in

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