Sinusoidal Endothelial Cells Research Articles

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

BackgroundBone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll–Plate–Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs).MethodsBy using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans’.ResultsThe NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the “stemness” of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.ConclusionsOverall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Phenotypical and biochemical characterization of murinepsoriasiform and fibrotic skin disease models inStabilin-deficient mice.

Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are scavenger receptors expressed by liver sinusoidal endothelial cells (LSECs). The Stabilin-mediated scavenging function is responsible for regulating the molecular composition of circulating blood in mammals. Stab1 and Stab2 have been shown to influence fibrosis in liver and kidneys and to modulate inflammation in atherosclerosis. In this context, circulating and localized TGFBi and POSTN are differentially controlled by the Stabilins as their receptors. To assess Stab1 and Stab2 functions in inflammatory and fibrotic skin disease, topical Imiquimod (IMQ) was used to induce psoriasis-like skin lesions in mice and Bleomycin (BLM) was applied subcutaneously to induce scleroderma-like effects in the skin. The topical treatment with IMQ, as expected, led to psoriasis-like changes in the skin of mice, including increased epidermal thickness and significant weight loss. Clinical severity was reduced in Stab2-deficient compared to Stab1-deficient mice. We did not observe differential effects in the skin of Stabilin-deficient mice after bleomycin injection. Interestingly, treatment with IMQ led to a significant increase of Stabilin ligand TGFBi plasma levels in Stab2-/- mice, treatment with BLM resulted in a significant decrease in TGFBi levels in Stab1-/- mice. Overall, Stab1 and Stab2 deficiency resulted in minor alterations of the disease phenotypes accompanied by alterations of circulating ligands in the blood in response to the disease models. Stabilin-mediated clearance of TGFBi was altered in these disease processes. Taken together our results suggest that Stabilin deficiency-associated plasma alterations may interfere with preclinical disease severity and treatment responses in patients.

In Vivo Endothelial Cell Gene Silencing by siRNA-LNPs Tuned with Lipoamino Bundle Chemical and Ligand Targeting.

Although small-interfering RNAs (siRNAs) are specific silencers for numerous disease-related genes, their clinical applications still require safe and effective means of delivery into target cells. Highly efficient lipid nanoparticles (LNPs) are developed for siRNA delivery, showcasing the advantages of novel pH-responsive lipoamino xenopeptide (XP) carriers. These sequence-defined XPs are assembled by branched lysine linkages between cationizable polar succinoyl tetraethylene pentamine (Stp) units and apolar lipoamino fatty acids (LAFs) at various ratios into bundle or U-shape topologies. Formulation of siRNA-LNPs using LAF4-Stp1 XPs as ionizable compounds led to robust cellular uptake, high endosomal escape, and successful in vitro gene silencing activity at an extremely low (150 picogram) siRNA dose. Of significance is the functional in vivo endothelium tropism of siRNA-LNPs with bundle LAF4-Stp1 XP after intravenous injection into mice, demonstrated by superior knockdown of liver sinusoidal endothelial cell (LSEC)-derived factor VIII (FVIII) and moderate silencing of hepatocyte-derived FVII compared to DLin-MC3-DMA-based LNPs. Optimizing lipid composition following click-modification of siRNA-LNPs with ligand c(RGDfK) efficiently silenced vascular endothelial growth factor receptor-2 (VEGFR-2) in tumor endothelial cells (TECs). The findings shed light on the role of ionizable XPs in the LNP in vivo cell-type functional targeting, laying the groundwork for future therapeutic applications.

Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

BackgroundAcute liver failure (ALF) is a life-threatening disorder that progresses from self-limiting acute liver injury (ALI). Microcirculatory disturbance characterized by sinusoidal hypercoagulation and subsequent massive hypoxic hepatocyte damage have been proposed to be the mechanism by which ALI deteriorates to ALF; however, the precise molecular pathway of the sinusoidal hypercoagulation remains unknown. Here, we analyzed ALI patients and mice models to uncover the pathogenesis of ALI with microcirculatory disturbance.MethodsWe conducted a single-center retrospective study for ALI and blood samples and liver tissues were analyzed to evaluate the microcirculatory disturbance in ALI patients (n = 120). Single-cell RNA sequencing analysis (scRNA-seq) was applied to the liver from the concanavalin A (Con A)‑induced mouse model of ALI. Interferon-gamma (IFNγ) and tumor necrosis factor-alpha knockout mice, and primary human liver sinusoidal endothelial cells (LSECs) were used to assess the mechanism of microcirculatory disturbance.ResultsThe serum IFNγ concentrations were significantly higher in ALI patients with microcirculatory disturbance than in patients without microcirculatory disturbance, and the IFNγ was upregulated in the Con A mouse model which presented microcirculatory disturbance. Hepatic IFNγ expression was increased as early as 1 hour after Con A treatment prior to sinusoidal hypercoagulation and hypoxic liver damage. scRNA-seq revealed that IFNγ was upregulated in innate lymphoid cells and stimulated hepatic vascular endothelial cells at the early stage of liver injury. In IFNγ knockout mice treated with Con A, the sinusoidal hypercoagulation and liver damage were remarkably attenuated, concomitant with the complete inhibition of CD40 and tissue factor (TF) upregulation in vascular endothelial cells. By ligand-receptor analysis, CD40-CD40 ligand interaction was identified in vascular endothelial cells. In human LSECs, IFNγ upregulated CD40 expression and TF was further induced by increased CD40-CD40 ligand interaction. Consistent with these findings, hepatic CD40 expression was significantly elevated in human ALI patients with microcirculatory disturbance.ConclusionWe identified the critical role of the IFNγ-CD40 axis as the molecular mechanism of microcirculatory disturbance in ALI. This finding may provide novel insights into the pathogenesis of ALI and potentially contribute to the emergence of new therapeutic strategies for ALI patients.

Influence of nuclear factor κB and adenosine monophosphate-activated protein kinase on the vascular bed of the liver under the conditions of modeling chronic alcoholic hepatitis

Chronic alcohol use activates the transcription factor κB (NF-κB) in liver sinusoidal endothelial cells (LSECs), macrophages and other cells of the liver parenchyma, which controls the regulation of the expression of pro-inflammatory cytokines that activate signaling pathways of immune regulation of liver inflammation and vascular tone. AMP-activated protein kinase (AMPK) is an important immunometabolic regulatory factor in macrophages and, as a metabolic sensor, AMPK in vascular cells may be involved in the regulation of blood flow. The aim of the work was to find out the effect of modulators of the transcription factor κB and AMP-activated protein kinase on the morphometric parameters of the vascular bed of the liver of rats under the conditions of modeling chronic alcoholic hepatitis. Simulation of chronic alcoholic hepatitis leads to a violation of the exchange of metabolites between the central and interlobular veins, which creates prerequisites for the development of hypoxic damage to hepatocytes, as evidenced by a decrease in the diameters of lobular arterioles and venules. Administration of ammonium pyrrolidinedithiocarbamate and bacterial lipopolysaccharide, which affect NF-κB signaling under the conditions of modeling chronic alcoholic hepatitis, helps to restore the exchange of metabolites between the central and interlobular veins, which is evidenced by an increase in the lumens of lobular arterioles and venules. The introduction of phenformin and doxorubicin, which affect AMPK under the conditions of modeling chronic alcoholic hepatitis, prevents the development of hypoxic damage to hepatocytes, as evidenced by an increase in the diameters of lobular arteries and veins, and leads to intensification of interlobular blood circulation.

A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells (ECs) that play an important role in liver development and regeneration. Additionally, it is involved in various pathological processes, including steatosis, inflammation, fibrosis and hepatocellular carcinoma. However, the rapid dedifferentiation of LSECs after culture greatly limits their use in vitro modeling for biomedical applications. In this study, we developed a highly efficient protocol to induce LSEC-like cells from human induced pluripotent stem cells (hiPSCs) in only 8 days. Using single-cell transcriptomic analysis, we identified several novel LSEC-specific markers, such as EPAS1, LIFR, and NID1, as well as several previously revealed markers, such as CLEC4M, CLEC1B, CRHBP and FCN3. These LSEC markers are specifically expressed in our LSEC-like cells. Furthermore, hiPSC-derived cells expressed LSEC-specific proteins and exhibited LSEC-related functions, such as the uptake of acetylated low density lipoprotein (ac-LDL) and immune complex endocytosis. Overall, this study confirmed that our novel protocol allowed hiPSCs to rapidly acquire an LSEC-like phenotype and function in vitro. The ability to generate LSECs efficiently and rapidly may help to more precisely mimic liver development and disease progression in a liver-specific multicellular microenvironment, offering new insights into the development of novel therapeutic strategies.

Liver Cell Mitophagy in Metabolic Dysfunction-Associated Steatotic Liver Disease and Liver Fibrosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately one-third of the global population. MASLD and its advanced-stage liver fibrosis and cirrhosis are the leading causes of liver failure and liver-related death worldwide. Mitochondria are crucial organelles in liver cells for energy generation and the oxidative metabolism of fatty acids and carbohydrates. Recently, mitochondrial dysfunction in liver cells has been shown to play a vital role in the pathogenesis of MASLD and liver fibrosis. Mitophagy, a selective form of autophagy, removes and recycles impaired mitochondria. Although significant advances have been made in understanding mitophagy in liver diseases, adequate summaries concerning the contribution of liver cell mitophagy to MASLD and liver fibrosis are lacking. This review will clarify the mechanism of liver cell mitophagy in the development of MASLD and liver fibrosis, including in hepatocytes, macrophages, hepatic stellate cells, and liver sinusoidal endothelial cells. In addition, therapeutic strategies or compounds related to hepatic mitophagy are also summarized. In conclusion, mitophagy-related therapeutic strategies or compounds might be translational for the clinical treatment of MASLD and liver fibrosis.

Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

BackgroundTransplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34+ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34+ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34+ isolated from cord blood (CB-CD34+) or fetal liver (FL-CD34+ and FL-CD34+CD14−) and their engraftment into immunocompromised NOD/Shi-scid Il2rgnull (NOG) mice.MethodsNOG mice were transplanted with either CB-CD34+, FL-CD34+ or FL-CD34+CD14− to generate CB-NOG, FL-NOG and FL-CD14−-NOG, respectively. After 15–20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles.ResultsWe show that CB-CD34+ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34+ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34+ isolates contain non-hematopoietic CD14+ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14+CD34+ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14+CD34+ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection.ConclusionOur results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

Beyond stiffness: Multiscale viscoelastic features as biomechanical markers for assessing cell types and states

Cell mechanics are pivotal in regulating cellular activities, diseases progression, and cancer development. However, the understanding of how cellular viscoelastic properties vary in physiological and pathological stimuli remains scarce. Here, we develop a hybrid self-similar hierarchical theory-microrheology approach to accurately and efficiently characterize cellular viscoelasticity. Focusing on two key cell types associated with livers fibrosis—the capillarized liver sinusoidal endothelial cells and activated hepatic stellate cells—we uncover a universal two-stage power-law rheology characterized by two distinct exponents, αshort and αlong. The mechanical profiles derived from both exponents exhibit significant potential for discriminating among diverse cells. This finding suggests a potential common dynamic creep characteristic across biological systems, extending our earlier observations in soft tissues. Using a tailored hierarchical model for cellular mechanical structures, we discern significant variations in the viscoelastic properties and their distribution profiles across different cell types and states from the cytoplasm (elastic stiffness E1 and viscosity η), to a single cytoskeleton fiber (elastic stiffness E2), and then to the cell level (transverse expansion stiffness E3). Importantly, we construct a logistic-regression-based machine-learning model using the dynamic parameters that outperforms conventional cell-stiffness-based classifiers in assessing cell states, achieving an area under the curve of 97% vs. 78%. Our findings not only advance a robust framework for monitoring intricate cell dynamics but also highlight the crucial role of cellular viscoelasticity in discerning cell states across a spectrum of liver diseases and prognosis, offering new avenues for developing diagnostic and therapeutic strategies based on cellular viscoelasticity.

Advanced three-dimensional in vitro liver models to study the activity of anticancer drugs.

The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell-cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.

Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, increases worldwide and associates with type 2 diabetes and other cardiometabolic diseases. Here we demonstrate that Sema3a is elevated in liver sinusoidal endothelial cells of animal models for obesity, type 2 diabetes and MASLD. In primary human liver sinusoidal endothelial cells, saturated fatty acids induce expression of SEMA3A, and loss of a single allele is sufficient to reduce hepatic fat content in diet-induced obese mice. We show that semaphorin-3A regulates the number of fenestrae through a signaling cascade that involves neuropilin-1 and phosphorylation of cofilin-1 by LIM domain kinase 1. Finally, inducible vascular deletion of Sema3a in adult diet-induced obese mice reduces hepatic fat content and elevates very low-density lipoprotein secretion. Thus, we identified a molecular pathway linking hyperlipidemia to microvascular defenestration and early development of MASLD.

Differential expression of HIF-1α and its hypoxia-related inducers in the spleens of plateau yaks and plain yellow cattle.

The present study aims to investigate the distribution and expression characteristics of HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of plateau yaks and plain yellow cattle and to speculate the possible regulatory role of HIF-1α and its related hypoxia-inducible factors in the adaptation of the yak spleen to the plateau hypoxic environment. Histological features were observed using H&amp;E and PAS stains. Immunohistochemical staining and optical density analysis were applied to investigate the distribution and differences in the expression of HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of yaks and cattle. The results showed that the area of splenic trabeculae and splenic nodules was significantly larger in the yak than in yellow cattle (P<0.05). Glycogen was mainly distributed in splenic arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblasts, and the distribution was significantly higher in the spleen of yaks than in cattle (P<0.05). HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 were mainly expressed in lymphocytes, arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblast cytoplasm, with higher expression in yak spleen (P<0.05). In conclusion, combining the differences in spleen tissue structure, glycogen distribution, and expression distribution of several hypoxia-related factors between yaks and cattle, we suggest that HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 may be important factors in the adaptation of yak spleen to the plateau environment, which provides a theoretical basis for further exploring the adaptation mechanism of plateau hypoxia in yaks.

Understanding the Liver’s Role in the Clearance of Aβ40

The clearance of peripheral beta amyloid (Aβ) is a potential target for the treatment of Alzheimer’s disease (AD). The liver has been implicated in the elimination of Aβ from the peripheral circulation. Here, the single-pass uptake of Aβ40 in perfused livers from young and old rats (6 to 10 rats per group) was investigated with the multiple indicator dilution technique. Aβ40 had volumes of distribution between those of the vascular marker Evans Blue and the extracellular marker sucrose. The hepatic extraction of Aβ40 was negligible, explained in part by the small permeability surface area products consistent with a high endothelial barrier to liver uptake. There were no substantial effects of age on any of these results. In vitro experiments with isolated hepatocytes and liver sinusoidal endothelial cells showed only very small amounts of Aβ uptake consistent with low intrinsic clearance. These results indicate that the hepatic clearance of Aβ is capacity-limited, explained by the low-permeability surface area products and hepatocyte uptake. However, this does not preclude an effect of aging in longer-term in vivo studies where age-related changes in liver blood flow and protein binding influence liver clearance.

Sphingosine-1-phosphate promotes liver fibrosis in metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most prevalent liver diseases and is characterized by steatosis and the accumulation of bioactive lipids. This study aims to understand the specific lipid species responsible for the progression of liver fibrosis in MASH. Changes in bioactive lipid levels were examined in the livers of MASH mice fed a choline-deficient diet (CDD). Additionally, sphingosine kinase (SphK)1 mRNA, which generates sphingosine 1 phosphate (S1P), was examined in the livers of patients with MASH. CDD induced MASH and liver fibrosis were accompanied by elevated levels of S1P and increased expression of SphK1 in capillarized liver sinusoidal endothelial cells (LSECs) in mice. SphK1 mRNA also increased in the livers of patients with MASH. Treatment of primary cultured mouse hepatic stellate cells (HSCs) with S1P stimulated their activation, which was mitigated by the S1P receptor (S1PR)2 inhibitor, JTE013. The inhibition of S1PR2 or its knockout in mice suppressed liver fibrosis without reducing steatosis or hepatocellular damage. S1P level is increased in MASH livers and contributes to liver fibrosis via S1PR2.

Extracellular Matrix Stiffness-Induced Mechanotransduction of Capillarized Liver Sinusoidal Endothelial Cells.

The mechanobiological response mechanism of the fenestrae of liver sinusoidal endothelial cells (LSECs) to the physical stiffness of the extracellular matrix (ECM) remains unclear. We investigated how the mechanical properties of their substrates affect the LSECs' fenestrae by the nitric oxide (NO)-dependent pathway and how they relate to the progression of hepatic sinus capillarization during liver fibrosis. We detected different stiffnesses of ECM in the progress of liver fibrosis (LF) and developed polyacrylamide hydrogel (PAM) substrates to simulate them. Softer stiffness substrates contributed to LSECs maintaining fenestrae phenotype in vitro. The stiffness of liver fibrosis tissue could be reversed in vivo via treatment with anti-ECM deposition drugs. Similarly, the capillarization of LSECs could be reversed by decreasing the ECM stiffness. Our results also indicate that the NO-dependent pathway plays a key regulatory role in the capillarization of ECM-LSECs. Our study reveals ECM-induced mechanotransduction of capillarized LSECs through a NO-dependent pathway via a previously unrevealed mechanotransduction mechanism. The elucidation of this mechanism may offer precise biomechanics-specific intervention strategies targeting liver fibrosis progression.

Xa inhibitor edoxaban ameliorates hepatic ischemia-reperfusion injury via PAR-2-ERK 1/2 pathway.

Hepatic ischemia-reperfusion injury causes liver damage during surgery. In hepatic ischemia-reperfusion injury, the blood coagulation cascade is activated, causing microcirculatory incompetence and cellular injury. Coagulation factor Xa (FXa)- protease-activated receptor (PAR)-2 signaling activates inflammatory reactions and the cytoprotective effect of FXa inhibitor in several organs. However, no studies have elucidated the significance of FXa inhibition on hepatic ischemia-reperfusion injury. The present study elucidated the treatment effect of an FXa inhibitor, edoxaban, on hepatic ischemia-reperfusion injury, focusing on FXa-PAR-2 signaling. A 60 min hepatic partial-warm ischemia-reperfusion injury mouse model and a hypoxia-reoxygenation model of hepatic sinusoidal endothelial cells were used. Ischemia-reperfusion injury mice and hepatic sinusoidal endothelial cells were treated and pretreated, respectively with or without edoxaban. They were incubated during hypoxia/reoxygenation in vitro. Cell signaling was evaluated using the PAR-2 knockdown model. In ischemia-reperfusion injury mice, edoxaban treatment significantly attenuated fibrin deposition in the sinusoids and liver histological damage and resulted in both anti-inflammatory and antiapoptotic effects. Hepatic ischemia-reperfusion injury upregulated PAR-2 generation and enhanced extracellular signal-regulated kinase 1/2 (ERK 1/2) activation; however, edoxaban treatment reduced PAR-2 generation and suppressed ERK 1/2 activation in vivo. In the hypoxia/reoxygenation model of sinusoidal endothelial cells, hypoxia/reoxygenation stress increased FXa generation and induced cytotoxic effects. Edoxaban protected sinusoidal endothelial cells from hypoxia/reoxygenation stress and reduced ERK 1/2 activation. PAR-2 knockdown in the sinusoidal endothelial cells ameliorated hypoxia/reoxygenation stress-induced cytotoxicity and suppressed ERK 1/2 phosphorylation. Thus, edoxaban ameliorated hepatic ischemia-reperfusion injury in mice by protecting against micro-thrombosis in sinusoids and suppressing FXa-PAR-2-induced inflammation in the sinusoidal endothelial cells.

Finding a Direct Method for a Dynamic Process: The DD (Direct and Dynamic) Cell-Tox Method.

The main focus of in vitro toxicity assessment methods is to assess the viability of the cells, which is usually based on metabolism changes. Yet, when exposed to toxic substances, the cell triggers multiple signals in response. With this in mind, we have developed a promising cell-based toxicity method that observes various cell responses when exposed to toxic substances (either death, division, or remain viable). Based on the collective cell response, we observed and predicted the dynamics of the cell population to determine the toxicity of the toxicant. The method was tested with two different conformations: In the first conformation, we exposed a monoculture model of blood macrophages to UV light, hydrogen peroxide, nutrient deprivation, tetrabromobisphenol A, fatty acids, and 5-fluorouracil. In the second, we exposed a coculture liver model consisting of hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells to rifampicin, ibuprofen, and 5-fluorouracil. The method showed good accuracy compared to established toxicity assessment methods. In addition, this approach provided more representative information on the toxic effects of the compounds, as it considers the different cellular responses induced by toxic agents.

Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV-driven sinusoidal remodeling.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB-dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

Role of adipocyte-hepatic stellate cell cross talk in liver fibrosis

Hepatic stellate cells (HSCs) are located in the space of Disse, between sinusoidal epithelial cells and hepatocytes. When the liver is injured chronically by alcohol, toxins, viruses, or nonalcoholic steatohepatitis, HSCs are activated. Activated HSCs are transdifferentiated into proliferative myofibroblasts which secrete extracellular matrix and exhibit contractile properties. HSCs may be an effective target to treat liver fibrosis. HSCs can communicate with neighboring cells such as hepatocytes, sinusoidal endothelial cells, Kupffer cells, and immune cells that regulate the activation of HSCs. The secretion of cytokines has led to HSC activation by the cell types mentioned, adipocytes are also known to release cytokines. The role of adipocytes in HSC activation has been rarely reported. This research aims to explore the roles of adipocyte-hepatic stellate cell crosstalk in liver fibrosis. At this annual meeting preliminary gene expression of activation markers, α-smooth muscle actin and type I collagen fiber, in HSCs will be discussed. Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #P20GM103424-21. Grambling State University Title 3 Program (Award #P01B220013). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potential contribution of liver sinusoidal endothelial cell expressing EphrinB1 in fibrogenesis during metabolic dysfunction-associated steatohepatitis in mice

Study Objective and Hypothesis: The pathogenesis of fibrosis during metabolic dysfunction-associated steatohepatitis (MASH) is not fully understood. Our previous work indicate that the Eph/Ephrin signaling could be a major cell-cell signaling hub regulating fibrogenesis in MASH. While cell-cell interaction between HSC and liver sinusoidal endothelial cells (LSEC) has long been proposed as a driver for liver fibrosis, molecular mechanisms of their interaction have not been elaborated. In this study, we tested the hypothesis that EphrinB ligands expressed in LSEC plays a role in the pathogenesis of MASH fibrosis. Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for Efnb1 and Efnb2 expression. Human liver tissues obtained from patients with MASH were assessed for pEphrinB expression. Plasma level of EphrinB2 was measured in MASH and healthy individuals. EphrinB1 deletion in LSEC were generated by crossing Efnb1fl/fl mice with CDH5(PAC)-CreERT2 mice. Experimental MASH was developed in LSEC-specific Efnb1 knockout and wild type mice by feeding them with a choline-deficient amino-acid defined (CDAA) high fat diet for 8 weeks. Histology, flow cytometry, qPCR, biochemical assays and immunohistochemistry were used to dissect the contribution of LSEC- Efnb1 function in MASH. Statistical analysis was performed using ANOVA and Mann Whitney U test and a p-value &lt; 0.05 was considered significant. Data: Single nuclear RNA sequencing showed that Efnb1 and Efnb2 are upregulated in LSEC of mice with MASH. Plasma level of Ephrin-B2 is significantly elevated in MASH patients compared to healthy individuals. Deletion of Efnb1 was achieved after tamoxifen administration, as depicted by reduced expression of Efnb1 mRNA level and EphrinB1 protein in the liver of LSEC Efnb1−/− mice compared to littermate ( Efnb1fl/fl) fed the CDAA-HFD. However, we noted an increased expression of Ephb receptors mRNA level in the liver of LSEC Efnb1−/− mice compared to littermate fed the CDAA-HFD. In addition, we noted an increased CDAA-HFD-mediated inflammation and fibrosis after deletion of Efnb1 in LSEC compared to littermates mice fed CDAA-HFD. Conclusions: Although EphrinB1 is upregulated in LSEC during advanced MASH, its deletion does not protect from MASH fibrosis. Given the functional redundancy between Ephrin-B ligands, deletion of one member of this family might not be suffcient to abolished EphB-EphrinB bidirectional signaling. NIDDK, NIAMS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.