Angiocrine Signaling in Sinusoidal Health and Disease

Abstract

Liver sinusoidal endothelial cells (LSECs) are key players in maintaining hepatic homeostasis. They also play crucial roles during liver injury by communicating with liver cell types as well as immune cells and promoting portal hypertension, fibrosis, and inflammation. Cutting-edge technology, such as single cell and spatial transcriptomics, have revealed the existence of distinct LSEC subpopulations with a clear zonation in the liver. The signals released by LSECs are commonly called “angiocrine signaling.” In this review, we summarize the role of angiocrine signaling in health and disease, including zonation in healthy liver, regeneration, fibrosis, portal hypertension, nonalcoholic fatty liver disease, alcohol-associated liver disease, aging, drug-induced liver injury, and ischemia/reperfusion, as well as potential therapeutic advances. In conclusion, sinusoidal endotheliopathy is recognized in liver disease and promising preclinical studies are paving the path toward LSEC-specific pharmacotherapies.

Graphical Abstract

Lay Summary

Liver sinusoidal endothelial cells (LSECs) are key players in maintaining hepatic homeostasis. They also play crucial roles during liver injury by sending signals, referred to as angiocrine signaling, to liver cell types as well as immune cells. In this review, we will summarize angiocrine signaling in health and disease.

Hepatic microvessels are called sinusoids due to their unique sinusoid alanatomical structure. These sinusoids are composed of liver sinusoidal endothelial cells (LSECs), which represent a subpopulation of endothelial cells in the liver with distinct features, such as lack of a basal membrane and the presence of fenestrae.^1–3 The fusion of the luminal and abluminal plasma membrane to form the sinusoids occurs at the level of LSEC fenestrae, differently from other endothelial cells where it usually happens at the level of cell junctions.⁴ The fenestrated cellular walls of LSECs are highly permeable enabling the rapid transport of nutrients, metabolites, and soluble angiocrine signaling (Asig) molecules between the circulation and the space of Disse for access to hepatocytes and resident non-parenchymal cells such as hepatic stellate cells (HSCs) and immune cells.^5–7 Recent technological advances, such as single cell RNA sequencing (scRNAseq) and spatial transcriptomics, have revealed LSEC angiocrine phenotype heterogeneity⁶ and allow studies of the angioimmune niche at unprecedented depth. LSEC contributions to liver health and disease were very recently extensively reviewed.⁶ Therefore, in this article, we will give an overview of the recent advances regarding Asig in liver health, regeneration, and disease, specifically.

Asig in Hepatic Health and Regeneration

The importance of hepatic zonation during health and injury has received significant attention recently, due in part to advances in scRNAseq and spatial transcriptomics/multiomics.^8,9 Furthermore, web-browsable versions of published datasets and species/organ atlases are increasingly available—a powerful resource to explore newly appreciated Asig molecules, cellular subtypes, and interaction partners (► Table 1).

Table 1.

Browsable datasets for angiocrine signaling

First author and year	Article title	Web site	Modality	Species
Inverso 202110	A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie-Wnt signaling axis in the liver	http://pproteomedb.dkfz.de	Transcriptome, proteome, phosphoproteome	Mouse
Kalucka 2020150	Single-cell transcriptome atlas of murine endothelial cells	https://carmelietlab.sites.vib.be/en/software-tools	Multiple	Mouse
Aizarani 2019151	A human liver cell atlas reveals heterogeneity and epithelial progenitors	http://human-liver-cellatlas.ie-freiburg.mpg.de	scRNAseq	Human
Drzewiecki 202178	GIMAP5 maintains liver endothelial cell homeostasis and prevents portal hypertension	https://cellbrowser.yalespace.org/gimap5_res0-3/?ds=gimap5	scRNAseq	Mouse
Ramachandran 201936	Resolving the fibrotic niche of human liver cirrhosis at single-cell level	http://www.livercellatlas.mvm.ed.ac.uk	scRNAseq	Human, mouse
McParland 201823	Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations	http://shiny.baderlab.org/HumanLiverAtlas/	scRNAseq	Human
Dobie 201934	Single-cell transcriptomics uncovers zonation of function in the mesenchyme during liver fibrosis	http://livermesenchyme.hendersonlab.mvm.ed.ac.uk	scRNAseq	Mouse
Pita-Juarez 2022152	A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients	Supplemental data	snRNAseq and nanostring DSP WTA	Human
Holland 2022153	Transcriptomic cross-species analysis of chronic liver disease reveals consistent regulation between humans and mice	https://saezlab.shinyapps.io/liverdiseaseatlas/	scRNAseq	Human, mouse
Bondareva 2022154	Single-cell profiling of vascular endothelial cells reveals progressive organ-specific vulnerabilities during obesity	https://obesity-ecatlas.helmholtz-muenchen.de/	scRNAseq	Mouse
Betsholtz 2022155	Human cell atlas: contains links to multiple browsable portals and datasets	https://www.humancellatlas.org/portals/	Multiple	Human
The Tabula Muris Consortium 2018156	Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris	http://tabula-muris.ds.czbiohub.org/	Multiple	Mouse
Paik 2020157	Single-cell RNA sequencing unveils unique transcriptomic signatures of organ-specific endothelial cells	Supplemental data	Multiple	Mouse
Massalha 2020158	A single cell atlas of the human liver tumor microenvironment	https://itzkovitzwebapps.weizmann.ac.il/webapps/home/	Multiple	Human
Cavalli 2020159	A multi-omics approach to liver diseases: integration of single nuclei transcriptomics with proteomics and HiCap bulk data in human liver	Supplemental data	Multiple	Human
Gainullina 202327	Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes	https://artyomovlab.wustl.edu/immgen-met/	Multiple	Mouse
Guilliams 202228	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches	https://www.livercellatlas.org/index.php	Multiple	Human, mouse
Andrews 202233	Single-cell, single-nucleus, and spatial RNA sequencing of the human liver identifies cholangiocyte and mesenchymal heterogeneity	https://macparlandlab.shinyapps.io/healthylivermapspatialgui/	Visium 10× Spatial	Human
Ben-Moshe 202238	The spatiotemporal program of zonal liver regeneration following acute injury	https://itzkovitzapapp.weizmann.ac.il/apap/	Multiple	Mouse
Kim 202147	Gene deconvolution reveals aberrant liver regeneration and immune cell infiltration in alcohol-associated hepatitis	Supplemental data	RNAseq	Human
Kostallari 202135	Stiffness is associated with hepatic stellate cell heterogeneity during liver fibrosis	Supplemental data	scRNAseq	Mouse
Furuta 202197	Lipid-induced endothelial vascular cell adhesion molecule 1 promotes nonalcoholic steatohepatitis pathogenesis	Supplemental data	Multiple	Mouse
Su 202124	Single-cell transcriptomics reveals zone-specific alterations of liver sinusoidal endothelial cells in cirrhosis	Supplemental data	scRNAseq	Mouse

Zonation in Healthy Liver by Asig

In addition to central vein-associated angiocrine Wnt signaling, endothelial tyrosine kinase with Ig-like and EGF-like domains 1/2 (Tie1/2),¹⁰ Notch,^11,12 c-Maf,^13–16 Heg,¹⁷ and periportal Dll4 and Esm1^16,18 also participate to hepatic and endothelial zonation.^5,6,19–22 Briefly, paired endothelial cell/hepatocyte sequencing was originally utilized to develop a panel of pericentral to periportal endothelial functional landmark genes (70 each).⁸ Subsequent studies have demonstrated that Stab2, Cd32b, Flt4, and Lyve1 are primarily expressed by Zone2/3 mid-central LSECs, while CD36 and Adam23 are highly expressed in the periportal zone 1 LSECs. This contrasts abundant expression of Wnt2, Wnt9b, Lhx6, and cKit in pericentral LSECs and central venous endothelial cells.^6,23,24 The field has integrated these findings into cell type-specific zonation marker panels critical to the analysis of single cell and spatial transcriptomic platforms.²⁴ However, zonation of protein phosphorylation, despite ubiquitous expression, was demonstrated as a significant mechanism for regulation of pericentral Wnt signaling by Tie1/2. The authors further demonstrated a global increase in tyrosine kinase activity pericentrally versus a periportal serine-threonine kinase activity, highlighting the importance of moving toward integrated multiomics studies, including posttranslational protein modification analyses,¹⁰ an important consideration for bioinformatic data interpretation.

Liver immune zonation is not developmentally determined, but rather driven by LSEC interaction with commensal gut-derived microbes and microbial products.⁷ Periportal LSECs increase C-X-C chemokine receptor (CXCR) CXCL9 expression and utilize sustained MyD88 signaling to alter pericellular ECM binding of CXCL9 to generate a chemokine gradient.⁷ Chemokine gradients are critical for regulating the spatial distribution of immune cells. During injury, LSEC-derived CXCL9 interaction with macrophage CXCR3 is a key example.²⁵ Other studies have further delved into the zonation of macrophages in health and disease, demonstrating a macrophage niche that is regulated by complex angiocrine interactions with neighboring LSEC and HSCs^26–31 and is recently reviewed.³² Furthermore, scRNAseq increasingly reveals that HSCs are far more heterogeneous than previously appreciated and zonated in both health and disease.^33–36

Asig in Liver Regeneration

LSECs, macrophages, and HSCs significantly contribute in the process of liver regeneration,^{5,6,9,10,12,18,22,28,32,33,37–54} supported by scRNAseq and spatial transcriptomics studies.⁵⁵ Regeneration is studied via acute injury models such as the two-thirds partial hepatectomy model (PHx)¹⁰ resulting in rapid regeneration of liver mass by 96 hours postinjury or the centrilobular necrosis with acetaminophen overdose (APAP)³⁸ (further discussed later). In the APAP model, Hgf expression and hepatocyte proliferation did not show zonation.³⁸ However, the pericentral endothelial cell-specific-secreted Wnt9b, Wnt2, and Rspo3 were shown to reprogram new hepatocytes in pericentral areas.^38,45 This was confirmed with single cell “omics,” further demonstrating that Wnt agonist administration late in APAP toxicity can support rodent liver regeneration by Wnt2 and Wnt9b induction.⁴⁵ In the PHx model, regeneration is a well-organized, LSEC-driven series of events that includes the inductive phase of hepatocyte proliferation, angiogenic phase, and terminal phase.^5,6 During the inductive phase, hepatocyte proliferation is stimulated via LSEC-derived nitric oxide (NO) signaling, which can be stimulated via multiple mechanisms including flow-induced sheer stress and LSEC-derived HGF.^6,56,57 Indeed, increased HGF secretion activates c/MET, Deptor, liver kinase B1, and subsequent adenosine monophosphate-activated protein kinase (AMPK) phosphorylation to increase NO release via phosphorylated endothelial nitric oxide synthase (eNOS).^54,58 In addition, Wnt2 release for hepatocyte proliferation can be regulated downstream of the vascular endothelial growth factor receptor (VEGFR)-2/inhibitor of the DNA binding 1 (ID1) axis.⁴² During the switch between the inductive and angiogenic phases, LSEC-derived angiopoietin-2 (Angpt2) is reduced initially, which lessens transforming growth factor-β (TGF-β) production and is permissive for hepatocyte proliferation. This is reversed at a later stage via an autocrine mechanism, as well as increased VEGFR-2 to begin the angiogenic phase.⁵⁹ Animal models have demonstrated that, as the angiogenic phase progresses, hepatocytes secrete hypoxia-induced angiogenic factors such as VEGF and angiopoietins that bolster LSEC proliferation until the population reaches its peak at around 3 to 4 days post PHx.^42,60 Finally, LSECs play a key role during the termination phase of liver regeneration by secreting TGF-β, which stimulates HSCs, contributes to reconstruction of the extracellular matrix (ECM) scaffold, and terminates hepatocyte regeneration.⁶¹ Asig is a critical driver of the regenerative response and thus, the focus of developing potential therapeutic targets for liver regeneration.

Diseases

Cirrhosis and Fibrosis

Dysregulated liver regeneration can result in fibrosis and ultimately cirrhosis, characterized by (1) HSC activation secreting profibrotic molecules and extracellular vesicles^62–67 and depositing primarily collagen 1 (COL I) into the Space of Disse; (2) LSECs depositing COL IV. These events are potentiated by the profibrotic angioimmune niche, which we define as the microenvironment where LSECs interact with immune cells to promote fibrosis.^5,68 Examples of the angioimmune niche in the liver include the sinusoidal microenvironment where interactions between LSEC and macrophages or neutrophils promote fibrosis.^68,69 LSEC NO signaling, critical for normal sinusoidal function, is impaired during injury (►Fig. 1).^70,71 Activated serine/threonine protein kinase B (PKB/AKT) promotes binding of endothelial NO synthase (eNOS) to G-protein–coupled receptor kinase-interacting protein-1 (GIT1) scaffold protein, leading to NO release.⁷² While important for vascular tone, LSEC-produced NO also protects the liver from fibrosis by preventing HSC activation.⁷³ HSCs are activated into myofibroblasts with injury and characterized by increased proliferation, migration, and ECM deposition. The stimuli, including stimuli from LSECs, responsible for myofibroblastic phenotypes, such as platelet-derived growth factor (PDGF) and TGF-β, are known, but the mechanisms leading to activated HSC myofibroblast persistence and subsequent fibrosis are not fully understood.^74,75 However, HSCs are not the only cell type depositing collagen into the ECM. Capillarized LSECs also deposit COL IV and significantly contribute to fibrosis.⁷⁶ Loss of the LSEC master regulator GATA4 permits sinusoidal capillarization and promotes perisinusoidal liver fibrosis via de novo expression of profibrotic angiocrine factors including PDGF receptor β (PDGFRB), secreted protein acidic and cysteine rich like 1 (SPARCL1), endothelial cell specific molecule 1 (ESM1), and insulin-like growth factor binding protein 5 (IGFBP5).⁷⁷ Furthermore, loss of GATA4 upstream regulator GTPase, IMAP family member 5 (GIMAP5), increases capillarization and vascular resistance during human portal hypertension (PH).⁷⁸ Immune cells can contribute to LSEC capillarization via leukocyte cell-derived chemotaxin 2 (LECT2) binding to the endothelial Tie1 to upregulate MAPK-dependent PPAR signaling and results in the release of matrix proteins such as collagen IV and fibronectin.^10,79 Modulation of LECT2 ameliorated the effect in CCl₄-induced liver fibrosis.⁷⁹

Fig. 1.

Role of LSECs and angiocrine signaling during fibrosis. LSEC NO signaling is critical for normal sinusoidal function. In a healthy environment, AKT activation induces eNOS binding to GIT1 leading to NO release, which in turn is involved in maintaining HSC quiescence. Injury and stress promote LSEC capillarization, dysfunction, and ultimately fibrosis. Injured LSECs produce profibrogenic molecules, such as PDGF, IGFBP5, and ESM1. During injury, eNOS is inhibited, which is accompanied by HSC activation and matrix deposition. Finally, immune cell–derived LECT2 binds to the endothelial Tie1 leading to the release of matrix proteins such as collagen IV and fibronectin. eNOS, endothelial nitric oxide synthase; LSEC, liver sinusoidal endothelial cell; NO, nitric oxide; PDGF, platelet-derived growth factor. (Created with BioRender.)

Portal Hypertension

Clinically significant PH is defined by an increased hepatic venous pressure gradient of at least 10 mm Hg.^80–83 PH stems from increased hepatic vascular resistance. This mechanism has two primary components: architectural due to maladaptive tissue remodeling and dynamic due to endothelial dysfunction and increased hepatic microcirculatory tone. Increased vasoconstrictors such as endothelin-1, cyclooxygenase 1 (COX1), and thromboxane A2 (TXA2) exaggerate contractile responses of LSEC, HSC, and sinusoidal vascular smooth muscle cells along with impaired synthesis of vasodilators like eNOS.^57,71 eNOS-derived NO inhibits the release of P-selectin containing Weibel-Palade bodies, which can contribute to leukocyte recruitment and increase vascular inflammation.⁸⁴ NO also blocks platelet adhesion and aggregation on LSECs to reduce microvascular thrombosis.⁸⁵ Mechanical stretch and stretch-induced glycolysis can induce LSEC expression of C-X-C chemokine ligand 1 (CXCL1), which recruits neutrophils to generate microthrombi and neutrophil extracellular traps (NETs) that contribute to vascular resistance.^69,86 In addition, LSEC-derived CCL2 is increased via tumor necrosis factor-α (TNF-α)-induced p300 epigenetic modification of the CCL2 enhancer and promoter regions and specific interactions with BRD4 and nuclear factor kappa B (NF-κB) to recruit CCR2⁺ macrophages.⁶⁸ Additionally, p300 activity in HSCs is critical for epigenetic regulation of IGFBP-3, which has been demonstrated to be critical for migration in vitro and promotes PH in vivo.⁶⁷

Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) has the highest prevalence among chronic liver diseases and refers to the accumulation of steatosis in the absence of alcohol.⁸⁷ A subset of patients with NAFLD develop nonalcoholic steatohepatitis (NASH), which is characterized by the release of toxic lipids, increase of inflammation, and development of liver fibrosis.⁶ LSEC phenotype can be affected in the lipotoxic hepatic microenvironment during the development of NAFLD toward NASH (►Fig. 2). Although one study reports that LSEC fenestrae are preserved at 20 weeks of high-fat diet,⁸⁸ several other studies confirm a loss of the fenestrae due to high-fat high-carbohydrate diet, choline-deficient high-fat diet, choline-deficient L-amino-acid–defined high-fat diet with 0.1% methionine, or 22 to 24 weeks of high-fat diet.^89–91 The loss of fenestration is also observed in human livers at the stage of steatosis as compared with steatohepatitis stage,⁹² suggesting that the change in fenestrae likely happens during the early development of NAFLD. During the progression of NAFLD, the mechanical stress induced by ballooned hepatocytes impairs NO endothelin 1 balance leading to LSEC dysfunction.⁹³ The association of NAFLD to decreased eNOS expression, NO release, and increased endothelin1expression^93,94suggests that blocking endothelin 1 receptor could be potential strategy to ameliorate LSEC dysfunction in NASH.⁹⁵ Notch, expressed on LSECs, is also involved in LSEC dysfunction and NASH. eNOS activator and NOTCH inhibitor ameliorate LSEC capillarization, steatosis, inflammation, and fibrosis.⁹⁶ During NASH progression, LSECs become proinflammatory and are characterized by an increased release of proinflammatory cytokines and chemokines and upregulation of adhesion molecule expression.^97–99 This change in phenotype and function is called endotheliopathy.¹⁰⁰ LSECs, which represent the interface between sinusoidal circulation and liver parenchyma, respond to toll-like receptors (TLRs) ligands by releasing TNF-α, IL-1β, IL-6, and IFN-β and, thus, participate in the host innate immune response.^101,102 ScRNA-seq and spatial mapping show that LSECs in the fibrotic niche of human liver with NASH cirrhosis express atypical chemokine receptor 1 (ACKR1), also known as Duffy antigen receptor for chemokines (DARC), which is involved in transendothelial migration of leukocytes.^36,103 The immunomodulatory abilities of LSECs seem to be regulated by autophagy as well. Indeed, patients with NASH present with reduced autophagic vacuoles in LSECs. In line with this, LSECs from mice with defective endothelial autophagy increase the expression of proinflammatory genes.¹⁰⁴ All these studies suggest that LSECs are the main immunomodulatory cells during NAFLD and NASH and restoring their homeostasis is beneficial to disease progression.

Fig. 2.

Role of LSECs and angiocrine signaling during NAFLD and NASH. During NAFLD progression, LSECs become capillarized and increase the expression of endothelin-1. Ballooned hepatocytes exert a mechanical stress toward capillarized LSECs, which increase Notch expression and decrease eNOS activity. Notch inhibitor and eNOS activator ameliorate LSEC capillarization, liver steatosis, inflammation, and fibrosis. Injured LSEC release proinflammatory molecules such as TNF-α, IL-6, IL-1β, IFN-β, and express ACKR1, which in turn promotes leukocyte transendothelial migration. eNOS, endothelial nitric oxide synthase; LSEC, liver sinusoidal endothelial cell; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NO, nitric oxide. (Created with BioRender.)

Aging

Aging impacts endothelial dysfunction and liver disease.^{40,59,105–108} Aging amplifies pathogenic injury mechanisms, increases inflammation, and impairs sinusoidal function. Age-related LSEC pseudocapillarization results in fenestrae loss, increased basement membrane thickness, similar but less severe than fibrosis,^105,109 and microvascular rarefication (MVR)¹¹⁰ which impedes the transport between the sinusoidal circulation and space of Disse.¹¹¹ VEGF was recently shown to ameliorate age-related MVR,¹¹² which contrasts with increases in VEGF signaling that induces angiogenesis and aggravate PH (see section “Portal Hypertension”). Paradoxically, aging increases the accumulation of steatosis-associated soluble VEGFR (sFLT1) in rodent liver vasculature and major vessels, but blockade ameliorates age-related effects.¹¹² LSEC cellular adhesion molecules such as ICAM-1 are increased, leading to neutrophils and macrophage recruitment, further increasing inflammatory cytokines like IL-6, TNF-α, and MPO.¹¹³ Prothrombotic (fibrinogen-2) and senescence-associated molecules (p16, IL-6, IL-10) are also increased in the aged liver, which are partially due to age-related impairment of angiocrine antioxidant signaling and NO production,⁵⁹ and contribute to a proinflammatory environment in the aged liver. LSEC regulation of HSC activation and quiescence via VEGF-stimulated NO production is critical for sinusoidal homeostasis,⁷³ but aging results in increased activation and transdifferentiation of HSCs into myofibroblasts, as well as HSC hyperplasia, lipid drop alterations, telomere attrition, impaired release of Asig proteins such as HGF, and finally impaired production of ECM components such as laminin.^108,114,115 This failure of LSEC to maintain HSC quiescence with age was also associated with significantly increased macrophage infiltration in rodent models and altered gene expression of sinusoidal protective pathways in young and aged cirrhosis patients.¹⁰⁹

Alcohol-Associated Liver Disease

Alcohol-associated liver disease (ALD) is a significant public health issue with extremely limited therapeutic options. Recent investigations into the role of Asig in ALD provide valuable insight for potential future therapeutics. While LSEC dysfunction has been previously known with ALD, the fact that LSECs are capable of metabolizing alcohol and express enzymes typically associated with hepatocytes including alcohol dehydrogenase 1 (ADH1) and cytochrome P450 2E1 (CYP2E1) has been more recently reported.^116,117 This also is important because CYP2E1 has been highlighted as a zonation landmark gene for pericentral hepatocytes for single cell studies, and while it’s expression is highest in hepatocytes, approximately 3.3-fold higher than LSECs, using this gene to assess for hepatocyte mRNA contamination and zonation of mixed liver cell types may not be appropriate.¹¹⁸ It is not currently known if LSEC CYP2E1 expression is zonated, but studies have shown that ethanol not only increases CYP2E1 gene expression and protein stability in hepatocytes but also chronically induces LSEC CYP2E1.^117,119 This CYP2E1 induction has been demonstrated to impair angiocrine NO signaling and potentiate liver injury via increasing the downstream production of the protein acetylation substrate acetyl-CoA, which promotes Hsp90 acetylation and decreases its activation of eNOS and subsequent NO production.^117,120 Recruitment of infiltrating immune cells in alcohol-associated hepatitis (AH), the most severe manifestation of ALD, is another Asig-mediated mechanism of liver injury. Cytokine activation and LSEC-derived chemokine release have been demonstrated to be critical for neutrophil (CXCL 1, 6, 8), monocyte/macrophage (CXCL 9, 10, 11), and lymphocyte recruitment (CXCL 9, 12) during injury.^47,121–123 Additionally, chemokine internalization by LSEC contributes to modulation of Asig and its importance in CD4+ T cell recruitment was demonstrated.¹²³ Chemokine production is also potentiated by clinically relevant simultaneous insults such as alcohol plus high-fat diet and Chang et al reported that this paradigm increased production of neutrophil-recruiting CXCL1 from endothelial cells, hepatocytes, and HSCs.¹²⁴ Finally, angiocrine-mediated neutrophil recruitment in AH is epigenetically regulated. A super-enhancer, which is a DNA element that activates the transcription of genes from a distance and independently of their orientation on DNA,¹²⁵ upstream of CXCL8, regulates CXCL 1, 2, 3, 6, and 8 expression in a TNF-α/NF-κB-dependent manner.¹²²

Drug-Induced Liver Injury

Drug-induced liver injury (DILI) is due to parenchymal cell, hepatocyte, or cholangiocyte injury. Acetaminophen toxicity has the highest occurrence among the DILIs, and it is characterized by a centrilobular necrosis. Acetaminophen-mediated DILI has been demonstrated to have a direct effect on LSECs. Indeed, mice treated with acetaminophen by oral gavage showed swollen LSECs starting from 30 minutes after injury, which precedes the hepatocellular injury.¹²⁶ In addition, in this same model, TNF-related apoptosis-inducing ligand (TRAIL) signaling pathway significantly amplifies LSEC death through apoptosis.¹²⁷ DILI is also promoted by the leukotriene pathway, from which cysteinyl leukotriene receptor 1 (CYSLTR1) is expressed in several liver cell types, including LSECs.¹²⁸ Anti-Cysltr1 small interfering RNA pretreatment, in combination with a G-protein-coupled bile acid receptor 1 (GPBAR1) agonist, reversed LSEC/monocyte interactions and liver injury.¹²⁸ The interaction of LSECs with Kupffer cells (KCs) is crucial to maintain liver health, as the depletion of KCs prior to acetaminophen insult aggravated DILI-mediated LSEC injury and enhanced the expression of cellular adhesion molecules in LSECs.¹²⁹ In line with these studies, the depletion of both KCs and macrophages leads to a delayed liver repair due to a prolonged vascular leakage caused by acetaminophen-mediated LSEC injury.¹³⁰ Finally, it has been shown that high cholesterol diet exacerbates acetaminophen-mediated hepatotoxicity through promoting free cholesterol accumulation in LSECs in a toll-like receptor 9 (TLR9) manner.¹³¹ These studies suggest that LSECs have a central role in DILI and acute liver injury.

Ischemia Reperfusion

Liver ischemia reperfusion (IR) injury is a blood circulation disorder following liver transplantation that involves the reestablishment of the hepatic circulation after the cold ischemia during organ preservation and warm ischemia period during the liver transplantation.^6,132 LSEC damage following IR injury includes LSEC detachment and sinusoidal wall destruction, which is followed by extravasated platelet aggregation,^133,134 most likely due to a change in Asig. LSECs and platelets produce platelet-activating factor which primes neutrophils for reactive oxygen species generation and leukotriene B4 release.^135,136 Exposure of LSECs to cold ischemia increases intracellular calcium concentration, calpain activity, and induces actin disassembly.¹³⁷ Reperfusion promotes autophagosome accumulation in LSECs and subsequent cell death and microvascular dysfunction.¹³⁸ In addition, IR downregulates several metabolic pathways in LSECs, such as carbohydrate, lipid, as well as mitochondrial metabolism, and this effect can be attenuated by adenosine A2a receptor (A2aR) agonist.¹³⁹ In aged rats, the effect of IR injury on LSEC damage are exacerbated. Indeed, warm IR increased LSEC capillarization and reduced vasodilation, which is associated with liver damage and cellular stress.¹³³ However, these effects were reduced when rats were treated with KLF2-inducer simvastatin.¹³³ In line with this study, simvastatin addition to the cold preservation solution ameliorated the hepatic IR injury in a rat experimental model.¹⁴⁰ Utilizing spatial transcriptomics, a recent study demonstrates that IR injury affects mainly the pericentral zone (zone 3) in the liver where it induces an increase in endothelial cell proportion.¹⁴¹ However, it remains to be discovered what specific Asig molecules are altered in zone 3 LSECs and whether this could be a therapeutic strategy to alleviate hepatic IR injury.

Therapeutics

Although sinusoidal endotheliopathy is recognized in several liver diseases, no LSEC-specific therapy exists. LSECs immunomodulatory Asig is a promising therapeutic approach in inflammatory liver diseases.^100,142 In this regard, vascular cell adhesion molecule 1 neutralizing antibody reduced monocyte/macrophage accumulation in the liver and ameliorated diet-induced liver damage in a mouse model of NASH.⁹⁷ Another study demonstrates that NASH can be ameliorated in methionine choline deficient diet-fed mice via in vivo targeted silencing of the Runt-related transcription factor (RUNX1) gene in LSECs. This was achieved by administrating RUNX1 siRNA encapsulated in VEGFR-3 antibody tagged immuno-nano-lipocarriers.¹⁴³ In a similar nanoparticle approach, LSEC-targeting and fenestrae-repairing nanoparticles (HA-NPs/SMV) containing hyaluronic acid were loaded with simvastatin. These HA-NPs/SMV repaired LSEC fenestrae and reduced liver fibrosis.¹⁴⁴ Recent studies have attributed the effects of simvastatin on PH to Kruppel-like factor 2 (KLF2), a zinc finger transcription factor.^145,146 In addition to nanoparticles, targeting Asig epigenetically by pharmacological inhibition of bromodomain and extraterminal (BET) proteins reduces chemokine production by LSECs in vitro, neutrophil infiltration in vivo, and ameliorates alcoholic hepatitis.¹²² Currently, the development of BET protein inhibitors is an active area of research¹⁴⁷ with high potential for liver disease therapeutics. NO is a crucial molecule in maintaining LSEC homeostasis. Treatment with the NO signaling enhancer sildenafil reduced liver inflammation.¹⁴⁸ A recent study suggests that chronic liver disease can be ameliorated by inducing capillarized LSEC apoptosis. The authors have developed a rationally designed protein (ProAgio), which is designed to target integrin α_vβ₃ at a novel site and induce HSC and LSEC apoptosis.¹⁴⁹ As a potential treatment for DILI, the combinatorial G-protein-coupled bile acid receptor 1 (GPBAR1) agonist and cysteinyl leukotriene receptor 1 (CYSLTR1) antagonist, namely CHIN117, affected chemokine production and attenuated liver damage.¹²⁸ All these studies testify of a quickly evolving field of LSEC biology. The recent technological advances, such as multiomics, single cell and spatial omics, as well as the development of CRISPR/Cas9 transgenic mouse models, will lead the field toward better therapeutic strategies.

Conclusion

Our knowledge of LSEC phenotypes, roles, and their Asig in liver diseases is rapidly evolving. LSECs damage during liver injury promotes disease progression through releasing proinflammatory, antiregeneration, and profibrotic molecules. Recent studies utilizing state-of-the-art technologies have brought granularity on zonation of LSEC subpopulations. However, specific Asig from each of these different LSEC subpopulations remains to be studied. LSECs are excellent candidates to target for treating liver disease as they are in the interface between circulation and liver parenchyma. Some promising options include exploring the immunomodulatory function of LSECs in the field of liver transplant, targeting LSEC luminal adhesion molecules in inflammatory liver diseases, and inhibiting shear-stress signaling pathways and subsequent proinflammatory molecule expression to ameliorate PH. With preclinical studies presenting encouraging results, we expect that LSEC-specific pharmacotherapies will be developed soon.