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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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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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Chemokine inlet
Media inlet
Cell culture port
CXCL1 concentration
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Fib
rin
inte
gra
ted
den
sity
Supplementary Figure 2.
*
Sham IVC
dsD
NA
(n
g/m
L)
A.
0
50
100
150
200 Control Cardiac cirrhosis
DA
PI
Fib
rin
MP
OM
erg
e
Ctl Cirrhosis
*
Ctl Cirrhosis
MP
O in
teg
rate
d d
ensi
ty
*
B.
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DA
PI
Fib
rin
MP
OM
erg
e
A. Pad4-/- IVCWT IVCWT Sham
Fib
rin
inte
gra
ted
den
sity
* *
WTSham WT IVC Pad4-/-
IVC
WTSham WT IVC Pad4-/-
IVC
MP
O in
teg
rate
d d
ensi
ty * *
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WT Sham WT BDL NE-/- BDL
aSM
Am
RN
A(f
old
ch
ang
e)
**
0
20
40
60
WT Sham WT BDL NE-/- BDL
TIM
P-1
mR
NA
(fo
ld c
han
ge)
*
*
WT Sham WT BDL NE-/- BDL
Co
l-1
mR
NA
(fo
ld c
han
ge)
*
Supplementary Figure 4.A.
C.
WT Sham WT BDL NE-/- BDL
*NE-/- BDLWT BDLWT Sham
C.
0
5
10
15
20
25
WT Sham WT BDL NE-/- BDL
*
Sir
ius
red
qu
anti
fica
tio
n(%
are
a)
0.0
0.1
0.2
0.3
Hyd
roxy
prol
ine
(ug/
ml)
Hyd
roxy
pro
line
(µg
/mL
)
B.
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Primary murine LSECs
Stretched (1 Hz, 14% strain).
RNA extraction.
Library preparation and sequencing.
A.
Static controls.
B. Control Stretch
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B.
C.
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CX
CL
1 m
RN
A(f
old
ch
ang
e)
Notchfl/fl Notchi∆EC
*
CX
CL
2 m
RN
A(f
old
ch
ang
e)
Notchfl/fl Notchi∆EC Notchfl/fl Notchi∆EC
CC
L2
mR
NA
(fo
ld c
han
ge)
Supplementary Figure 8.
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A. IgG Notch
Piezo 1
Notch 1
Input
IgG Notch
Pie
zo1
den
sito
met
ry(f
old
ch
ang
e)
*
Supplementary Figure 9.
B. Notchfl/fl
Sham
DA
PI
Fib
rin
MP
OM
erg
e
Notchfl/fl
IVCNotchi∆ECl
ShamNotchi∆ECl
IVC
Notchfl/fl Notchfl/flNotch1i∆ECNotchi∆EC
Sham IVC Sham IVCF
ibri
n in
teg
rate
d d
ensi
ty
* *
*
Notchfl/fl Notchfl/flNotch1i∆ECNotchi∆EC
Sham IVC Sham IVC
MP
O in
teg
rate
d d
ensi
ty * *
*
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