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Endothelial GATA4 controls liver fibrosis and regeneration by preventing a pathogenic switch in angiocrine signaling

  • Author Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Manuel Winkler
    Correspondence
    Corresponding authors. Address: Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. Tel.: +49 621 383 2280.
    Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
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  • Author Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Theresa Staniczek
    Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
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  • Author Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Sina Wietje Kürschner
    Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
    Search for articles by this author
  • Author Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Christian David Schmid
    Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
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  • Hiltrud Schönhaber
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
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  • Julio Cordero
    Affiliations
    Department of Anatomy and Developmental Biology, European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Linda Kessler
    Affiliations
    Department of Anatomy and Developmental Biology, European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Arthur Mathes
    Affiliations
    Department of Anatomy and Developmental Biology, European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Carsten Sticht
    Affiliations
    Core Facility Next Generation Sequencing, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Michelle Neßling
    Affiliations
    Central Unit Electron Microscopy, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • Alexey Uvarovskii
    Affiliations
    Center for Molecular Biology (ZMBH), Heidelberg University, Heidelberg, Germany
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  • Simon Anders
    Affiliations
    Center for Molecular Biology (ZMBH), Heidelberg University, Heidelberg, Germany
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  • Xue-jun Zhang
    Affiliations
    Department of Surgery, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Department of Orthopedic Surgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China
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  • Guido von Figura
    Affiliations
    II. Medical Clinic and Policlinic, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
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  • Daniel Hartmann
    Affiliations
    Department of Surgery, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
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  • Carolin Mogler
    Affiliations
    Institute of Pathology, School of Medicine, Technical University of Munich, Munich, Germany
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  • Gergana Dobreva
    Affiliations
    Department of Anatomy and Developmental Biology, European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Kai Schledzewski
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany
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  • Author Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Cyrill Géraud
    Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany

    Section of Clinical and Molecular Dermatology, Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

    European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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  • Author Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Philipp-Sebastian Koch
    Correspondence
    Corresponding authors. Address: Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. Tel.: +49 621 383 2280.
    Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany

    European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
    Search for articles by this author
  • Author Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Sergij Goerdt
    Footnotes
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
    Affiliations
    Department of Dermatology, Venereology and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, and Center of Excellence in Dermatology, Mannheim, Germany

    European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
    Search for articles by this author
  • Author Footnotes
    † These first authors contributed equally to the work: Manuel Winkler, Theresa Staniczek, Sina Wietje Kürschner, Christian David Schmid.
    ‡ These senior authors contributed equally to the work: Cyrill Géraud, Philipp-Sebastian Koch, Sergij Goerdt.
Open AccessPublished:September 08, 2020DOI:https://doi.org/10.1016/j.jhep.2020.08.033

      Highlights

      • Genetic endothelial Gata4 deletion leads to liver fibrosis and hepatopathy.
      • Gata4 deficiency causes profibrotic angiocrine signaling via endothelial PDGFB.
      • Increased chromatin accessibility and activated MYC mediate Pdgfb expression in LSECs.
      • Dietary liver fibrosis causes dysregulation of the endothelial GATA4/MYC/PDGFB axis.
      • In human cirrhosis, single-cell data implicate the GATA4/PDGFB axis.

      Background & Aims

      Angiocrine signaling by liver sinusoidal endothelial cells (LSECs) regulates hepatic functions such as growth, metabolic maturation, and regeneration. Recently, we identified GATA4 as the master regulator of LSEC specification during development. Herein, we studied the role of endothelial GATA4 in the adult liver and in hepatic pathogenesis.

      Methods

      We generated adult Clec4g-icretg/0xGata4fl/fl (Gata4LSEC-KO) mice with LSEC-specific depletion of Gata4. Livers were analyzed by histology, electron microscopy, immunohistochemistry/immunofluorescence, in situ hybridization, and LSECs were isolated for gene expression profiling, ChIP- and ATAC-sequencing. Partial hepatectomy was performed to assess regeneration. We used choline-deficient, l-amino acid-defined (CDAA) diet and chronic carbon tetrachloride exposure to model liver fibrosis. Human single cell RNA-seq data sets were analyzed for endothelial alterations in healthy and cirrhotic livers.

      Results

      Genetic Gata4 deficiency in LSECs of adult mice caused perisinusoidal liver fibrosis, hepatopathy and impaired liver regeneration. Sinusoidal capillarization and LSEC-to-continuous endothelial transdifferentiation were accompanied by a profibrotic angiocrine switch involving de novo endothelial expression of hepatic stellate cell-activating cytokine PDGFB. Increased chromatin accessibility and amplification by activated MYC mediated angiocrine Pdgfb expression. As observed in Gata4LSEC-KO livers, CDAA diet-induced perisinusoidal liver fibrosis was associated with GATA4 repression, MYC activation and a profibrotic angiocrine switch in LSECs. Comparison of CDAA-fed Gata4LSEC-KO and control mice demonstrated that endothelial GATA4 indeed protects against dietary-induced perisinusoidal liver fibrosis. In human cirrhotic livers, GATA4-positive LSECs and endothelial GATA4 target genes were reduced, while non-LSEC endothelial cells and MYC target genes including PDGFB were enriched.

      Conclusions

      Endothelial GATA4 protects against perisinusoidal liver fibrosis by repressing MYC activation and profibrotic angiocrine signaling at the chromatin level. Therapies targeting the GATA4/MYC/PDGFB/PDGFRβ axis offer a promising strategy for prevention and treatment of liver fibrosis.

      Lay summary

      The liver vasculature is supposed to play a major role in the development of liver fibrosis and cirrhosis, which can lead to liver failure and liver cancer. Herein, we discovered that structural and transcriptional changes induced by genetic deletion of the transcription factor GATA4 in the hepatic endothelium were sufficient to cause liver fibrosis. Activation of the transcription factor MYC and de novo expression of the “angiocrine” growth factor PDGFB were identified as downstream drivers of fibrosis and as potential therapeutic targets for this potentially fatal disease.

      Graphical abstract

      Keywords

      Introduction

      Among the microvascular beds of different organs, the hepatic sinusoids are a prime model of structural and functional angiodiversity.
      • Augustin H.G.
      • Koh G.Y.
      Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology.
      Liver sinusoidal endothelial cells (LSECs) are discontinuous and exert specialized functions in regulating liver immunity
      • Shetty S.
      • Lalor P.F.
      • Adams D.H.
      Liver sinusoidal endothelial cells - gatekeepers of hepatic immunity.
      and inflammation, and by clearing a variety of blood factors from the circulation via endocytic receptors such as the mannose receptor, CD32b, LYVE-1, Stabilin-1 and Stabilin-2.
      • Schledzewski K.
      • Géraud C.
      • Arnold B.
      • Wang S.
      • Gröne H.-J.
      • Kempf T.
      • et al.
      Deficiency of liver sinusoidal scavenger receptors stabilin-1 and -2 in mice causes glomerulofibrotic nephropathy via impaired hepatic clearance of noxious blood factors.
      In addition, LSECs are sub-specialized and seem to be co-zonated with hepatocytes allowing a spatially defined paracrine crosstalk between the cells in the hepatic vascular niche.
      • Halpern K.B.
      • Shenhav R.
      • Massalha H.
      • Toth B.
      • Egozi A.
      • Massasa E.E.
      • et al.
      Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells.
      LSEC-derived organ-specific cytokines, so called “angiocrine factors”, are involved in liver development, homeostasis and disease pathogenesis.
      • Rafii S.
      • Butler J.M.
      • Ding B.-S.
      Angiocrine functions of organ-specific endothelial cells.
      Angiocrine BMP-2 and BMP-6 signaling regulates iron metabolism by controlling secretion of hepcidin from hepatocytes.
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      • Sticht C.
      • Demory A.
      • Leibing T.
      • et al.
      Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis.
      Angiocrine Wnt-2, Wnt-9b and R-spondin-3 signaling towards pericentral hepatocytes is indispensable for liver growth and maturation as well as metabolic liver zonation and liver regeneration.
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      • Géraud C.
      • Augustin I.
      • Boutros M.
      • Augustin H.G.
      • Okun J.G.
      • et al.
      Angiocrine Wnt signaling controls liver growth and metabolic maturation in mice.
      ,
      • Wang B.
      • Zhao L.
      • Fish M.
      • Logan C.Y.
      • Nusse R.
      Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver.
      Regarding angiocrine control of liver fibrosis, important endothelial signaling systems in toxic liver fibrosis and/or bile duct ligation are the VEGF/SDF-1/CXCR-4/CXCR-7/FGFR-1 pathway,
      • Ding B.-S.
      • Cao Z.
      • Lis R.
      • Nolan D.J.
      • Guo P.
      • Simons M.
      • et al.
      Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.
      the eNOS/sGC axis
      • Xie G.
      • Wang X.
      • Wang L.
      • Wang L.
      • Atkinson R.D.
      • Kanel G.C.
      • et al.
      Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats.
      and S1P1 signaling. Notably, HB-EGF has recently been identified as an angiocrine factor that maintains hepatic stellate cell (HSC) quiescence and whose secretion in liver fibrosis is impaired due to a lack of proteolytic processing.
      • Maretti-Mira A.C.
      • Wang X.
      • Wang L.
      • DeLeve L.D.
      Incomplete differentiation of engrafted bone marrow endothelial progenitor cells initiates hepatic fibrosis in the rat.
      Conversely, HSC-derived signaling towards LSECs is also involved in regulating liver fibrosis.
      • Desroches-Castan A.
      • Tillet E.
      • Ricard N.
      • Ouarné M.
      • Mallet C.
      • Belmudes L.
      • et al.
      Bone morphogenetic protein 9 is a paracrine factor controlling liver sinusoidal endothelial cell fenestration and protecting against hepatic fibrosis.
      Therefore, it seems important to further scrutinize LSEC-HSC crosstalk in the hepatic vascular niche to decipher the pathogenesis of liver fibrosis and to identify novel therapeutic targets.
      In contrast, the molecular regulators driving organ-specific differentiation of LSECs have not yet been comprehensively elucidated. While ID-1, LXR-α, endothelial Notch signaling,
      • Wohlfeil S.A.
      • Häfele V.
      • Dietsch B.
      • Schledzewski K.
      • Winkler M.
      • Zierow J.
      • et al.
      Hepatic endothelial notch activation protects against liver metastasis by regulating endothelial-tumor cell adhesion independent of angiocrine signaling.
      KLF-2, and ERG
      • Dufton N.P.
      • Peghaire C.R.
      • Osuna-Almagro L.
      • Raimondi C.
      • Kalna V.
      • Chauhan A.
      • et al.
      Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis.
      contribute to the molecular differentiation program of LSECs, we have recently identified the transcription factor GATA4 as the molecular master regulator of LSEC specification during early liver development, wherein it controls embryonic stem cell migration and fetal hematopoiesis.
      • Géraud C.
      • Koch P.-S.
      • Zierow J.
      • Klapproth K.
      • Busch K.
      • Olsavszky V.
      • et al.
      GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.
      Herein, we sought to analyze the role of endothelial GATA4 in the adult liver and in hepatic disease pathogenesis.

      Materials and methods

      Ethical compliance

      The experimental protocols used in this study complied with national and international ethical guidelines and, in case of animal models, were approved by the animal welfare commission of the Regierungspräsidium Karlsruhe (Karlsruhe, Germany).

      Animal models

      Female and male mice aged 1 day to 12 months were used in this study. Mice were hosted under specific-pathogen-free conditions in single ventilated cages in a 12 h/12 h day/night cycle and fed ad libitum with a standard rodent diet (V1534-000, Ssniff, Soest, Germany) with free access to water. For the generation of LSEC-specific conditional knockout mice Clec4g-icre driver mice
      • Wohlfeil S.A.
      • Häfele V.
      • Dietsch B.
      • Schledzewski K.
      • Winkler M.
      • Zierow J.
      • et al.
      Hepatic endothelial notch activation protects against liver metastasis by regulating endothelial-tumor cell adhesion independent of angiocrine signaling.
      (C57BL/6N-Tg(Clec4g-icre)1.1Sgoe, MGI:6280453) were crossed to Gata4 floxed mice (STOCK Gata4tm1.1Sad/J, JAX:008194), bearing a loxP site downstream of exon 3 and a loxP site upstream of exon 5. Mice bearing the genotype Clec4g-icretg/0 x Gata4fl/fl indicating homozygous recombination were denoted as Gata4LSEC-KO. Littermates bearing the genotypes Clec4g-icre0/0 x Gata4fl/fl or Clec4g-icre0/0 x Gata4wt/fl were used as controls. Genetic background analysis of crossed mice used in this study is provided in Table S1. Wild-type C57BL/6N mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and were habituated for at least 1 week in the local animal facility before the start of experiments.

      Statistical analysis

      Statistical analyses were performed using R 3.6.1
      R Core Team
      R: A Language and Environment for Statistical Computing.
      and Prism 8 (GraphPad Software, La Jolla, CA, USA). Sample size determination was performed separately for each experiment according to experience from previous experiments using an α-level of 0.05 and a β-level of 0.2. For statistical testing Welch's t test, paired t test, one- or two-way ANOVA, Fisher's exact test, and Mann-Whitney U test were used. A p value of <0.05 was considered statistically significant. The appropriate statistical test was chosen according to the requirements of each test (e.g. normal distribution or equal variance). Normal distribution was assessed using the Shapiro-Wilk test, equal variance using the F-test of equality of variances.

      Additional methodological details

      For further information on animal models, LSEC isolation, microarray and ATAC-seq analysis, and other materials and methods, please refer to the CTAT table and supplementary information.

      Results

      Endothelial Gata4 deficiency causes perisinusoidal liver fibrosis and hepatopathy

      To investigate the role of endothelial GATA4 in the adult liver, we established a novel genetic model of Gata4 deficiency in adult hepatic endothelium. As Stab2-icre-driven liver endothelial-selective Gata4 deficiency leads to embryonic lethality in mice between E14.5 and E17.5,
      • Géraud C.
      • Koch P.-S.
      • Zierow J.
      • Klapproth K.
      • Busch K.
      • Olsavszky V.
      • et al.
      GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.
      we used Clec4g-icre driver mice in which Cre is not fully active in liver endothelial cells until E17.5.
      • Wohlfeil S.A.
      • Häfele V.
      • Dietsch B.
      • Schledzewski K.
      • Winkler M.
      • Zierow J.
      • et al.
      Hepatic endothelial notch activation protects against liver metastasis by regulating endothelial-tumor cell adhesion independent of angiocrine signaling.
      Clec4g-icretg/0 x Gata4fl/fl (Gata4LSEC-KO) mice were born at a normal Mendelian ratio and their life expectancy was not reduced (Fig. S1A and B). PCR analysis confirmed recombination of Gata4 in the genome and on the mRNA level in Gata4LSEC-KO mice (Fig. S1C and D).
      Macroscopically, the livers of 3-month-old Gata4LSEC-KO mice revealed signs of liver fibrosis (Fig. 1A). Liver weight and liver-to-body weight ratio were significantly reduced in Gata4LSEC-KO mice (Fig. 1B, Fig. S1E). Upon histology and picrosirius red staining (PSR), Gata4LSEC-KO mice showed marked perisinusoidal liver fibrosis without any signs of bridging fibrosis (Fig. 1C and D). Deposition of collagens type I, III and IV was demonstrated by immunohistochemical staining confirming increased extracellular matrix protein synthesis (Fig. 1C, Fig. S1F). Accordingly, procollagen III N-terminal propeptide (PIIINP) levels were increased in the peripheral blood of Gata4-deficient mice (Fig. 1E). In liver fibrosis, HSCs are the main source of collagens. In Gata4LSEC-KO mice, HSC activity was found to be increased as measured by a reverse transcription quantitative PCR (RT-qPCR) panel including Acta2, Col1a1, Col3a1, Des and Pdgfrb (Fig. 1F). Moreover, the number of HSCs was increased as analyzed by in situ hybridization (ISH) of Pdgfrb (Fig. 1G, H).
      Figure thumbnail gr1
      Fig. 1Hepatic endothelial Gata4 deficiency causes hepatopathy.
      (A–B) Macroscopic liver phenotype (n = 13, 15). (C–D) PSR staining and collagen immunohistochemistry (n = 6). (E) PIIINP ELISA (n = 12). (F) RT-qPCR for HSC activation markers (n = 6). (G–H) Pdgfrb in situ hybridization (n = 6). (J–L) Liver laboratory values (n = 12, 19). (M–P) Immunofluorescence of c-CASP-3, Ki-67 and cell density (n = 6). Scale bars: (C, G) 50 μm; (M) 100 μm. n.s.p ≥0.05; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; (B, D, F, H, P) Mean ± SD, Welch's t test; (E, J–L, N–O) Mean ± SD, Mann-Whitney U test. HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; KO, knockout; PIIINP, procollagen type III N-terminal propeptide; PSR, Picrosirius red; RT-qPCR, reverse transcription quantitative PCR.
      Basic liver function tests showed significant elevations of aspartate aminotransferase, alanine aminotransferase and glutamate dehydrogenase as well as reduction of whole serum protein in Gata4LSEC-KO mice, indicating impaired hepatocyte integrity and function (Fig. 1J–L, Fig. S1G). Moreover, a metabolic profile revealed reduction of serum glucose, cholesterol and triglycerides (Fig. S1G). Hepatopathy was accompanied by enhanced apoptosis and proliferation of hepatocytes and endothelial cells as demonstrated by significantly increased detection of cleaved Caspase-3 and Ki-67 in Gata4LSEC-KO mice (Fig. 1M-O). Notably, there was a significant increase in DAPI cell counts (cells per mm2 area) in Gata4LSEC-KO livers, indicating that hepatocytes were smaller in Gata4LSEC-KO mice (Fig. 1P).
      Upon further histological analysis of Gata4LSEC-KO livers, we observed a slightly enhanced inflammatory infiltrate consisting of CD3+ lymphocytes and CD68+ macrophages, increased numbers of mitoses and binucleated hepatocytes, as well as a significant degree of sinusoidal dilatation and focal regenerative changes of the surrounding liver tissue, while PAS, Prussian Blue and Oil Red O revealed no significant alterations (Fig. S2A–C). Likewise, a colorimetric assay did not show significant changes in hepatic triglyceride levels (Fig. S2D).
      Macroscopic and histological analysis of P1, P8 and 6-week-old Gata4LSEC-KO livers did not show major defects in liver development nor prenatal onset of liver fibrosis. While P1 livers did not have any fibrotic changes upon PSR staining, P8 and 6-week-old Gata4LSEC-KO livers showed gradually increasing PSR values. However, these PSR values were considerably lower than the PSR values obtained from 3-month-old Gata4LSEC-KO livers (Fig. S3A–C; Fig. 1C, D). A significant reduction in liver-to-body weight ratio was only observed in 6-week-old Gata4LSEC-KO mice, but not in P1 or P8 mice (Fig. S3A–C).

      Endothelial Gata4 deficiency impairs liver regeneration

      As hepatocyte apoptosis was accompanied by continuously elevated hepatocyte proliferation and reduced hepatocyte cell size in Gata4LSEC-KO mice, we hypothesized that the regenerative capacity of the liver might be impaired in Gata4LSEC-KO mice. Liver regeneration in a two-thirds partial hepatectomy model (PHx; 70%) is mediated by hepatocyte proliferation (regenerative hyperplasia, early phase/day 3–4) and by hepatocyte enlargement (regenerative hypertrophy, later time points). After PHx, the predefined endpoints for euthanasia were reached significantly earlier in Gata4LSEC-KO mice compared to control animals (Fig. S4A–D). All Gata4LSEC-KO mice that did not recover after PHx failed to do so in the early phase indicating a defect in regenerative hypertrophy (Fig. 2A). In the group of the PHx Gata4LSEC-KO mice that survived to the end of the experiment, liver-to-body weight ratio and liver weight failed to increase significantly between 48 h and 144 h after PHx in contrast to the control animals (Fig. 2B). Similarly, the liver weight had nearly doubled in the control group at 144 h after PHx, while the liver weight did not significantly increase in the Gata4LSEC-KO mice at this late time point, indicating severe impairment rather than delay of liver regeneration (Fig. S4A).
      Figure thumbnail gr2
      Fig. 2Hepatic endothelial Gata4 deficiency impairs liver regeneration after PHx.
      (A) Kaplan–Meier curve after PHx (n = 15 males). (B) Liver weight to body weight ratio (n = 4–7). (C–D) Immunofluorescent staining for Ki-67, Podocalyxin and DAPI (n = 4–7). (E–F) Immunofluorescent staining for c-CASP-3, Podocalyxin and DAPI (n = 4–7). (G) Quantification of liver cell density in (C). Scale bars: 100 μm. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.0001; (A) Log-rank test; (B, D, F, G) Mean ± SD, two-way ANOVA with Tukey's post hoc test. LSEC, liver sinusoidal endothelial cell; KO, knockout; PHx, partial hepatectomy.
      To test for regenerative hyperplasia, we studied cell proliferation. Baseline proliferation as measured by Ki-67 positivity was already elevated in Gata4LSEC-KO mice (Fig. 1M, O and Fig. 2C, D). Nevertheless, the numbers of proliferating cells were significantly increased after 48 h and declined after 144 h in both Gata4LSEC-KO and control mice (Fig. 2C, D) indicating that regenerative hyperplasia was not defective. Similarly, hepatocyte apoptosis was slightly, but significantly increased at baseline in Gata4LSEC-KO mice and remained on a similar level during PHx-induced regeneration (Fig. 2E, F). To test for regenerative hypertrophy, we assessed hepatocyte size by measuring cell numbers per area. While hepatocyte size significantly increased at 48 and 144 h after PHx in control animals, this increase did not occur in Gata4LSEC-KO mice (Fig. 2G), indicating that hepatocytes are not able to undergo regenerative hypertrophy in Gata4LSEC-KO mice. In conclusion, regenerative hypertrophy of the liver and as a consequence liver regeneration as a whole seem to be grossly impaired in Gata4LSEC-KO mice, while a mere delay in regeneration seems less likely.

      Endothelial Gata4 deficiency causes sinusoidal capillarization

      Regarding the vasculature, hepatic microvessels in Gata4LSEC-KO mice showed a remarkable degree of sinusoidal capillarization. Using immunofluorescent staining, ISH, or immunoblot analysis, respectively, sinusoidal endothelial cell-specific markers such as Stab2
      • Schledzewski K.
      • Géraud C.
      • Arnold B.
      • Wang S.
      • Gröne H.-J.
      • Kempf T.
      • et al.
      Deficiency of liver sinusoidal scavenger receptors stabilin-1 and -2 in mice causes glomerulofibrotic nephropathy via impaired hepatic clearance of noxious blood factors.
      and LYVE-1 were shown to be downregulated while continuous endothelial cell-specific markers Cd34 and Endomucin were upregulated (Fig. 3A–C). In line with loss of Stab2 from capillarized LSECs, hyaluronic acid levels were enhanced in the peripheral blood of Gata4LSEC-KO mice (Fig. 3D). Transmission electron microscopy showed deposition of large bundles of collagen in the space of Disse and apposition of a basement membrane beneath the capillarized LSECs (Fig. 3E). In addition, a considerable loss of hepatocyte villi was observed in the space of Disse (Fig. 3E).
      Figure thumbnail gr3
      Fig. 3Hepatic endothelial Gata4 deficiency causes sinusoidal capillarization.
      (A–B) In situ hybridization for Stab2, Cd34, and immunofluorescent staining for Endomucin and LYVE-1 (n = 6). Scale bars: in situ hybridization 50 μm; immunofluorescence 100 μm. (C) Western Blot of LYVE-1 in LSECs (n = 6). (D) Hyaluronic acid in plasma (n = 12). (E) TEM of livers (n = 2–3 males). Red arrowheads: basement membrane. Red arrows: endothelial fenestrae. Scale bars: 3 μm. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; Mean ± SD; (B–C) Welch's t test; (D) Mann-Whitney U test. Col, collagen; LSEC, liver sinusoidal endothelial cell; KO, knockout; MV, microvilli; SoD, space of Disse.

      Endothelial Gata4 deficiency causes LSEC-to-continuous endothelial cell transdifferentiation

      For in-depth analysis of the molecular alterations in LSECs of Gata4LSEC-KO mice, we performed gene expression profiling of isolated LSECs. Altogether, 403 genes were significantly dysregulated in Gata4LSEC-KO compared to control mice (Fig. 4A, Table S3). Performing overrepresentation analysis (ORA) and gene set-enrichment analysis (GSEA) with established gene sets for continuous vs. sinusoidal endothelial differentiation,
      • Wohlfeil S.A.
      • Häfele V.
      • Dietsch B.
      • Schledzewski K.
      • Winkler M.
      • Zierow J.
      • et al.
      Hepatic endothelial notch activation protects against liver metastasis by regulating endothelial-tumor cell adhesion independent of angiocrine signaling.
      ,
      • Géraud C.
      • Koch P.-S.
      • Zierow J.
      • Klapproth K.
      • Busch K.
      • Olsavszky V.
      • et al.
      GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.
      ,
      • Géraud C.
      • Schledzewski K.
      • Demory A.
      • Klein D.
      • Kaus M.
      • Peyre F.
      • et al.
      Liver sinusoidal endothelium: a microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1.
      we systematically confirmed that sinusoidal and continuous endothelial cell genes were suppressed or induced, respectively, indicating LSEC-to-continuous endothelial transdifferentiation in Gata4LSEC-KO mice (Fig. 4A, B, C). Furthermore, we performed GSEA of MSigDB hallmark gene sets to identify other altered endothelial processes. Among the most regulated gene sets, we found enrichment of angiogenesis-associated genes and MYC target genes in the Gata4LSEC-KO transcriptomic data (Fig. 4D). In another approach, ORA of the 403 significantly dysregulated genes was carried out using Enrichr. In the Gene ontology (GO) Biological Process 2018 library, ‘Extracellular matrix organization’ was identified as the most significant GO term in LSECs of Gata4LSEC-KO mice (Fig. 4E). As GATA4 is a transcription factor (TF), we also performed ORA of the library “TF Pertubations Followed by Expression” and found GATA6 and MYC among the most strongly enriched transcription factors in LSEC of Gata4LSEC-KO mice (Fig. 4F). Notably, MYC target genes were enriched in both GSEA and ORA approaches (Fig. 4D, F) and upregulation of MYC target genes paralleled upregulation of continuous endothelial cell genes in Gata4-deficient LSEC (Fig. 4C, G). Myc was also among the significantly upregulated transcription factors in Gata4LSEC-KO LSECs (Fig. 4H). Regarding angiocrine factors, we observed an upregulation of angiogenesis-related genes such as Pdgfb, Apln and Vegfa (Fig. 4J). Moreover, angiocrine factors known to control hepatocyte function, such as Bmp2 and Wnt2, were significantly downregulated in Gata4LSEC-KO LSECs (Fig. 4J).
      Figure thumbnail gr4
      Fig. 4Hepatic endothelial Gata4 deficiency causes molecular transdifferentiation of LSECs comprising a profibrotic angiocrine switch.
      (A–B) Significant genes (Padj <0.05) and annotation for LSEC (p = 0.0200) and CEC (p = 0.0011) gene sets. (C) Enrichment plots of LSEC-associated (p = 0.0018, NES = −2.58) and CEC-associated (p = 0.0021, NES = 2.65) genes (n = 5). (D) GSEA using MSigDB hallmark gene sets. (E–F) ORA of selected gene ontology and transcription factor libraries. (G) Enrichment plot of MYC target genes (Padj = 0.0005, NES = 2.24). Heat maps of (H) transcription and (J) angiocrine factors. (A) Fisher's exact test. CEC, continuous endothelial cell; LSEC, liver sinusoidal endothelial cell; KO, knockout; GSEA, gene set-enrichment analysis; NES, normalized enrichment score; ORA, overrepresentation analysis.

      Endothelial Gata4 deficiency mediates a profibrotic angiocrine switch and HSC activation

      To confirm the transcriptomic alterations seen in our microarray data (Fig. 5A), we performed RT-qPCR and ISH of selected genes of interest. Myc and Pdgfb were significantly upregulated in Gata4-deficient LSECs and therefore represent novel endothelial candidate genes involved in liver fibrogenesis (Fig. 5B). Angiocrine factors Apln, Esm1 and Igfbp5 were upregulated in Gata4-deficient LSECs, while Bmp2 and Wnt2 were downregulated (Fig. 5C, Fig. S5A). Analysis of extracellular matrix genes including Sparcl1, Collagens and Laminins further confirmed a pro-fibrotic switch in Gata4LSEC-KO LSECs (Fig. 5D). Duplex ISH confirmed that Pdgfb was predominantly expressed de novo by endothelial cells in Gata4LSEC-KO livers (Fig. 5E–H and Fig. S5B, C). Likewise, endothelial Myc was significantly upregulated in Gata4LSEC-KO livers (Fig. 5J, Fig. S5A). Sparcl1 was expressed de novo by both endothelial cells and HSCs in Gata4LSEC-KO livers (Fig. S5D, E). In line with the higher number of Pdgfrb-expressing HSCs (Fig. 1G, H), Col1a1 was expressed mainly by HSCs, indicating HSC activation (Fig. S5F).
      Figure thumbnail gr5
      Fig. 5Angiocrine PDGFB signaling in Gata4LSEC-KO mice mediates stellate cell activation.
      (A) Volcano plot of LSEC transcriptomic data (n = 5 males). (B–D) RT-qPCR of LSEC for selected genes (n = 5 males). (E–H) In situ hybridization of liver tissue for Pdgfb with Cdh5 or Pdgfrb (n = 6 females). (J) Myc in situ hybridization of livers. (K–L) Genome tracks of GATA4-ChIP-Seq, MYC-ChIP-Seq (L), histone marker-ChIP-seq, and ATAC-Seq on the Myc and Pdgfb loci. (M) ATAC-Seq reads mapped around transcription-start-sites of upregulated and downregulated genes in Gata4LSEC-KO. (N) ChIP-qPCR to analyze MYC occupancy at Pdgfb promoter in LSECs (n = 3); Scale bars: 50 μm. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.0001; (B–D, G, N) Mean ± SD; Welch's t test; (H) Mean ± SD; two-way ANOVA with Tukey's post hoc test. ATAC-seq, assay for transposase-accessible chromatin using sequencing; ChIP-seq, chromatin immunoprecipitation sequencing; KO, knockout; LSEC, liver sinusoidal endothelial cell; RT-qPCR, reverse transcription PCR.

      Increased chromatin accessibility and amplification by activated MYC mediate de novo endothelial Pdgfb expression

      To investigate how endothelial Gata4 deficiency leads to activation of the transcription factor MYC and its downstream targets, we performed genome-wide analysis of open chromatin landscapes using ATAC-seq of LSECs isolated from Gata4LSEC-KO and control mice. Importantly, we found that GATA4 controls gene expression by regulating chromatin accessibility. ATAC-seq revealed increased chromatin accessibility at a multitude of gene loci in Gata4LSEC-KO LSECs, including the Myc and the Pdgfb gene loci that were directly bound by GATA4 (Fig. 5K, L).
      In a more global approach, we intersected the gene expression profiling data and ATAC-seq data obtained from Gata4LSEC-KO LSECs and found that chromatin accessibility was highly increased for the genes significantly upregulated in Gata4LSEC-KO LSECs (Fig. 5M). Conversely, no changes in chromatin accessibility were found for genes significantly downregulated in Gata4LSEC-KO LSECs (Fig. 5M). These results indicate that the repressive function of GATA4 is executed on the chromatin level, whereas its activating function is independent of alterations in chromatin accessibility.
      Notably, we identified MYC binding sites at the promotor region of the Pdgfb gene using the ATAC-seq peaks differentially regulated in Gata4-deficient LSECs combined with the published MYC-ChIP-seq data of the liver
      • Kress T.R.
      • Pellanda P.
      • Pellegrinet L.
      • Bianchi V.
      • Nicoli P.
      • Doni M.
      • et al.
      Identification of MYC-dependent transcriptional programs in oncogene-addicted liver tumors.
      (Fig. 5L). To further study the binding of the transcription factor MYC at the promoter region of the angiocrine factor Pdgfb, we performed MYC ChIP-qPCR. MYC binding at Pdgfb was increased in LSECs from Gata4LSEC-KO mice compared to control mice in 3 biologically independent ChIP experiments. These results suggest that MYC directly augments Pdgfb expression in Gata4LSEC-KO LSECs (Fig. 5N).

      Endothelial Gata4 deficiency causes downregulation of angiocrine Wnt signaling, impairing metabolic liver zonation and liver regeneration

      Furthermore, we confirmed the regulation of other angiocrine factors targeting hepatocytes in endothelial Gata4-deficiency. Wnt2 was downregulated in Gata4-deficient LSECs (Fig. S6A), whereas Wnt9b, Rspo3 and Hgf were slightly increased or unchanged (Fig. S6A–C). As a consequence, angiocrine-mediated β-catenin signaling was impaired in hepatocytes, as demonstrated by reduced hepatocyte Axin2 expression (Fig. S6A). Consequently, metabolic liver zonation was disturbed as shown by reduced glutamine synthetase expression in Gata4LSEC-KO mice (Fig. S6A). To further corroborate these results, we performed gene expression analysis of whole livers from Gata4LSEC-KO mice vs. control animals. GSEA of Gata4LSEC-KO livers showed that major metabolic pathways were affected, including fatty acid metabolism, bile acid metabolism, oxidative phosphorylation and xenobiotic metabolism (Fig. S6D).
      As angiocrine factors Wnt-2 and HGF are known to contribute to liver regeneration,
      • Ding B.-S.
      • Nolan D.J.
      • Butler J.M.
      • James D.
      • Babazadeh A.O.
      • Rosenwaks Z.
      • et al.
      Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.
      we analyzed these factors in Gata4LSEC-KO mice after PHx. After an initial increase of Hgf in both, Gata4LSEC-KO and control LSEC, Hgf remained elevated only in Gata4LSEC-KO LSECs at 144 h after PHx, indicating that regeneration was not successful despite Hgf induction and hepatocyte proliferation, i.e. regenerative hyperplasia (Fig. S7A). In contrast to control animals, angiocrine Wnt2 expression did not increase in Gata4LSEC-KO LSECs at 48 h after PHx (Fig. S7B). As induction of angiocrine Wnt-2 signaling is a hallmark of the early phase of liver regeneration, impaired induction of Wnt2 in Gata4LSEC-KO mice likely contributes to insufficient regeneration in these animals by impairing regenerative hypertrophy.

      Endothelial GATA4 protects against CDAA diet-associated perisinusoidal liver fibrogenesis

      As the liver fibrosis seen in Gata4LSEC-KO mice resembles the perisinusoidal fibrosis of non-alcoholic steatohepatitis, we studied the hepatic endothelium in choline-deficient, l-amino acid-defined (CDAA) diet-induced liver fibrosis.
      In CDAA diet-fed mice, perisinusoidal liver fibrosis developed after 10 weeks (Fig. 6A, B) accompanied by development of hepatic steatosis and moderate hepatopathy (Fig. S8A, B). RT-qPCR analysis of whole liver specimens demonstrated activation of HSCs (Fig. 6C). Gene expression profiling of LSECs isolated from CDAA diet-fed mice (CDAA LSECs) showed similar downregulation of LSEC-specific genes (Fig. 6D–F) as seen in LSECs of Gata4LSEC-KO mice (Fig. 4A–C). Similarly, GSEA of CDAA LSECs revealed results partially overlapping with those found in Gata4LSEC-KO LSECs, identifying MYC target genes as the top enriched gene set (Fig. 6G). ORA of 3,890 significantly dysregulated genes revealed ‘Cell migration’ and ‘Extracellular matrix organization’ as the most strongly enriched gene sets (Fig. 6H). We also performed ORA of the library “TF Pertubations Followed by Expression” and found SOX9 and GATA6 gene sets to be the most significantly regulated gene sets (Fig. 6J). Comparing microarray data from Gata4LSEC-KO and CDAA LSECs revealed a remarkable overlap of up- and downregulated genes (Fig. 6K, L). Accordingly, many transcription and angiocrine factors were regulated in the same direction including Myc and Pdgfb (Fig. 6M, N). These results demonstrate congruent alterations in the endothelial transcriptomic profile of Gata4LSEC-KO LSECs and CDAA LSECs compared to control animals or standard-fed mice, respectively.
      Figure thumbnail gr6
      Fig. 6Diet-induced perisinusoidal liver fibrosis recapitulates loss of GATA4 and the angiocrine switch.
      (A–B) PSR staining of livers. Scale bars: 100 μm. (n = 3). (C) RT-qPCR for HSC activation markers (n = 3). (D–E) Significant genes (Padj < 0.05) and annotation for LSEC (p = 0.0015) and CEC (p = 0.2787) gene sets. (F) Enrichment plot of LSEC-associated (p = 0.0019, NES = −2.11) and CEC-associated (p = 0.15, NES = 1.27) genes. (G) GSEA using the MSigDB hallmark gene sets. (H–J) ORA of selected gene ontology and transcription factor libraries. (K–L) Overlap between genes in Gata4LSEC-KO and CDAA diet. Heat maps of (M) transcription and (N) angiocrine factors significantly regulated in Gata4LSEC-KO with annotation for their expression and significance upon CDAA diet. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; (B–C) Welch's t test. (D) Fisher's exact test. CDAA, choline-deficient, l-amino acid-defined; CEC, continuous endothelial cell; GSEA, gene set-enrichment analysis; HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; KO, knockout; NES, normalized enrichment score; ORA, overrepresentation analysis; PSR, Picrosirius red; RT-qPCR, reverse transcription PCR.
      To investigate whether endothelial GATA4 function is affected in CDAA-fed mice, we defined a GATA4 gene set as an indicator for GATA4 function. To this end, we combined transcriptional and regulatory data from 3 different sources (Fig. 7A): (i) significantly downregulated genes from Gata4LSEC-KO LSECs from this study, and ChIP-seq data for GATA4 of whole liver from (ii) ENCODE (ENCSR194GNY) and (iii) GEO (GSM1194141). 73 genes were identified that were significantly downregulated in Gata4LSEC-KO LSECs and exhibited ≥2 binding sites for GATA4 in either or both ChIP-seq data sets (Table S4). The gene set of these 73 genes was regarded as a reporter for GATA4 activity and named “Endothelial GATA4 gene set”. Using the “Endothelial GATA4 gene set” for GSEA on endothelial cells from CDAA-fed mice, we found enrichment for the gene set in LSECs of standard-fed mice indicating a reduction of GATA4-dependent genes in the CDAA model of liver fibrosis (Fig. 7B, C). On the contrary, MYC target genes were enriched in endothelial cells from CDAA-fed mice (Fig. 7D). These findings imply antagonistic functions of endothelial GATA4 and MYC in liver fibrogenesis. Confirming the results regarding the regulation of the GATA4 and MYC target gene sets in CDAA LSECs, Gata4 and Myc themselves were differentially expressed in CDAA LSECs and LSECs from standard-fed control animals, respectively. While GATA4 was significantly downregulated (Fig. 7E, F), Myc and Pdgfb were significantly upregulated (Fig. 7E).
      Figure thumbnail gr7
      Fig. 7Endothelial GATA4 serves as protective factor preventing perisinusoidal liver fibrosis in CDAA diet.
      (A) Approach to defining the endothelial Gata4-gene-set. (B) Overlap of downregulated genes upon CDAA diet and endothelial Gata4-gene-set. (C) Enrichment plot of endothelial Gata4-gene-set (p = 0.0018, NES = −3.08), and (D) MYC target genes (Padj = 0.0007, NES = 1.98) in LSEC. (E) RT-qPCR analysis of LSEC (n = 5). (F) Immunoblot for GATA4 of whole liver (n = 3). (G–H) PSR staining of liver (n = 5–6), Scale bars: 100 μm. (J–K) Liver laboratory values. (L) RT-qPCR for HSC activation markers (n = 5–6). (E–F, H–L) Mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.0001; (E-F) Welch's t test; (H–L) two-way ANOVA with Tukey's post-hoc test. CDAA, choline-deficient, l-amino acid-defined; HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; KO, knockout; NES, normalized enrichment score; PSR, Picrosirius red; RT-qPCR, reverse transcription PCR.
      Based on the findings that acquired endothelial GATA4 downregulation may contribute to CDAA-associated perisinusoidal liver fibrosis, we asked whether complete genetic endothelial Gata4 deficiency further aggravates CDAA-induced liver fibrogenesis. When Gata4LSEC-KO mice were fed the CDAA diet (Fig. S9A), the amount of perisinusoidal liver fibrosis was significantly increased compared to both CDAA-fed control mice and standard-fed Gata4LSEC-KO mice (Fig. 7G, H). While hepatopathy was not greatly different in CDAA-fed control and Gata4LSEC-KO mice (Fig. 7J, K), HSC activity as measured by expression of extracellular matrix molecules Acta2, Col1a1 and Col3a1 was significantly higher in CDAA-fed Gata4LSEC-KO mice compared to both CDAA-fed control mice and standard-fed Gata4LSEC-KO mice (Fig. 7L). In contrast to the aggravation of perisinusoidal liver fibrosis in CDAA-fed Gata4LSEC-KO mice, no significant changes in the extent of bridging fibrosis were seen in Gata4LSEC-KO mice upon induction of toxic liver fibrosis in the carbon tetrachloride model (Fig. S9C–F).
      In conclusion, downregulation of GATA4 and its downstream target genes seems to be an important pathogenic factor in the development of CDAA-associated perisinusoidal fibrosis. Although there might be additional mechanisms that add to the complex phenotype in CDAA-fed mice, our results show that endothelial GATA4 protects against dietary perisinusoidal liver fibrosis.

      Analysis of single cell RNA-seq data reveals that the endothelial GATA4/PDGFB axis is also affected in human cirrhosis

      To validate our results, we analyzed a published single cell transcriptome sequencing (scRNA-seq) dataset of human liver non-parenchymal cells including endothelial cells from patients with cirrhosis and healthy control individuals.
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      Louvain clustering yielded 33 different cell clusters from which endothelial cells were identified by the expression of PECAM1, CDH5 and the endothelial transcription factor ERG (Fig. 8A–E).
      Figure thumbnail gr8
      Fig. 8Single cell transcriptome sequencing in human liver fibrosis reveals that the endothelial GATA4/PDGFB axis is affected.
      (A) Louvain clustering of 66,135 non-parenchymal cells from liver cirrhosis patients and healthy individuals (n = 5). Scaled gene expression of endothelial genes (B) PECAM1, (C) CDH5 and (D) ERG. (E) Annotation of endothelial cells in blue. Annotation of endothelial cells by (F) condition and (G) EC subtype. (H) Count of non-LSEC EC and LSEC. (J) Frequency of LSEC in livers (n = 5). Error bars: 95% CI. Scaled expression of (K) STAB2, (L) GATA4 and (M) PDGFB. EC, endothelial cell; LSEC, liver sinusoidal endothelial cell.
      Endothelial cells with expression of markers GATA4 and STAB2 or GATA4 and WNT2 were defined as LSECs. While the cluster of endothelial cells from healthy livers mainly consisted of LSECs, endothelial cells from cirrhotic livers were mainly non-LSEC endothelial cells (Fig. 8F–J). These results demonstrate that endothelial cells from cirrhotic patients have undergone a transdifferentiation process with loss of STAB2 and GATA4 expression (Fig. 8 K, L). Notably, PDGFB was induced in the endothelial cells of cirrhotic patients (Fig. 8M).
      We performed GSEA using the MSigDB hallmark gene sets and compared LSEC vs. non-LSEC endothelial cell data. “MYC targets” and “Angiogenesis” were the top 2 enriched gene sets in non-LSEC endothelial cells (Fig. S10A, B). Meanwhile, the endothelial GATA4 gene set defined in this study was enriched in LSECs (Fig. S10C).
      In conclusion, these data support the notion of a remarkable congruence between murine and human endothelial cell alterations during liver fibrosis/cirrhosis. This indicates that the antagonistic counterplay of endothelial GATA4 and MYC represents a common pathway contributing to liver fibrogenesis in mice and humans.

      Discussion

      Our data show that compromising LSEC differentiation leads to hepatopathy, sinusoidal capillarization, and perisinusoidal liver fibrosis. Protection against liver fibrosis is mediated by endothelial expression of the sinusoidal master regulator GATA4. Loss of GATA4 expression in LSECs is permissive for induction of the sinusoidal capillarization program and allows de novo expression of profibrotic angiocrine factors including Pdgfb, Sparcl1, Esm1 and Igfbp5.
      Notably, the regulation of MYC and GATA4 was antagonistic. As a consequence, endothelial GATA4 and MYC target gene sets were also counter-regulated and this was further paralleled by counter-regulation of LSEC genes and continuous endothelial cell genes at large. Preferentially, MYC is not an on-off specifier of gene activity, but is a non-linear amplifier of expression, acting universally at active genes.
      • Nie Z.
      • Hu G.
      • Wei G.
      • Cui K.
      • Yamane A.
      • Resch W.
      • et al.
      c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.
      By performing ATAC-seq of LSECs from Gata4LSEC-KO and control mice, we demonstrate here that GATA4 deficiency in LSECs leads to de-repression of a general (continuous) endothelial cell differentiation program mediated by increased chromatin accessibility. Improved chromatin accessibility may then allow activation of the respective genes by MYC for which chromatin accessibility and expression are also increased upon Gata4 depletion in LSECs. Interestingly, the significant enrichment of MYC at the Pdgfb promoter region in Gata4LSEC-KO LSECs indicates a GATA4- and MYC-dependent activation of Pdgfb in our model. Notably, Pdgfb is expressed in the microvasculature of most organs except the liver, making it a constitutive angiokine of continuous endothelial cells. This is in line with the results from ATAC-seq which suggested that GATA4 acts as a repressor of Pdgfb and other continuous endothelial cell genes in LSECs. Functionally, angiocrine PDGFB signaling is indispensable for pericyte maintenance,
      • Bjarnegård M.
      • Enge M.
      • Norlin J.
      • Gustafsdottir S.
      • Fredriksson S.
      • Abramsson A.
      • et al.
      Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities.
      a cell type not found in the liver sinusoids.
      De novo expression of angiocrine Pdgfb in LSECs alone seems to be sufficient to induce perisinusoidal fibrosis, as has been demonstrated by artificial Pdgfb overexpression in hepatocytes.
      • Czochra P.
      • Klopcic B.
      • Meyer E.
      • Herkel J.
      • Garcia-Lazaro J.F.
      • Thieringer F.
      • et al.
      Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice.
      Vice versa, loss of Pdgfb activity increases hepatic permeability, prevents sinusoidal capillarization, and improves insulin sensitivity
      • Raines S.M.
      • Richards O.C.
      • Schneider L.R.
      • Schueler K.L.
      • Rabaglia M.E.
      • Oler A.T.
      • et al.
      Loss of PDGF-B activity increases hepatic vascular permeability and enhances insulin sensitivity.
      indicating the importance of balanced PDGFB signaling for the homeostasis of the hepatic vascular niche.
      Notably, endothelial Gata4 deficiency caused non-fatal, perisinusoidal liver fibrosis, but not progressive periportal or bridging-type liver fibrosis in Gata4LSEC-KO mice. This may be due to the fact that only CD248+ HSCs, that are induced by hepatocyte damage, are permissive for overall activation by PDGFB comprising proliferation and migration.
      • Wilhelm A.
      • Aldridge V.
      • Haldar D.
      • Naylor A.J.
      • Weston C.J.
      • Hedegaard D.
      • et al.
      CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism.
      Furthermore, a recent study using single cell RNA-seq shows that HSCs are zonated along the liver sinusoids from pericentral to periportal
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Henderson B.E.P.
      • Smith J.R.
      • Matchett K.P.
      • Portman J.R.
      • et al.
      Single-cell transcriptomics uncovers zonation of function in the mesenchyme during liver fibrosis.
      ; central vein-associated HSCs were identified as the major HSC subpopulation driving liver fibrosis. This differential reactivity of HSCs was also confirmed in a recent study demonstrating that loss of hepatocyte-derived ECM-1 induced moderate perisinusoidal, but massive pericentral liver fibrosis.
      • Fan W.
      • Liu T.
      • Chen W.
      • Hammad S.
      • Longerich T.
      • Hausser I.
      • et al.
      ECM1 prevents activation of transforming growth factor β, hepatic stellate cells, and fibrogenesis in mice.
      Therefore, additional hits may be necessary to allow angiocrine PDGFB to exert its full profibrotic effects and to finally mediate progressive portal and bridging fibrosis.
      • Xu M.
      • Xu H.-H.
      • Lin Y.
      • Sun X.
      • Wang L.-J.
      • Fang Z.-P.
      • et al.
      LECT2, a ligand for Tie1, plays a crucial role in liver fibrogenesis.
      Regarding the other members of the profibrotic angiocrine panel identified in LSECs of Gata4LSEC-KO mice, SPARCL1 may interact with PDGFB-activated HSCs to enhance collagen I fibril formation.
      • Sullivan M.M.
      • Barker T.H.
      • Funk S.E.
      • Karchin A.
      • Seo N.S.
      • Höök M.
      • et al.
      Matricellular hevin regulates decorin production and collagen assembly.
      Interestingly, Sparcl1 is also induced in HSCs in non-alcoholic steatohepatitis (NASH), but not toxic liver fibrosis.
      • Xiong X.
      • Kuang H.
      • Ansari S.
      • Liu T.
      • Gong J.
      • Wang S.
      • et al.
      Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis.
      Esm1 and Igfbp5 were also significantly regulated angiocrine factors in LSECs of Gata4LSEC-KO mice and they are known biomarkers for liver fibrosis.
      • Toshikuni N.
      • Ozaki K.
      • George J.
      • Tsutsumi M.
      Serum endocan as a survival predictor for patients with liver cirrhosis.
      ,
      • Sokolović A.
      • Sokolović M.
      • Boers W.
      • Elferink R.P.O.
      • Bosma P.J.
      Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells.
      Perisinusoidal liver fibrosis is a special feature in the early stage of NASH which is caused by a cascade of pathogenic hits that lead to hepatocyte and non-parenchymal liver cell damage. Although data have been grossly lacking, LSECs have been suggested to play a pivotal role among the non-parenchymal cells of the hepatic vascular niche in NASH.
      • Hammoutene A.
      • Rautou P.-E.
      Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease.
      Notably, in-depth analysis of the single cell RNA-seq data of human cirrhotic liver specimens published by Ramachandran and colleagues
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      regarding LSEC differentiation has largely confirmed our experimental findings in Gata4LSEC-KO mice and CDAA-induced, dietary liver fibrosis. Therefore, our data provide proof of principle that dysregulation of antagonistically acting endothelial transcriptional regulators GATA4 and MYC leads to downstream disturbance of the PDGFB/PDGFRβ axis and contributes to murine and human liver fibrosis.
      In conclusion, we have shown here that liver endothelial cells actively drive and determine perisinusoidal liver fibrogenesis, with the transcription factor GATA4 acting as the master regulator of chromatin accessibility. Therefore, therapies targeting the GATA4/MYC/PDGFB/PDGFRβ axis represent a promising strategy for the prevention and treatment of liver fibrosis.
      • Malehmir M.
      • Pfister D.
      • Gallage S.
      • Szydlowska M.
      • Inverso D.
      • Kotsiliti E.
      • et al.
      Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.

      Abbreviations

      ATAC-seq, assay for transposase-accessible chromatin using sequencing; CDAA, choline-deficient, l-amino acid-defined; ChIP-seq, chromatin immunoprecipitation sequencing; GSEA, gene set-enrichment analysis; HSC, hepatic stellate cell; ISH, in situ hybridization; KO, knockout; LSEC, liver sinusoidal endothelial cell; MSigDB, molecular signatures database; NES, normalized enrichment score; ORA, overrepresentation analysis; PHx, partial hepatectomy; PIIINP, procollagen type III N-terminal propeptide; PSR, Picrosirius red; RT-qPCR, reverse transcription quantitative PCR.

      Financial support

      The authors gratefully acknowledge the data storage service [email protected] supported by the Ministry of Science, Research and the Arts Baden-Württemberg (MWK) and the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by the Deutsche Forschungsgemeinschaft, Germany (DFG, German Research Foundation) – Project numbers 259332240 – RTG/GRK 2099; 394046768 – CRC/SFB 1366; 314905040 – CRC/SFB-TR 209; 413262200 – ICON/EB 187/8-1.

      Authors' contributions

      Conceptualization, M.W., T.S., S.W.K., C.G., P.-S.K., S.G.; Investigation, M.W., T.S., S.W.K., C.D.S., H.S., J.C., L.K., A.M., C.S., M.N., X.-J.Z.; Data Curation, M.W., C.D.S., J.C., C.S., G.D.; Writing – Original Draft, C.G., P.-S.K., S.G.; Writing – Review & Editing, M.W., T.S., S.W.K., C.D.S., H.S., J.C., L.K., A.M., C.S., M.N., A.U., S.A., X.-J.Z., G.v.F., D.H., C.M., G.D., K.S., C.G., P.-S.K., S.G.; Visualization, M.W., T.S., S.W.K., C.D.S, A.U., K.S.; Supervision, C.G., P.-S.K., S.G.; Funding Acquisition, M.W., S.A., C.M., G.D., C.G., P.-S.K., S.G.

      Data availability statement

      The raw and normalized gene expression profiling data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE141004 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141004). ATAC (‘assay for transposase-accessible chromatin’)-sequencing (ATAC-seq) and chromatin immunoprecipitation (ChIP)-sequencing (ChIP-seq) data are accessible through accession number GSE154828 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154828).

      Conflict of interest

      The authors declare no competing interests.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgments

      We thank Stephanie Riester and Carolina De La Torre for excellent technical support.

      Supplementary data

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