Accepted Manuscript

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal EndothelialCells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote PortalHypertension

Moira B. Hilscher, Tejasav Sehrawat, Juan Pablo Arab Verdugo, Zhutian Zeng,Jinhang Gao, Mengfei Liu, Enis Kostallari, Yandong Gao, Douglas A. Simonetto,Usman Yaqoob, Sheng Cao, Alexander Revzin, Arthur Beyder, Rong Wang, PatrickS. Kamath, Paul Kubes, Vijay H. Shah

PII: S0016-5085(19)33554-1DOI: https://doi.org/10.1053/j.gastro.2019.03.013Reference: YGAST 62521

To appear in: GastroenterologyAccepted Date: 5 March 2019

Please cite this article as: Hilscher MB, Sehrawat T, Arab Verdugo JP, Zeng Z, Gao J, Liu M, KostallariE, Gao Y, Simonetto DA, Yaqoob U, Cao S, Revzin A, Beyder A, Wang R, Kamath PS, Kubes P, ShahVH, Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to RecruitNeutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension, Gastroenterology(2019), doi: https://doi.org/10.1053/j.gastro.2019.03.013.

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Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells

to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal

Hypertension

Short title: Angiocrine signals drive portal hypertension

Moira B. Hilscher1, Tejasav Sehrawat1, Juan Pablo Arab Verdugo1, Zhutian Zeng2,

Jinhang Gao1, Mengfei Liu1, Enis Kostallari1, Yandong Gao3, Douglas A. Simonetto1,

Usman Yaqoob1, Sheng Cao1, Alexander Revzin1,3, Arthur Beyder1, Rong Wang4, Patrick

S. Kamath1, Paul Kubes2, Vijay H. Shah1

1 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester MN, USA

2 Department of Immunology, University of Calgary, Alberta CA

3 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester MN, USA

4 Department of Surgery, University of California at San Francisco, San Francisco, CA, USA

Grant support: Supported by National Institutes of Health (NIH), USA R01 DK59615 and R01

AA21171 (VHS)

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Acknowledgements: Mayo Center for Cell Signaling in Gastroenterology (NIDDK

P30DK084567)

RNA sequencing accession number: GSE119547

Abbreviations: PHTN, portal hypertension; LSEC, liver sinusoidal endothelial cell; pIVCL,

partial inferior vena cava ligation; NET, neutrophil extracellular trap; NE, neutrophil elastase;

BDL, bile duct ligation; CH, congestive hepatopathy; DMSO, dimethyl sulfoxide; HUVEC,

human umbilical vein endothelial cells; extDNA, extracellular DNA; PAD4, protein arginine

deiminase 4; dsDNA, double-stranded DNA; cit-Histone, citrullinated histone; MPO,

myeloperoxidase; α-SMA, alpha smooth muscle actin; CAMKII, calcium/calmodulin-dependent

protein kinase II; Hes1, Hairy and Enhancer of Split 1; RGD peptide, arginine-glycine-aspartate

peptide; EC, endothelial cell.

Correspondence:

Vijay H. Shah

200 1st Street SW

Rochester, MN, 55905 USA

Disclosures: The authors have declared that no conflicts of interest exist.

Author Contributions:

MBH contributed study concept and design, acquisition of data, analysis and interpretation of

data, statistical analysis, and drafting of the manuscript. JPAV and TS contributed acquisition of

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data and analysis and interpretation of data. ZZ, EK, JG and YG contributed acquisition of data.

DAS contributed critical revision of the manuscript for important intellectual content. UY

contributed acquisition of data and technical support. SC, AR, AB, PSK, DS, ML, and PK

contributed critical revision of the manuscript for important intellectual content. VHS

contributed study concept and design, critical revision of the manuscript for important

intellectual content, and study supervision.

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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 PAD4 (Pad4–/–) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice

with LSEC-specific deletion of Notch1 (Notch1i∆EC). 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 upregulated in primary

LSECs exposed to mechanical stretch, compared to 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 to 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

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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.

KEY WORDS: Congestive hepatopathy, mouse model, chemokine, extracellular matrix

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Introduction

Portal hypertension (PHTN) is a common sequelae of chronic liver disease which

constitutes the principle driver of mortality and liver transplantation in patients with cirrhosis.

The pathophysiology of PHTN is complex and is regulated at multiple levels, including

paracrine signaling within sinusoids, alterations in vasoconstrictive tone, and more grossly, by

angio-architectural distortion of the liver 1. Chronic hepatic congestion, or congestive

hepatopathy (CH), is a cause of PHTN that occurs in conditions such as congestive heart

failure and Budd-Chiari syndrome which perturb efficient blood flow in the liver. Despite the

increasing prevalence of CH in an aging population, the mechanism by which chronic hepatic

congestion promotes PHTN and fibrosis has been poorly understood. Our partial inferior vena

cava ligation (pIVCL) murine model of CH revealed that chronic congestion drives fibrosis

through sinusoidal thrombosis and mechanical forces 2. However, the molecular pathways

that mediate and integrate these processes remain incompletely defined. Furthermore,

sinusoidal thrombosis has been implicated in chronic liver disease progression in human

studies 3, 4, suggesting that sinusoidal thrombogenesis may be broadly applicable to other

etiologies of fibrosis-related PHTN.

Recent studies implicate neutrophils in the formation and propagation of thrombosis 5,

6. Neutrophils accumulate early in the formation of thrombosis 7, promote propagation of the

coagulation cascade, and aggregate with other thrombogenic mediators, most notably, platelets

5, 8. Neutrophil interaction with platelets can contribute to thrombosis through the formation of

neutrophil extracellular traps, or NETs 9, 10. NETs are composed of a backbone of

extracellular DNA fibers bound to histones and granular proteins such as myeloperoxidase and

neutrophil elastase (NE) 11. Various cellular components of neutrophils and NETs, including

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histone and granular proteins, can initiate or propagate coagulation, and NETs have been

identified as pro-thrombotic structures in the setting of sepsis and deep vein thrombosis 9, 10, 12-

14. The role of NETs in the development of PHTN has not been mechanistically explored.

Tissue-specific endothelial cells, including LSECs, secrete “angiocrine” factors which

serve as critical regulators of metabolism, organ homeostasis and regeneration 15.

Mechanosensitive proteins contribute to a paradigm of “mechanocrine” signaling where

changes in mechanical forces and the physical environment are transduced into secretion of

angiocrine signals which impact neighboring cells. Integrins are transmembrane proteins that

link extracellular matrix molecules with the cellular cytoskeleton and thereby sense and

respond to a spectrum of biochemical and mechanical signals 16. Studies have shown

reciprocal interactions between integrins and the Notch pathway 17, a mechanosensitive

signaling pathway which is critical to endothelial function and hepatic vasculature 18, 19. Mice

with selective deletion of the Notch1 receptor in LSECs have distorted vascular architecture,

dilated sinusoids, and increased portal pressures, suggesting that the Notch1 receptor regulates

sinusoidal structure and tone 18. Piezo proteins are components of mechanosensitive

nonselective ion channels which have also been implicated in modulation of vascular tone 20,

21. However comprehensive RNA-sequencing based analysis of pathways activated by

mechanical stretch in LSECs has not been previously explored.

In this study, we first examined the impact of pathologic levels of mechanical stretch on

LSEC signaling. We used non-biased high-throughput screening with microarray and RNA-

sequencing technology to identify novel mechanocrine signals generated by LSECs. These

signals were further tested in hypothesis-driven in vivo and in vitro studies to elucidate events in

the sinusoidal microenvironment which contribute to the pathophysiology of PHTN. We

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demonstrate that mechanical stretch of LSECs induces Notch-dependent upregulation and

secretion of the neutrophil chemotactic chemokine CXCL1. CXCL1-mediated neutrophil

chemotaxis propagates PHTN by interacting with platelets to promote extrusion of NETs, and

inhibition of NET formation attenuates PHTN. We show that integrins and piezo channels serve

as mechanosensors which activate the Notch signaling pathway to upregulate CXCL1. These

results have the potential to augment the spectrum of therapeutic targets to treat PHTN and its

complications in CH and other forms of chronic liver injury.

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Materials and Methods

More detailed Materials and Methods are included in the Supplementary Materials.

Animal experiments

Partial inferior vena cava ligation

C57BL/6 mice (8-10 weeks) were purchased from Envigo Laboratories, and NE-/- and

Pad4-/- mice were purchased from Jackson Laboratories (Bar Harbor, ME). Notch1flox/flox mice

were crossed with Cdh5(PAC)-CreERT mice to generate mice with LSEC-specific deletion of

Notch1 (Notch1i∆EC). Mice were subjected to pIVCL for 4 or 6 weeks to induce PHTN and

fibrosis as previously described 2, 22. In other protocols, after pIVCL, C57BL/6 mice were

injected subcutaneously with sivelestat (30 mg/kg) or equal volume of dimethyl sulfoxide

(DMSO) control. All animal work was performed under Mayo Institutional Animal Care and

Use Committee oversight.

Bile duct ligation

C57BL/6 mice (8-10 weeks) were subjected to bile duct ligation (BDL) for 4 weeks as

previously described 22.

Portal pressure measurements

Portal pressure was measured using a digital blood pressure analyzer (Digi-Med) 23.

After calibration of the analyzer, a 16-guage catheter attached to a pressure transducer was

inserted into the portal vein. The average portal pressure (mm Hg) was then recorded.

Intravital imaging

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Intravital imaging of the liver was done using an inverted spinning-disk confocal

microscopy system (Olympus IX81)24. Please refer to the Supplementary Materials and Methods

for more detail.

Statistical analysis

Means are expressed as means ± standard error. Significance was established using the

Student’s t-test and analysis of variance when appropriate.

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Results

Liver sinusoidal endothelial cells (LSECs) subjected to cyclic stretch secrete the neutrophil

chemoattractant CXCL1

CH is characterized by passive hepatic congestion and sinusoidal dilatation which

imparts mechanical stretch on LSECs. Given the critical role of LSECs in mechanical sensing of

sinusoidal forces as well as recent studies implicating angiocrine signaling in diverse liver

functions and diseases 25, 26, we aimed to elucidate the role of “mechanocrine” signaling

mechanisms in the pathogenesis of CH. We isolated primary murine LSECs and subjected them

to cyclic biaxial stretch with a Flexcell device. Cyclic stretch was imposed at an intensity and

frequency (20% strain, 1 Hz) intended to recapitulate the cardiac cycle and therefore mimic the

forces experienced by LSECs during CH 27. We then performed microarray screening of genes

related to endothelial cell function. Microarray screening of LSECs subjected to cyclic stretch

showed transcriptional upregulation of a number of cytokines which impact inflammatory cell

chemotaxis, including CXCL1, CXCL2, and Ccl2 (Figure 1a). We pursued neutrophil

chemotactic signals given the prominent role that neutrophils play in formation of thromboses,

which we hypothesize are integral to the pathophysiology of CH-induced PHTN. We confirmed

an increase in CXCL1 in LSECs subjected to cyclic biaxial stretch by quantitative PCR (Figure

1b, upper panel), and ELISA analysis (Figure 1b, lower panel). These findings suggest that

LSECs subjected to cyclic stretch generate angiocrine signals which have the potential to recruit

neutrophils and possibly propagate microthrombus formation, fibrosis, and PHTN. Prior studies

suggest that CXCL1 induces neutrophil chemotaxis 28, 29. To confirm a functional role of

CXCL1 as a neutrophil chemoattractant, a microfluidic gradient generator was utilized to create

a gradient of CXCL130 (Supplementary Figure 1). Neutrophils were plated on a surface coated

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with fetal bovine serum, and their migration was studied over the ensuing hour. Neutrophils

migrated towards higher concentrations of CXCL1 (Figure 1c), confirming the ability of CXCL1

to promote neutrophil chemotaxis. We next tested this proposed pathway in vivo.

Pro-thrombotic NETs form in the sinusoids in congestive hepatopathy

Our in vitro results thus far indicate that LSEC responses to mechanical force leads to

angiocrine signals that can recruit neutrophils. To directly examine the role of neutrophils in CH

in vivo, we performed intravital imaging 24 hours after pIVCL and sham procedures. Intravital

microscopy of livers 24 hours after sham procedures revealed normal sinusoidal architecture

with sparse neutrophils which traverse through the sinusoids and rapidly exit via the hepatic vein

(Figure 2a, left panel; Supplementary Movie 1, left panel). Visualization of livers 24 hours after

pIVCL revealed significant sinusoidal dilatation and vascular accumulation of neutrophils which

aggregate with platelets (Figure 2a, right panel; Supplementary Movie 1, right panel). We next

performed transmission electron microscopy which confirmed early recruitment of neutrophils

within the liver sinusoids (Figure 2b) and spatial association with erythrocytes and platelets after

pIVCL (Figures 2b and 2c). These findings suggest that neutrophil stasis within the sinusoids

may serve as a nidus of platelet interaction and thrombosis in the setting of CH.

Platelet interaction with activated neutrophils is a potent inducer of NET formation 13, 31,

and recent studies suggest that endothelial cells also play a key role in neutrophil chemotaxis and

induction of NET formation32, 33. NET components, including histones and granular proteins,

have the capability to then initiate or propagate thrombosis and coagulation 9, 34. Having

visualized extensive neutrophil-platelet aggregation lining liver sinusoids 24 hours after pIVCL,

we hypothesized that this interaction may promote the formation of NETs which then propagate

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microvascular thrombosis, fibrosis, and PHTN in CH. We utilized combination staining with

intravital microscopy to visualize colocalization of three key NET components: extracellular

DNA (extDNA), neutrophil elastase (NE), and histones. Visualization of livers 24 hours after

pIVCL revealed colocalization of these structures within the lining of the liver sinusoids which

was absent after the sham procedure (Figure 2d). Furthermore, neutrophils were visualized

during the process of NET formation (Figure 2c) aggregating with platelets on TEM. This

morphological data confirms that neutrophils are recruited into the sinusoids early after pIVCL

and rapidly form NETs after engaging with LSECs and platelets. Consistent with prior

histologic observations of CH, few neutrophils were observed within the parenchyma after

pIVCL35, 36.

Given the role of sinusoidal thrombosis in portal hypertension and CH, we next used

complementary microscopy techniques to ascertain the relationship of NETs with sinusoidal

thrombosis. Six weeks after surgery, livers of mice who had undergone pIVCL had significantly

increased levels of citrullinated histone 3, the protein byproduct of the peptidyl arginine

deiminase 4 (PAD4) enzyme which is a critical mediator of NET formation 37 (Figure 2e). The

formation of NETs results in the release of double-stranded DNA (dsDNA) which is commonly

measured as a marker of NET formation 38. Indeed, serum levels of circulating dsDNA were

significantly increased in mice which had undergone pIVCL when compared with sham-operated

animals (Supplementary Figure 2a). Finally, we then performed additional immunostaining

which revealed increased peri-sinusoidal deposition of myeloperoxidase (MPO), a neutrophil

granular protein, after pIVCL (Figure 2f). Importantly, MPO was spatially associated with

fibrin, suggesting that NET formation serves as a nidus of sinusoidal thrombosis in our pIVCL

model of CH. These findings confirm the formation of NETs after the pIVCL procedure and

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their contribution to thrombosis. To further test our hypothesis, liver samples were obtained

from patients who had undergone corrective surgery (Fontan procedure) for congenital heart

disease in addition to healthy controls. The Fontan procedure creates an anastomosis between

the vena cava or right atrium with the pulmonary arteries and imposes chronic venous passive

congestion on the liver as a result39. We performed immunostaining which confirmed that livers

from patients with Fontan physiology had increased sinusoidal fibrin which was spatially

associated with myeloperoxidase (Supplementary Figure 2b). Serum obtained from patients with

cardiac cirrhosis contained higher levels of circulating dsDNA-MPO and dsDNA-cit-Histone

complexes compared with healthy controls (Figure 2g), confirming the relevance of our findings

in patients with CH. This in total suggests that NET formation contributes to thrombogenesis in

patients with CH.

Genetic deletion of neutrophil elastase impairs thrombosis, fibrosis, and PHTN in CH.

Sinusoidal microvascular thromboses are critical mediators of PHTN and fibrosis in CH4.

Our studies thus far indicate that pIVCL induces neutrophil accumulation within sinusoids and

the formation of NETs. Furthermore, spatial association of NET components with fibrin suggest

that NETs contribute to the formation of microvascular thrombosis after the pIVCL procedure 14.

To determine the impact of NET formation on PHTN and fibrosis, neutrophil elastase (NE-/-)

deficient mice were subjected to pIVCL and sham procedures and then compared to wild type

mice. NE is a granular protein which is a key component of and driver of NET formation 40. Six

weeks after pIVCL, NE-/- mice had significantly lower portal pressures (Figure 3a) when

compared with wild type mice undergoing pIVCL. Expression of hepatic fibrosis markers α-

SMA, collagen 1, and fibronectin were also significantly decreased in NE-/- mice compared to

wild type controls as demonstrated by PCR and immunoblotting (Figure 3b and 3c).

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Hydroxyproline assay confirmed decreased liver collagen content in NE-/- mice (Figure 3d).

Next, we utilized immunofluorescent studies to confirm that NE deficiency attenuates

intrahepatic thrombosis in the setting of CH. These studies revealed decreased fibrin

immunostaining as well as decreased spatial association with collagen (Figure 3e). These results

suggest that NE deficiency attenuates PHTN and fibrosis in the setting of CH by preventing

thrombosis.

NET formation has been identified despite inhibition of NE in certain sterile forms of

injury41. Citrullination of histones by Pad4 is a critical step in NET formation, and genetic

inhibition of Pad4 prevents NET formation42. To verify the role of NETs in thrombosis and

PHTN in CH, peptidyl arginine deaminase 4 (Pad4-/-) deficient mice or wild type controls were

subjected to pIVCL or sham. Pad4-/- mice had significantly attenuated portal pressure increases

when compared with wild type mice after pIVCL (Figure 3f). Immunofluorescent studies

confirmed that fibrin formation and sinusoidal MPO deposition was also attenuated after pIVCL

in Pad4-/- mice (Supplementary Figure 3a), indicating that Pad4 deficiency prevents NET

formation and fibrin clot. These results corroborate the critical role of NETs in driving

thrombosis and PHTN in CH.

NE deficiency decreases portal pressure after BDL

To further test our hypothesis that NETs promote PHTN in CH through thrombogenesis,

we studied the impact of NE deficiency after BDL, a second model of PHTN. BDL is a

cholestatic model of liver cirrhosis which induces hepatocellular injury, cholangiocyte

proliferation, and parenchymal infiltration of inflammatory cells, including neutrophils and

Kuppfer cells, which culminates in PHTN 43, 44. To test our hypothesis that sinusoidal neutrophil

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recruitment impacts portal pressures, BDL was performed on wild-type (WT) and NE-/- mice.

Four weeks after BDL, NE-/- mice had attenuated portal pressure increase compared with WT

controls (Figure 4a). NE deficiency also attenuated fibrin formation after BDL, as demonstrated

by immunofluorescence (Figure 4b). In contrast to CH, deficiency of NE and NETosis did not

interrupt fibrogenesis after BDL, as assessed by PCR, biochemical, and microscopic techniques

(Supplementary Figures 4a-4c). We concluded that NE inhibition attenuates portal pressure

increases in two model of liver disease by impairing thrombogenesis.

To corroborate our genetically derived findings, we next employed pharmacologic NE

inhibition after pIVCL. Sivelestat is an inhibitor of NE which has been safely employed in

specific clinical scenarios, including interstitial pneumonia 45 and acute lung injury 46. Sivelestat

was administered via subcutaneous injection three times a week for six weeks following pIVCL.

Sivelestat-treated mice had significantly lower portal pressures 6 weeks after pIVCL when

compared with vehicle-treated mice (Figure 4c). Consistent with our other models of PHTN,

livers from sivelestat-treated mice demonstrated less fibrin formation compared with DMSO-

treated mice (Figure 4d). Sivelestat treatment also decreased sinusoidal myeloperoxidase (MPO)

deposition, suggesting that it disrupts NET formation (Figure 4D).

Cyclic stretch activates integrin-dependent Notch signaling

Given the aforementioned evidence that LSEC responses to mechanical stretch and

sinusoidal thrombosis contribute to the pathophysiology of PHTN, we next returned to our in

vitro models to explore the mechanosensitive pathways which transduce mechanical stretch in

LSECs. For this purpose, we performed RNA-sequencing (RNA-seq) to compare gene

expression profiles of stretched and unstretched control primary murine LSECs (Supplementary

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Figure 5a). Based on two selection criteria (fold change ≥ 2 & FDR < 0.05), 561 genes were

identified as transcriptionally regulated by cyclic stretch (Supplementary Figure 5b). This

analysis confirmed upregulation of CXCL1 observed from the earlier targeted microarray

analysis (logFc 0.981). While CXCL1 is produced by multiple liver cell types in a species and

stimulus dependent manner47, 48, RNAseq from cell lines generated from resident liver cell

populations demonstrated that LSEC are a major source of CXCL1 under basal conditions

(Supplementary Figure 6a) and that CXCL1 production predominates over CXCL2 and CCL2 in

LSEC (Supplementary Figure 6b). This was confirmed from in silico analysis of publically

accessible RNAseq data from FANTOM as well (Supplementary Figure 6c). Among the larger

network of genes impacted by cyclic stretch in LSECs were those related to cellular morphology

and function. Network analysis of genes related to cellular morphology and function revealed a

network of mechanosensitive cell signaling pathways and molecules, including integrin subunits,

the Notch pathway, and molecules related to calcium signaling such as calcium/calmodulin-

dependent protein kinase II (CAMKII) and sarco/endoplasmic reticulum calcium-ATPase

(SERCA) (Supplementary Figure 7a). Ingenuity Pathway Analysis confirmed that genes related

to integrin-linked kinase (ILK) and integrin signaling were significantly impacted by cyclic

stretch, including actin subunits, vinculin, and the integrin subunits (Figure 5a). This analysis

also confirmed upregulation of the transcriptional target of the Notch pathway, Hairy and

Enhancer of Split 1 (Hes1, logFC 1.213), which was verified at the protein level via Western

Blot (Figure 5c). Prior studies demonstrate that interactions between the Notch pathway and

integrins regulate development and carcinogenesis 17, 49, 50, and our network analysis of genes

related to morphology and function suggested a potential interaction with calcium signaling

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(Supplementary Figure 7a). We therefore hypothesized that the integrin-Notch interaction may

intersect with calcium-based signaling to mediate CXCL1-dependent angiocrine signaling.

Studies suggest that integrins enhance Notch signaling by regulating intracellular

processing and endosomal trafficking of the Notch receptor 51. We hypothesized that the Notch

signaling pathway serves as a mechanotransducer downstream of stretch-induced integrin

activation to culminate in CXCL1 release. To test this hypothesis, we treated HUVEC with

arginine-glycine-aspartate (RGD) peptide which inhibits integrin-mediated signaling 52.

Inhibition of integrin signaling prevented stretch-induced upregulation of the Notch

transcriptional targets Hes1 and Hey1 (Figure 5b). Integrin inhibition also prevented

upregulation of CXCL1 by stretch, suggesting that integrins activate the Notch pathway to

promote CXCL1 expression. Indeed, upregulation of Hes1 was verified at the protein level by

Western Blot (Figure 5c), and incubation of HUVEC with the Notch agonist Jagged-1 increased

mRNA levels of CXCL1 (Figure 5d). Conversely, transfection of pooled siRNA to Notch1

(siNotch1) decreased CXCL1 upregulation in the setting of stretch (Figure 5e), and primary

LSEC isolated from mice with LSEC-specific deletion of Notch1 (Notch1i∆EC) have decreased

expression of CXCL1 (Supplementary Figure 8a). Finally, pharmacologic inhibition of Notch

signaling with the gamma-secretase inhibitor DAPT also abrogated the stretch-induced

stimulation of CXCL1 secretion (Figure 5f). These data suggest that initial integrin

mechanosensation activates CXCL1 mechanocrine signaling through the Notch pathway in

LSECs.

The Notch pathway interacts with piezo channels to mediate stretch-induced CXCL1

secretion

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Our RNA-sequencing suggests interaction of integrins with calcium metabolism in

response to mechanical stretch (Supplementary Figure 7a). Integrin activation has been shown to

act upstream of calcium channel activation to modulate cell formation and function 53, and recent

studies suggest that integrins regulate activity of calcium-permeable mechanosensitive ion

channels through traction imposed by myosin 54, 55. We found that our network analysis of genes

related to cell morphology and function impacted by cyclic stretch includes multiple molecules

which have been linked to calcium signaling through piezo ion channels, including ERK1/2 56,

SERCA57, Akt58, and CAMKII 59. Given the aforementioned evidence of integrin interaction

with the Notch pathway, we hypothesized that the Notch pathway may link integrins to piezo

channels to impact downstream mechanocrine signaling. To test this hypothesis, we treated

HUVEC with the specific piezo1 activator Yoda1 60 which increased mRNA expression of the

Notch transcriptional targets Hes1 and Hey1 as well as CXCL1 (Figure 6a). Conversely,

inhibition of piezo1 activity with the pharmacologic inhibitor ruthenium red prevented stretch-

induced Notch activation and CXCL1 upregulation (Figure 6b). Transfection of HUVEC with

siRNA to piezo1 similarly abrogated this response (Figure 6c). To verify the role of the Notch

signaling pathway in piezo1-induced CXCL1 upregulation, HUVEC were transfected with

pooled siRNA to Notch1 and then treated with Yoda1. Transfection with siNotch attenuated

CXCL1 upregulation in response to Yoda1 (Figure 6d). To ascertain for a biochemical

interaction between piezo channels and the Notch pathway, immunoprecipitation assays were

performed on protein lysates from primary murine ECs. The cleaved Notch1 receptor was

immunoprecipitated and prepared for western blot for piezo1. Indeed, Notch1 receptor and

piezo1 protein co-precipitated suggesting a physical interaction between these two proteins

(Supplementary Figure 9a). In summary, our in vitro studies construct a multifaceted pathway of

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mechanocrine signaling which engages integrins, mechanosensitive piezo ion channels, and the

Notch pathway in a functional engagement that induces CXCL1 secretion.

To verify the relevance of our in vitro model of mechanical stretch to the pIVCL model

of CH, primary murine ECs were isolated from mice 48 hours after pIVCL. Primary murine ECs

isolated 48 hours after pIVCL also demonstrate transcriptional upregulation of the Notch

pathway and CXCL1, confirming the relevance of Notch signaling and downstream CXCL1

upregulation in the setting of CH (Figure 6e). Finally, to test the in vivo role of Notch pathway,

we performed IVC ligation and sham procedures in mice with LSEC-specific deletion of Notch1

(Notch1i∆EC). Four weeks after pIVCL, Notch1i∆EC mice had attenuated portal pressure increases

compared with WT controls (Figure 6f). Endothelial deletion of Notch1 also blunted fibrin

formation after pIVCL, as demonstrated by immunofluorescence microscopy (Supplementary

Figure 9b). These results elucidate a novel pathway of endothelial mechanocrine signaling

which drives PHTN and fibrosis in CH (Figure 7).

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Discussion

PHTN constitutes a common final pathway of chronic liver diseases and is a significant

driver of morbidity and mortality. While significant progress has been made in identifying and

treating the etiologies of specific liver diseases, few therapies exist to ameliorate their ultimate

consequence of PTHN. In the current study, we identify pathways of mechanocrine signaling

which are instigated by mechanical stretch and which culminate in microvascular thrombosis,

fibrosis, and PHTN (Figure 7). This study contains the following novel findings: 1) mechanical

stretch induces Notch-dependent upregulation and secretion of the CXCL1 chemokine by

LSECs, 2) CH instigates early vascular recruitment of neutrophils and aggregation with platelets

and LSECs via sinusoidal CXCL1 secretion 3) neutrophils/NETs promote microvascular

thrombosis and fibrosis in CH, 4) microvascular thrombosis drives PHTN through volume effect

in the sinusoids, and 5) interaction of integrins with piezo channels activates the Notch signaling

pathway, upregulates CXCL1, and drives CH-induced PHTN. Together, our results identify a

novel pathway of endothelial mechanocrine signaling and demonstrate the downstream

functional impact of this signaling mechanism on the pathophysiology of PHTN.

CH is characterized by scant parenchymal infiltration of neutrophils, contributing to the

traditional description of CH as a non-inflammatory form of fibrosis 36, 61. However, our results

elucidate a previously unrecognized but critical role that neutrophils within the hepatic sinusoids

play in the pathophysiology of CH. We observed that deficiency of NE abrogates the

development of fibrosis and microvascular thrombosis in our murine model of CH, suggesting

that sinusoidal recruitment of neutrophils is in fact a key component of the pathophysiology of

CH. Although neutrophil infiltration into liver tissue is more prominent histologically in

cholestatic liver diseases than in CH, we found that NE deficiency decreased portal pressure but

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did not impact fibrosis after BDL. This observation suggests that thrombosis may not be as

critical to the development of fibrosis in cholestatic liver diseases. In CH, fibrosis may result

from ischemia and thrombosis induced PHTN which is not the case in cholestatic fibrosis. Thus,

neutrophil recruitment to the sinusoids after pIVCL, as opposed to neutrophil recruitment to the

parenchyma after BDL, may account for the differences we observed62, 63. Importantly, genetic

and pharmacologic inhibition of NE impacts portal pressures in different models of chronic liver

injury, suggesting that neutrophil recruitment and NE may constitute a broad novel therapeutic

target in PHTN.

NETs are thrombogenic structures implicated in vascular thrombosis. Recent studies

suggest that endothelial-derived factors promote neutrophil chemotaxis and NET formation 32, 33.

Platelets have also been well-described as mediators of NET formation via interaction of platelet

toll-like receptor 4 (TLR4) with neutrophils 12,64, 65. We postulate that interactions between

neutrophils, platelets, and LSECs that we visualize in the liver sinusoids early after IVC ligation

instigate NET formation in CH. Hemodynamic forces such as shear stress modulate NET

formation in the setting of thrombosis 66, suggesting that sinusoidal mechanical forces may

further impact NETosis in CH. NETs capture red blood cells and promote clot maturation and

stability through interactions with fibronectin, fibrinogen, and von Willebrand Factor 14, 67. The

release of histones and proteases from NETs further propagates endothelial injury and thrombin

generation 68. Neutrophil serine proteases such as NE are particularly thrombogenic components

of NETs given their ability to promote degradation of tissue factor pathway inhibitor 9. We

found that genetic and pharmacologic inhibition of NE mitigates portal pressure increases in two

models of chronic liver injury by decreasing fibrin formation. We postulate that fibrin physically

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modulates portal pressures through volume and pressure impact within sinusoids and thereby

highlight a critical role of NETs-induced fibrin thrombi in PHTN pathogenesis.

Dilatation of the hepatic sinusoids as occurs in CH confers biaxial stretch on cells lining

the sinusoids, notably LSECs and hepatic stellate cells (HSCs). LSECs are the primary sensors

of disturbances in portal and arterial circulation. Despite their susceptibility to hemodynamic

changes, the mechanisms of mechano-sensation and -transduction in LSECs have not been well-

studied. Our RNA-seq analysis of LSECs subjected to cyclic stretch revealed upregulation of

genes related to integrin signaling. Integrins interact with cytoskeletal proteins and transmit

signals to the cell interior which impact cell differentiation, homeostasis, morphology, and

survival 69. Integrin subunits have also been implicated in collagen remodeling, suggesting that

integrins both sense and modulate mechanical forces 70, 71. Our study suggests that endothelial

integrins may contribute to pressure changes in CH by transducing changes in the sinusoidal

environment to generate inflammatory and fibrotic mechanocrine signals. Recent studies of

LSEC gene expression in a fibrotic matrix also revealed changes of genes related to chemokine

signaling and cytokine receptor interaction 70. Generation of chemokines such as CXCL1 occurs

in multiple liver cell types in addition to LSEC including hepatocytes and inflammatory cells 72.

This is likely dependent upon species as well as the physiologic or pathophysiologic stimulus.

CXCL1 production may also occur as part of a larger chemokine program given redundancy of

function and signal activation of multiple CXC chemokines 73. Nonetheless, our study provides

evidence that mechanical sensation by LSEC can drive chemokine dependent changes in

vascular structure and function with CXCL1 serving as a prototype.

We found that integrin activation contributes to a mechanosensitive pathway which

entails functional engagement of the Notch pathway with piezo proteins to generate CXCL1

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release in response to mechanical stretch. Studies suggest that the Notch1 receptor is a pivotal

mediator of liver sinusoidal structure and portal pressure 18, 74, 75. Prior studies report that mice

with disruption of Notch1 signaling in LSECs and hepatocytes develop a phenotype resembling

nodular regenerative hyperplasia (NRH) with prominent angio-architectural distortions 18,76. Our

data suggest that the Notch1 receptor modulates sinusoidal tone and portal pressure in response

to mechanical forces by regulating formation of fibrin within liver sinusoids demonstrating the

multifaceted role of Notch signaling in modulation of portal pressure. We also found that Notch

activation by mechanical stretch relies on integrin mechanosensation. Integrins exert traction

forces on piezo channels likely through myosin which enhance their activation. Piezo1 channels

then physically engage with the Notch1 receptor to modulate CXCL1 expression. While piezo1

expression has been described in human liver endothelium 77, its role as a calcium channel in the

development of liver disease has not been explored. Calcium influx impacts endothelial

vasodilation and vascular tone through several mechanisms 78, including calcium-dependent

modulation of vasodilatory molecules such as eNOS and endothelium-derived hyperpolarization

(factor) (EDH(F)) 20 and through activation of tissue transglutaminases and endothelial

remodeling 79. Recent studies implicate piezo channels in the upregulation of blood pressure

during physical activity 20. Our results suggest that piezo channels also contribute to vascular

tone by participating in mechanocrine signaling pathways whose downstream targets impact

portal pressure.

In total our work identifies a series of new pathophysiologic pathways and associated

potential therapeutic targets in PHTN and other diseases within hepatic sinusoids which are

attributed to mechanical forces.

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Figure Legends

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. 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

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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. 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

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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. 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. 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

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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. 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 (Notch1i∆EC) have lower portal

pressures when compared with Notchfl/fl mice 4 weeks after IVC ligation (n=7-11; ANOVA

P≤0.05).

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

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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 myosin 54, 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.

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References (author names in bold designate shared co-first authorship)

1. Bosch J, Groszmann RJ, Shah VH. Evolution in the understanding of the

pathophysiological basis of portal hypertension: How changes in paradigm are leading to

successful new treatments. J Hepatol 2015;62:S121-30.

2. Simonetto DA, Yang HY, Yin M, et al. Chronic passive venous congestion drives hepatic

fibrogenesis via sinusoidal thrombosis and mechanical forces. Hepatology 2015;61:648-

59.

3. Villa E, Camma C, Marietta M, et al. Enoxaparin prevents portal vein thrombosis and

liver decompensation in patients with advanced cirrhosis. Gastroenterology

2012;143:1253-60 e1-4.

4. Cerini F, Vilaseca M, Lafoz E, et al. Enoxaparin reduces hepatic vascular resistance and

portal pressure in cirrhotic rats. J Hepatol 2016;64:834-42.

5. Sreeramkumar V, Adrover JM, Ballesteros I, et al. Neutrophils scan for activated

platelets to initiate inflammation. Science 2014;346:1234-8.

6. Wang H, Wang Q, Wang J, et al. Proprotein convertase subtilisin/kexin type 9 (PCSK9)

Deficiency is Protective Against Venous Thrombosis in Mice. Sci Rep 2017;7:14360.

7. von Bruhl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets

cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med

2012;209:819-35.

8. Pak S, Kondo T, Nakano Y, et al. Platelet adhesion in the sinusoid caused hepatic injury

by neutrophils after hepatic ischemia reperfusion. Platelets 2010;21:282-8.

9. Massberg S, Grahl L, von Bruehl ML, et al. Reciprocal coupling of coagulation and

innate immunity via neutrophil serine proteases. Nat Med 2010;16:887-96.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

31

10. Martinod K, Demers M, Fuchs TA, et al. Neutrophil histone modification by

peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad

Sci U S A 2013;110:8674-9.

11. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill

bacteria. Science 2004;303:1532-5.

12. Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4 activates neutrophil extracellular

traps to ensnare bacteria in septic blood. Nat Med 2007;13:463-9.

13. McDonald B, Urrutia R, Yipp BG, et al. Intravascular neutrophil extracellular traps

capture bacteria from the bloodstream during sepsis. Cell Host Microbe 2012;12:324-33.

14. Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis.

Proc Natl Acad Sci U S A 2010;107:15880-5.

15. Rafii S, Butler JM, Ding BS. Angiocrine functions of organ-specific endothelial cells.

Nature 2016;529:316-25.

16. Gauthier NC, Roca-Cusachs P. Mechanosensing at integrin-mediated cell-matrix

adhesions: from molecular to integrated mechanisms. Curr Opin Cell Biol 2018;50:20-26.

17. LaFoya B, Munroe JA, Mia MM, et al. Notch: A multi-functional integrating system of

microenvironmental signals. Dev Biol 2016;418:227-41.

18. Cuervo H, Nielsen CM, Simonetto DA, et al. Endothelial notch signaling is essential to

prevent hepatic vascular malformations in mice. Hepatology 2016;64:1302-1316.

19. Hofmann JJ, Iruela-Arispe ML. Notch signaling in blood vessels: who is talking to whom

about what? Circ Res 2007;100:1556-68.

20. Rode B, Shi J, Endesh N, et al. Piezo1 channels sense whole body physical activity to

reset cardiovascular homeostasis and enhance performance. Nat Commun 2017;8:350.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

32

21. Coste B, Xiao B, Santos JS, et al. Piezo proteins are pore-forming subunits of

mechanically activated channels. Nature 2012;483:176-81.

22. Cao S, Yaqoob U, Das A, et al. Neuropilin-1 promotes cirrhosis of the rodent and human

liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells. J Clin Invest

2010;120:2379-94.

23. Semela D, Das A, Langer D, et al. Platelet-derived growth factor signaling through

ephrin-b2 regulates hepatic vascular structure and function. Gastroenterology

2008;135:671-9.

24. Zeng Z, Surewaard BG, Wong CH, et al. CRIg Functions as a Macrophage Pattern

Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

Cell Host Microbe 2016;20:99-106.

25. Ding BS, Nolan DJ, Butler JM, et al. Inductive angiocrine signals from sinusoidal

endothelium are required for liver regeneration. Nature 2010;468:310-5.

26. Sackey-Aboagye B, Olsen AL, Mukherjee SM, et al. Fibronectin Extra Domain A

Promotes Liver Sinusoid Repair following Hepatectomy. PLoS One 2016;11:e0163737.

27. Anwar MA, Shalhoub J, Lim CS, et al. The effect of pressure-induced mechanical stretch

on vascular wall differential gene expression. J Vasc Res 2012;49:463-78.

28. Cummings CJ, Martin TR, Frevert CW, et al. Expression and function of the chemokine

receptors CXCR1 and CXCR2 in sepsis. J Immunol 1999;162:2341-6.

29. Sawant KV, Poluri KM, Dutta AK, et al. Chemokine CXCL1 mediated neutrophil

recruitment: Role of glycosaminoglycan interactions. Sci Rep 2016;6:33123.

30. Gao Y, Sun J, Lin WH, et al. A compact microfluidic gradient generator using passive

pumping. Microfluid Nanofluidics 2012;12:887-895.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

33

31. Kim SJ, Jenne CN. Role of platelets in neutrophil extracellular trap (NET) production

and tissue injury. Semin Immunol 2016;28:546-554.

32. Gupta AK, Joshi MB, Philippova M, et al. Activated endothelial cells induce neutrophil

extracellular traps and are susceptible to NETosis-mediated cell death. FEBS Lett

2010;584:3193-7.

33. Gollomp K, Kim M, Johnston I, et al. Neutrophil accumulation and NET release

contribute to thrombosis in HIT. JCI Insight 2018;3.

34. Semeraro F, Ammollo CT, Morrissey JH, et al. Extracellular histones promote thrombin

generation through platelet-dependent mechanisms: involvement of platelet TLR2 and

TLR4. Blood 2011;118:1952-61.

35. Akiyoshi H, Terada T. Centrilobular and perisinusoidal fibrosis in experimental

congestive liver in the rat. J Hepatol 1999;30:433-9.

36. Arcidi JM, Jr., Moore GW, Hutchins GM. Hepatic morphology in cardiac dysfunction: a

clinicopathologic study of 1000 subjects at autopsy. Am J Pathol 1981;104:159-66.

37. Wang Y, Li M, Stadler S, et al. Histone hypercitrullination mediates chromatin

decondensation and neutrophil extracellular trap formation. J Cell Biol 2009;184:205-13.

38. Margraf S, Logters T, Reipen J, et al. Neutrophil-derived circulating free DNA (cf-

DNA/NETs): a potential prognostic marker for posttraumatic development of

inflammatory second hit and sepsis. Shock 2008;30:352-8.

39. Asrani SK, Asrani NS, Freese DK, et al. Congenital heart disease and the liver.

Hepatology 2012;56:1160-9.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

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40. Papayannopoulos V, Metzler KD, Hakkim A, et al. Neutrophil elastase and

myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol

2010;191:677-91.

Please see supplement for additional references.

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Supplemental Materials and Methods

Cell isolation and culture

Primary murine LSECs were isolated from wild type mice using an immunomagnetic bead

isolation protocol as previously described 80 and were used in all experiments unless indicated

otherwise. Human umbilical vein endothelial cells (HUVEC) at early passage were used for

selected experiments as indicated in which the proposed molecular interventions could not

technically be performed in primary murine LSECs. In selected experiments, cell supernatant

was collected and ELISA performed to assess concentration of secreted CXCL1 (R&D,

MKC00B).

Real Time Polymerase Chain Reaction

Messenger RNA (mRNA) levels were quantified by real-time reverse transcription polymerase

chain reaction. Human Endothelial Cell Biology microarray was performed on cDNA isolated

from primary murine LSECs (RT2 Profiler PCR Array, PAHS-015A, Qiagen Sciences,

Maryland, USA). RNA-seq analysis was done with the Mayo Clinic Center for Individualized

Medicine Medical Genomics Facility.

Cyclic stretch

Primary murine EC and HUVEC were seeded on 6-well plates with flexible silicone bottoms

coated with collagen type IV (FLEXI® culture plates). After incubation for 24 hours, cells were

serum-starved for 6 hours and then subjected to cyclic stretch for 4 hours as previously described

81. A strain amplitude of 20% was used at a rate of 60 cycles/minute at 37̊C and 5% CO2 in a

humidified incubator.

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Intravital imaging

Intravital imaging of mouse liver was conducted as previously described 82. Briefly, mice were

anesthetized by intraperitoneal administration of ketamine (200 mg per kg body weight; Bayer

Animal Health) and Xylazine (10 mg per kg body weight; Bimeda-MTC). Tail vein cannulation

was then performed to deliver additional anesthetics and fluorescence conjugated antibodies (PE-

Ly6G clone 1A8, 1.6µg/mouse). The mouse was placed in the supine position, and a midline

incision was made in the abdomen after applying mineral oil onto the skin. All the visible vessels

underlying the abdominal skin were cauterized. The skin was cut along the costal margin to the

mid-axillary line, and the underlying muscles were removed using cautery to expose the liver.

The falciform ligament was cut to separate the liver from the diaphragm. The mouse was then

placed in a right lateral position, the largest lobe of the liver was carefully isolated and

externalized onto a glass coverslip on the inverted microscope stage, which is kept at 37°C. The

isolated liver lobe was covered with Kimwipes and kept moist with saline. The liver was then

imaged using an inverted spinning-disk confocal microscopy system (Olympus IX81) which is

equipped with four laser excitation wavelengths (491 nm, 561 nm, 643 nm and 730nm; Cobalt)

in combination with bandpass emission filters (Semrock). The fluorescent signals were recorded

by a back thinned electron-multiplying charge-coupled device camera (512 × 512 pixel, C9100-

13; Hamamatsu). Images were acquired and analyzed using Volocity software 6.1 (Improvision).

Immunofluorescence

Frozen liver tissues were cut into 7 µm sections and fixed in 4% paraformaldehyde for 5

minutes. Samples were blocked in 10% fetal bovine serum and 1% BSA in PBS for one hour at

room temperature. Mouse on Mouse (M.O.M) blocking reagent (Vector Laboratories, MKB-

2213, 2 drops in 2.5 ml PBS) was then used to block endogenous mouse antibody for 1 hour at

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room temperature. Samples were then incubated overnight at 4°C with goat anti-collagen

(Southern Biotech, 1:100), rabbit anti-fibrin(ogen) (Molecular Innovations, MFBGN, 1:500), or

anti-mouse myeloperoxidase (Abcam, ab90812, 1:100), followed by incubation for one hour at

room temperature with Alexa Fluor 568 conjugated donkey anti-goat, Alexa Fluor 546

conjugated donkey anti-rabbit, or Alexa Fluor 488 conjugated goat anti-mouse. For experiments

utilizing biotinylated anti-mouse secondary antibody (Vector Laboratories, BA-9200, 1:100) an

additional blocking step with Avidin/Biotin Blocking Kit (Vector Laboratories, SP-2001) was

performed according to manufacturer’s instructions. This was followed by incubation for 30

minutes with streptavidin-Cy3 (Jackson ImmunoResearch, 016-160-084, 1:100). Nuclei were

counterstained with DAPI at a 1:2500 concentration for 10 minutes. Images were acquired using

epifluorescence microscopes (Axio Observer and Axiovert 200; Carl Zeiss MicroImaging) or an

inverted spinning-disk confocal microscopy system (Olympus IX81) as described above.

siRNA transfection and Yoda 1 treatment

HUVEC were seeded on 6-well plates with flexible silicone bottoms coated with collagen type

IV (FLEXI® culture plates). After incubation for 24 hours, cells were serum-starved for 6 hours

and transfected with Notch siRNA (OnTargetplus SmartPool siRNA, Dharmacon) according to

manufacturer’s instructions. Forty-eight hours later, cells were serum starved for 6 hours and

then treated with Yoda 1 (10 uM) for 6 hours.

dsDNA-MPO quantification

Subject samples were kindly provided by the Center for Cell Signaling in Gastroenterology,

Mayo Clinic. dsDNA-MPO complexes were identified as a measure of NETosis using capture

ELISA. 100 µl of 5 µg/ml capturing MPO antibody (Bio-Rad, 0400-0002) in coating buffer was

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incubated on 96-well microtiter plate overnight at 4°C on a rocking plate. The coating solution

was washed off by patting dry and washed thrice with 300 µl PBS. Blocking was done in 1 %

BSA (200 µl/well) for 1 hour. The blocking solution was patted dry and washed with PBS thrice.

50 µl of serum was then added to each well and each subject’s samples were run as technical

triplicates. Peroxidase-labeled anti-DNA monoclonal antibody (component 2, Cell Death

Detection ELISA Plus, Roche, 1174425001) was used according to manufacturer’s instructions.

Samples were left to mix on rocker plate for 1 hour at room temperature. Following three 300 µl

PBS washes, 200 µl of the peroxidase substrate (ABTS) from the kit was added. This was left to

incubate for 15 minutes in dark and then reaction stopped using the stop buffer from kit.

Absorbance was measured using a spectrophotometer (Molecular Devices, Spectramax Plus 384)

at 405 nm wavelength.

dsDNA-cis-Histone quantification

Subject samples were kindly provided by the Center for Cell Signaling in Gastroenterology,

Mayo Clinic. Capture ELISA was performed using the Cell Death Detection ELISA Plus

(Roche, 1174425001) using manufacturer’s instructions. This was left to incubate for 15 minutes

in dark, reaction stopped using ABTS stop buffer and absorbance was measured using a

spectrophotometer at 405 nm wavelength.

Neutrophil isolation and migration

Human neutrophils were isolated from whole blood using an immunomagnetic separation

technique (MACSxpress 130-104-434). Following isolation, cells were suspended in HBSS

supplemented with 10% fetal bovine serum (FBS). Prior to cell seeding, a microfluidic device

was coated with a 10% FBS solution for 30 minutes. This was removed and the device washed

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twice with ice cold phosphate buffered saline (PBS). Cells were imaged for one hour following

seeding using a Lionhart FX microscope, and their migration in response to CXCL1 (100 ng/mL

in PBS) or vehicle control was quantified using Metamorph software.

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Supplementary Figure Legends

Supplementary Figure 1. Diagram of microfluidic device used to study neutrophil chemotaxis.

Highest concentrations of vehicle and CXCL1 are located at the top on the device.

Supplementary Figure 2. (A) Serum from mice which had undergone pIVCL have significantly

increased levels of circulating dsDNA. (B) Liver samples were obtained from healthy

individuals (controls) and from patients with congestive hepatopathy after corrective surgery

(Fontan procedure) for complex congenital heart disease. Fibrin (green) and MPO (red)

immunofluorescence were significantly increased in CH compared to controls. DAPI

counterstain was used to stain cell nuclei. Quantification was performed with ImageJ and

displayed in the adjacent graphs (n = 5-7; *P ≤ 0.05 for all panels).

Supplementary Figure 3. (A) Immunofluorescent staining (10x) shows decreased fibrin (green)

and MPO (red) content in Pad4-/- mice after pIVCL compared to livers from WT mice.

Quantification was performed with ImageJ and displayed in the adjacent graph (n=4-6; *P ≤

0.05).

Supplementary Figure 4. (A) NE deficiency does not impact mRNA expression of activating

fibrogenic genes after BDL. Fibrosis was assessed by (B) hydroxyproline assay and (C) Sirius

red staining (5x) (n = 5-7; *P ≤ 0.05 for all panels).

Supplementary Figure 5. (A) Schematic of procedure for RNA isolation and sequencing. (B)

Heatmap of gene expression depicting differences in the expression profile between control and

stretched primary murine EC.

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Supplementary Figure 6. (A) CXCL1 production in various human liver cell types. Cells were

purchased commercially, including HepG2 cells, hepatic stellate cells, human intrahepatic biliary

cells, and human hepatic sinusoidal endothelial cells. Expression was analyzed by RNA

sequencing in triplicates. RPKM values of CXCL1 were normalized to the RPKM values of

multiple house-keeping genes including C1orf43 CHMP2A GPI PSMB2 PSMB4 RAB7A VCP

VPS29, as described by Eisenberg et. Al 83. (B) Expression of various chemokines in human

LSEC. RNA sequencing was performed as described above. (C) Transcriptome analysis of

CXCL1 expression of isolated primary liver cells from FANTOM database. RNA sequencing

data of hepatic sinusoidal endothelial cells, hepatic stellate cells, and hepatocytes were obtained

from three donors. Adult liver and fetal liver samples were each obtained from one donor.

Supplementary Figure 7. (A) Network analysis of genes related to cellular morphology and

function reveals interaction of integrin subunits (red box) with the Notch pathway (blue box) and

genes related to calcium metabolism including CAMKII (black box) and SERCA (green box)

(ITGAV, integrin subunit alpha V; ITGB3, integrin subunit beta 3; CAMKII,

calcium/calmodulin-dependent protein kinase II).

Supplementary Figure 8. (A) Primary LSECs were isolated from Notchfl/fl and Notch1i∆EC mice.

Quantitative reverse transcription PCR showed decreased mRNA levels of CXCL1 in LSEC

from Notch1i∆EC mice, while mRNA levels of CXCL2 and CCl2 were not significantly different

(n = 4, *P ≤ 0.05 for all panels).

Supplementary Figure 9. (A) Immunoprecipitation performed using anti-Notch 1 tagged

agarose beads, and Western blot using anti-piezo 1 antibody shows that piezo 1 channels bind to

the Notch receptor (n = 3; *P ≤ 0.05 for all panels). (B) Immunofluorescent staining (10x) shows

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decreased fibrin (green) and MPO (red) content in Notch1∆EC mice after pIVCL compared to

livers from WT mice. Quantification was performed with ImageJ and displayed in the adjacent

graph (n=5-7; *P ≤ 0.05).

Supplementary Movie 1. Intravital imaging of livers after sham (left) procedure reveals intact

sinusoidal architecture and neutrophils which traverse and rapidly exit the liver sinusoids.

Neutrophils are labelled with red-conjugated anti-Ly6G antibody, platelets with blue-labelled

anti-CD49b, and extracellular DNA was detected with Sytox Green. pIVCL (right) induces

sinusoidal dilation, neutrophil accumulation within sinusoids, and their aggregation with

platelets.

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References (author names in bold designate shared co-first authorship)

41. Martinod K, Witsch T, Farley K, et al. Neutrophil elastase-deficient mice form neutrophil

extracellular traps in an experimental model of deep vein thrombosis. J Thromb Haemost

2016;14:551-8.

42. Li P, Li M, Lindberg MR, et al. PAD4 is essential for antibacterial innate immunity mediated by

neutrophil extracellular traps. J Exp Med 2010;207:1853-62.

43. Georgiev P, Jochum W, Heinrich S, et al. Characterization of time-related changes after

experimental bile duct ligation. Br J Surg 2008;95:646-56.

44. Rautou PE, Tatsumi K, Antoniak S, et al. Hepatocyte tissue factor contributes to the

hypercoagulable state in a mouse model of chronic liver injury. J Hepatol 2016;64:53-9.

45. Kubo N, Araki K, Yamanaka T, et al. Perioperative management of hepatectomy in patients with

interstitial pneumonia: a report of three cases and a literature review. Surg Today 2017.

46. Zeiher BG, Artigas A, Vincent JL, et al. Neutrophil elastase inhibition in acute lung injury:

results of the STRIVE study. Crit Care Med 2004;32:1695-702.

47. Chang B, Xu MJ, Zhou Z, et al. Short- or long-term high-fat diet feeding plus acute ethanol binge

synergistically induce acute liver injury in mice: an important role for CXCL1. Hepatology

2015;62:1070-85.

48. Brempelis KJ, Yuen SY, Schwarz N, et al. Central role of the TIR-domain-containing adaptor-

inducing interferon-beta (TRIF) adaptor protein in murine sterile liver injury. Hepatology

2017;65:1336-1351.

49. Suh HN, Han HJ. Collagen I regulates the self-renewal of mouse embryonic stem cells through

alpha2beta1 integrin- and DDR1-dependent Bmi-1. J Cell Physiol 2011;226:3422-32.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

50. Mo JS, Kim MY, Han SO, et al. Integrin-linked kinase controls Notch1 signaling by down-

regulation of protein stability through Fbw7 ubiquitin ligase. Mol Cell Biol 2007;27:5565-74.

51. Gomez-Lamarca MJ, Cobreros-Reguera L, Ibanez-Jimenez B, et al. Integrins regulate epithelial

cell differentiation by modulating Notch activity. J Cell Sci 2014;127:4667-78.

52. Ruoslahti E, Pierschbacher MD. Arg-Gly-Asp: a versatile cell recognition signal. Cell

1986;44:517-8.

53. Jacquemet G, Baghirov H, Georgiadou M, et al. L-type calcium channels regulate filopodia

stability and cancer cell invasion downstream of integrin signalling. Nat Commun 2016;7:13297.

54. Nourse JL, Pathak MM. How cells channel their stress: Interplay between Piezo1 and the

cytoskeleton. Semin Cell Dev Biol 2017;71:3-12.

55. Pathak MM, Nourse JL, Tran T, et al. Stretch-activated ion channel Piezo1 directs lineage choice

in human neural stem cells. Proc Natl Acad Sci U S A 2014;111:16148-53.

56. Gudipaty SA, Lindblom J, Loftus PD, et al. Mechanical stretch triggers rapid epithelial cell

division through Piezo1. Nature 2017;543:118-121.

57. He L, Si G, Huang J, et al. Mechanical regulation of stem-cell differentiation by the stretch-

activated Piezo channel. Nature 2018;555:103-106.

58. Wang S, Chennupati R, Kaur H, et al. Endothelial cation channel PIEZO1 controls blood pressure

by mediating flow-induced ATP release. J Clin Invest 2016;126:4527-4536.

59. Hung WC, Yang JR, Yankaskas CL, et al. Confinement Sensing and Signal Optimization via

Piezo1/PKA and Myosin II Pathways. Cell Rep 2016;15:1430-1441.

60. Syeda R, Xu J, Dubin AE, et al. Chemical activation of the mechanotransduction channel Piezo1.

Elife 2015;4.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

61. Dai DF, Swanson PE, Krieger EV, et al. Congestive hepatic fibrosis score: a novel histologic

assessment of clinical severity. Mod Pathol 2014;27:1552-8.

62. Gujral JS, Farhood A, Bajt ML, et al. Neutrophils aggravate acute liver injury during obstructive

cholestasis in bile duct-ligated mice. Hepatology 2003;38:355-63.

63. Gujral JS, Liu J, Farhood A, et al. Functional importance of ICAM-1 in the mechanism of

neutrophil-induced liver injury in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol

2004;286:G499-507.

64. Maugeri N, Campana L, Gavina M, et al. Activated platelets present high mobility group box 1 to

neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J

Thromb Haemost 2014;12:2074-88.

65. Konstantopoulos K, Neelamegham S, Burns AR, et al. Venous levels of shear support neutrophil-

platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.

Circulation 1998;98:873-82.

66. Yu X, Tan J, Diamond SL. Hemodynamic force triggers rapid NETosis within sterile thrombotic

occlusions. J Thromb Haemost 2018;16:316-329.

67. Ward CM, Tetaz TJ, Andrews RK, et al. Binding of the von Willebrand factor A1 domain to

histone. Thromb Res 1997;86:469-77.

68. Saffarzadeh M, Juenemann C, Queisser MA, et al. Neutrophil extracellular traps directly induce

epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012;7:e32366.

69. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673-87.

70. Liu L, You Z, Yu H, et al. Mechanotransduction-modulated fibrotic microniches reveal the

contribution of angiogenesis in liver fibrosis. Nat Mater 2017;16:1252-1261.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

71. Henderson NC, Arnold TD, Katamura Y, et al. Targeting of alphav integrin identifies a core

molecular pathway that regulates fibrosis in several organs. Nat Med 2013;19:1617-24.

72. Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology 2014;147:577-594 e1.

73. Brown JD, Lin CY, Duan Q, et al. NF-kappaB directs dynamic super enhancer formation in

inflammation and atherogenesis. Mol Cell 2014;56:219-231.

74. Duan JL, Ruan B, Yan XC, et al. Endothelial Notch activation reshapes the angiocrine of

sinusoidal endothelia to aggravate liver fibrosis and blunt regeneration. Hepatology 2018.

75. Wang L, Wang CM, Hou LH, et al. Disruption of the transcription factor recombination signal-

binding protein-Jkappa (RBP-J) leads to veno-occlusive disease and interfered liver regeneration

in mice. Hepatology 2009;49:268-77.

76. Dill MT, Rothweiler S, Djonov V, et al. Disruption of Notch1 induces vascular remodeling,

intussusceptive angiogenesis, and angiosarcomas in livers of mice. Gastroenterology

2012;142:967-977 e2.

77. Li J, Hou B, Tumova S, et al. Piezo1 integration of vascular architecture with physiological

force. Nature 2014;515:279-82.

78. Bataller R, Gasull X, Gines P, et al. In vitro and in vivo activation of rat hepatic stellate cells

results in de novo expression of L-type voltage-operated calcium channels. Hepatology

2001;33:956-62.

79. Retailleau K, Duprat F, Arhatte M, et al. Piezo1 in Smooth Muscle Cells Is Involved in

Hypertension-Dependent Arterial Remodeling. Cell Rep 2015;13:1161-71.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

80. Huebert RC, Vasdev MM, Shergill U, et al. Aquaporin-1 facilitates angiogenic invasion

in the pathological neovasculature that accompanies cirrhosis. Hepatology 2010;52:238-

48.

81. Stroetz RW, Vlahakis NE, Walters BJ, et al. Validation of a new live cell strain system:

characterization of plasma membrane stress failure. J Appl Physiol (1985) 2001;90:2361-

70.

82. Zeng Z, Surewaard BG, Wong CH, et al. CRIg Functions as a Macrophage Pattern

Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

Cell Host Microbe 2016;20:99-106.

83. Eisenberg E, Levanon EY. Human housekeeping genes, revisited. Trends Genet

2013;29:569-74.

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ACCEPTED MANUSCRIPTSupplementary Figure 1.

Chemokine inlet

Media inlet

Cell culture port

CXCL1 concentration

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Fib

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Supplementary Figure 2.

*

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ACCEPTED MANUSCRIPTSupplementary Figure 3.

DA

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WT Sham WT BDL NE-/- BDL

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ACCEPTED MANUSCRIPTSupplementary Figure 5.

Primary murine LSECs

Stretched (1 Hz, 14% strain).

RNA extraction.

Library preparation and sequencing.

A.

Static controls.

B. Control Stretch

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ACCEPTED MANUSCRIPTSupplementary Figure 6.A.

B.

C.

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A. IgG Notch

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Notch 1

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ACCEPTED MANUSCRIPTSupplementary Movie 1.