Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury.

Liver sinusoidal endothelial cells (LSECs) rapidly clear lipopolysaccharide (LPS) from the bloodstream and establish intimate contact with immune cells. However, their role in regulating liver inflammation remains poorly understood. We show that LSECs modify their chemokine expression profile driven by LPS or interferon-γ (IFN-γ), resulting in the production of the myeloid- or lymphoid-attracting chemokines CCL2 and CXCL10, respectively, which accumulate in the serum of LPS-challenged animals. Natural killer (NK) cell exposure to LSECs in vitro primes NK cells for higher production of IFN-γ in response to interleukin-12 (IL-12) and IL-18. In livers of LPS-injected mice, NK cells are the major producers of this cytokine. In turn, LSECs require exposure to IFN-γ for CXCL10 expression, and endothelial-specific Cxcl10 gene deletion curtails NK cell accumulation in the inflamed livers. Thus, LSECs respond to both LPS and immune-derived signals and fuel a positive feedback loop of immune cell attraction and activation in the inflamed liver tissue.

ALK1 controls hepatic vessel formation, angiodiversity, and angiocrine functions in hereditary hemorrhagic telangiectasia of the liver.

BACKGROUND AND AIMS: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. APPROACH AND RESULTS: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. CONCLUSIONS: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.

Lyve-1 deficiency enhances the hepatic immune microenvironment entailing altered susceptibility to melanoma liver metastasis.

BACKGROUND: Hyaluronan receptor LYVE-1 is expressed by liver sinusoidal endothelial cells (LSEC), lymphatic endothelial cells and specialized macrophages. Besides binding to hyaluronan, LYVE-1 can mediate adhesion of leukocytes and cancer cells to endothelial cells. Here, we assessed the impact of LYVE-1 on physiological liver functions and metastasis. METHODS: Mice with deficiency of Lyve-1 (Lyve-1-KO) were analyzed using histology, immunofluorescence, microarray analysis, plasma proteomics and flow cytometry. Liver metastasis was studied by intrasplenic/intravenous injection of melanoma (B16F10 luc2, WT31) or colorectal carcinoma (MC38). RESULTS: Hepatic architecture, liver size, endothelial differentiation and angiocrine functions were unaltered in Lyve-1-KO. Hyaluronan plasma levels were significantly increased in Lyve-1-KO. Besides, plasma proteomics revealed increased carbonic anhydrase-2 and decreased FXIIIA. Furthermore, gene expression analysis of LSEC indicated regulation of immunological pathways. Therefore, liver metastasis of highly and weakly immunogenic tumors, i.e. melanoma and colorectal carcinoma (CRC), was analyzed. Hepatic metastasis of B16F10 luc2 and WT31 melanoma cells, but not MC38 CRC cells, was significantly reduced in Lyve-1-KO mice. In vivo retention assays with B16F10 luc2 cells were unaltered between Lyve-1-KO and control mice. However, in tumor-free Lyve-1-KO livers numbers of hepatic CD4+, CD8+ and regulatory T cells were increased. In addition, iron deposition was found in F4/80+ liver macrophages known to exert pro-inflammatory effects. CONCLUSION: Lyve-1 deficiency controlled hepatic metastasis in a tumor cell-specific manner leading to reduced growth of hepatic metastases of melanoma, but not CRC. Anti-tumorigenic effects are likely due to enhancement of the premetastatic hepatic immune microenvironment influencing early liver metastasis formation.

[Assessment of an e­learning platform for dermatosurgery]. / Beurteilung einer E­Learning-Plattform für Dermatochirurgie.

BACKGROUND: Dermatosurgical (DS) teaching is based on a combination of reading/understanding textbooks and applying surgical procedures (±â€¯supervision). Most textbooks are primarily text-centered. The text is visually supported by photos/sketches (S) and possibly videos (V). A learning goal of this teaching should be that the learner is confident to perform a procedure independently. METHODS: We have developed an online-based platform, the FlapFinder (FF; www.skin-surgery.org ), which teaches the user DS in the facial region primarily in the form of S + V. These are supported by a short text (T) and bonus material (B). B contains personal recommendations from the FF authors. A SurveyMonkey® (Survey Monkey, San Mateo, CA, USA) analysis should clarify how this is assessed by the user. RESULTS: In all, 62 participants completed the questionnaire in full. This was a heterogeneous group (27 dermatologists vs. 35 non-dermatologists; 32â€¯× clinic vs. 30â€¯× non-clinic) with different prior experience. The majority of users found that the combination of T + S + V helped them to understand (55/62; 88.7%), remember (53/62, 85.5%), and perform the procedures independently (43/62; 69.3%). While S + V were most frequently used (22/62; 35.5% and 27/62; 43.6%), users reported having benefited most from this (20/62; 32.3% and 24/62; 38.7%), T + B were used less (0/62, 0.0% and 2/62; 3.2%). Nevertheless, the majority would not want to do without either S, V, T, or B (49/62; 79%). CONCLUSION: The combination of S + V + T + B is rated positively by DS learners. S + V are rated as particularly helpful. Future studies must clarify whether the learning objective of the concrete practical performance of DS is changed by e­media.

Angiogenic and molecular diversity determine hepatic melanoma metastasis and response to anti-angiogenic treatment.

BACKGROUND: Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10-20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition. METHODS: Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment. RESULTS: WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization. CONCLUSION: Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Imbalanced Activation of Wnt-/ß-Catenin-Signaling in Liver Endothelium Alters Normal Sinusoidal Differentiation.

Endothelial wingless-related integration site (Wnt)-/ß-catenin signaling is a key regulator of the tightly sealed blood-brain barrier. In the hepatic vascular niche angiokine-mediated Wnt signaling was recently identified as an important regulator of hepatocyte function, including the determination of final adult liver size, liver regeneration, and metabolic liver zonation. Within the hepatic vasculature, the liver sinusoidal endothelial cells (LSECs) are morphologically unique and functionally specialized microvascular endothelial cells (ECs). Pathological changes of LSECs are involved in chronic liver diseases, hepatocarcinogenesis, and liver metastasis. To comprehensively analyze the effects of endothelial Wnt-/ß-catenin signaling in the liver, we used endothelial subtype-specific Clec4g-iCre mice to generate hepatic ECs with overexpression of Ctnnb1. In the resultant Clec4g-iCre tg/wt ;Ctnnb1(Ex3) fl/wt (Ctnnb1 OE-EC ) mice, activation of endothelial Wnt-/ß-catenin signaling resulted in sinusoidal transdifferentiation with disturbed endothelial zonation, that is, loss of midzonal LSEC marker lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve1) and enrichment of continuous EC genes, such as cluster of differentiation (CD)34 and Apln. Notably, gene set enrichment analysis revealed overrepresentation of brain endothelial transcripts. Activation of endothelial Wnt-/ß-catenin signaling did not induce liver fibrosis or alter metabolic liver zonation, but Ctnnb1 OE-EC mice exhibited significantly increased plasma triglyceride concentrations, while liver lipid content was slightly reduced. Ctnnb1 overexpression in arterial ECs of the heart has been reported previously to cause cardiomyopathy. As Clec4g-iCre is active in a subset of cardiac ECs, it was not unexpected that Ctnnb1 OE-EC mice showed reduced overall survival and cardiac dysfunction. Altogether, balanced endothelial Wnt-/ß-catenin signaling in the liver is required for normal LSEC differentiation and for maintenance of normal plasma triglyceride levels.

Angiodiversity and organotypic functions of sinusoidal endothelial cells.

'Angiodiversity' refers to the structural and functional heterogeneity of endothelial cells (EC) along the segments of the vascular tree and especially within the microvascular beds of different organs. Organotypically differentiated EC ranging from continuous, barrier-forming endothelium to discontinuous, fenestrated endothelium perform organ-specific functions such as the maintenance of the tightly sealed blood-brain barrier or the clearance of macromolecular waste products from the peripheral blood by liver EC-expressed scavenger receptors. The microvascular bed of the liver, composed of discontinuous, fenestrated liver sinusoidal endothelial cells (LSEC), is a prime example of organ-specific angiodiversity. Anatomy and development of LSEC have been extensively studied by electron microscopy as well as linage-tracing experiments. Recent advances in cell isolation and bulk transcriptomics or single-cell RNA sequencing techniques allowed the identification of distinct LSEC molecular programs and have led to the identification of LSEC subpopulations. LSEC execute homeostatic functions such as fine tuning the vascular tone, clearing noxious substances from the circulation, and modulating immunoregulatory mechanisms. In recent years, the identification and functional analysis of LSEC-derived angiocrine signals, which control liver homeostasis and disease pathogenesis in an instructive manner, marks a major change of paradigm in the understanding of liver function in health and disease. This review summarizes recent advances in the understanding of liver vascular angiodiversity and the functional consequences resulting thereof.

Exploring the transcriptomic network of multi-ligand scavenger receptor Stabilin-1- and Stabilin-2-deficient liver sinusoidal endothelial cells.

The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-ß/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood.

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

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/PDGFRß 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.

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.

Angiocrine Hepatocyte Growth Factor Signaling Controls Physiological Organ and Body Size and Dynamic Hepatocyte Proliferation to Prevent Liver Damage during Regeneration.

Liver sinusoidal endothelial cells (LSECs) control organ functions, metabolism, and development through the secretion of angiokines. LSECs express hepatocyte growth factor (Hgf), which is involved in prenatal development, metabolic homeostasis, and liver regeneration. This study aimed to elucidate the precise contribution of LSEC-derived Hgf in physiological homeostasis and liver regeneration. Stab2-iCretg/wt;Hgffl/fl (HgfΔLSEC) mice were generated to abrogate Hgf expression selectively in LSECs from early fetal development onwards, to study global development, metabolic and endothelial zonation, and organ functions as well as liver regeneration in response to 70% partial hepatectomy (PH). Although zonation and liver/body weight ratios were not altered, total body weight and total liver weight were reduced in HgfΔLSEC. Necrotic organ damage was more marked in HgfΔLSEC mice, and regeneration was delayed 72 hours after PH. This was associated with decreased hepatocyte proliferation at 48 hours after PH. Molecularly, HgfΔLSEC mice showed down-regulation of Hgf/c-Met signaling and decreased expression of Deptor in hepatocytes. In vitro knockdown of Deptor was associated with decreased proliferation. Therefore, angiocrine Hgf controls hepatocyte proliferation and susceptibility to necrosis after partial hepatectomy via the Hgf/c-Met axis involving Deptor to prevent excessive organ damage.

Reconstruction of Nasal Defects With Dermal Skin Substitutes-A Retrospective Study of 36 Defects.

OBJECTIVES: It is uncertain whether dermal regeneration templates (DRTs) are helpful to reconstruct nasal defects. The aim of this study was to assess whether the aesthetic subunits determine the outcome. METHODS: In this unicentric, retrospective study, the surgical procedures and outcomes of patients who received DRTs to reconstruct nasal defects were assessed and compared with the involved aesthetic subunits. RESULTS: DRTs were used for reconstruction of 36 nasal defects in 35 patients with involvement of 76 aesthetic subunits: nasal sidewall (n = 21), nasal ala (n = 13), nasal tip/columella (n = 12, n = 1, respectively), nasal dorsum (n = 12), and extranasal aesthetic areas (n = 17). Fifty-eight nasal and 8 extranasal aesthetic subunits were reconstructed with DRTs, 10 subunits with a flap. Twenty-nine of 36 defects healed without any complications (80.5%). All reconstructed nasal tips/columella and the nasal dorsa healed without any complications. Region-specific complications were retraction of the ala rim (4/12; 33.3% of the patients with involvement of the nasal ala) and the formation of a fistula in the nasal sidewall (1/21; 4.8%). Region-specific complications of extranasal subunits were the development of an ectropium (2/3; 66.7% of the patients with involvement of the lower lid). CONCLUSIONS: DRTs can be helpful to reconstruct nasal defects. However, if the defect involves the aesthetic subunits nasal ala or the infraorbital region, different techniques should be preferred.

Hepatic Endothelial Notch Activation Protects against Liver Metastasis by Regulating Endothelial-Tumor Cell Adhesion Independent of Angiocrine Signaling.

The interaction of tumor cells with organ-specific endothelial cells (EC) is an important step during metastatic progression. Notch signaling in organ-specific niches has been implicated in mediating opposing effects on organotropic metastasis to the lungs and the liver, respectively. In this study, we scrutinized the role of endothelial Notch activation during liver metastasis. To target hepatic EC (HEC), a novel EC subtype-specific Cre driver mouse was generated. Clec4g-Cretg/wt mice were crossed to Rosa26N1ICD-IRES-GFP to enhance Notch signaling in HEC (NICDOE-HEC). In NICDOE-HEC mice, hepatic metastasis of malignant melanoma and colorectal carcinoma was significantly reduced. These mice revealed reduced liver growth and impaired metabolic zonation due to suppression of hepatic angiocrine Wnt signaling. Hepatic metastasis, however, was not controlled by angiocrine Wnt signaling, as deficiency of the Wnt cargo receptor Wls in HEC of WlsHEC-KO mice did not affect hepatic metastasis. In contrast, the hepatic microvasculature in NICDOE-HEC mice revealed a special form of sinusoidal capillarization, with effacement of endothelial zonation functionally paralleled by reduced tumor cell adhesion in vivo. Notably, expression of endothelial adhesion molecule ICAM1 by HEC was significantly reduced. Treatment with an anti-ICAM1 antibody significantly inhibited tumor cell adhesion to HEC in wild-type mice confirming that Notch controls hepatic metastasis via modulation of HEC adhesion molecules. As endothelial Notch activation in the lung has been shown to promote lung metastasis, tumor therapy will require approaches that target Notch in an organ-, cell type-, and context-specific manner. SIGNIFICANCE: Manipulation of Notch signaling in the endothelium has opposing, organ-specific effects on metastasis to the lung and the liver, demonstrating that this pathway should be targeted in a cell- and context-specific fashion.

GPR182 is a novel marker for sinusoidal endothelial differentiation with distinct GPCR signaling activity in vitro.

Endothelial cells (EC) along the vascular tree exhibit organ-specific angiodiversity. Compared to most other ECs, liver sinusoidal endothelial cells (LSEC) that constitute the organ-specific microvasculature of the liver are morphologically and functionally unique. Previously, we showed that transcription factor Gata4 acts as a master regulator controlling LSEC differentiation. Upon analysis of the molecular signature of LSEC, we identified GPR182 as a potential LSEC-specific orphan G-protein coupled receptor (GPCR). Here, we demonstrate that GPR182 is expressed by LSEC and by EC with sinusoidal differentiation in spleen, lymph node and bone marrow in healthy human tissues. In a tissue microarray analysis of human hepatocellular carcinoma (HCC) samples, endothelial GPR182 expression was significantly reduced in tumor samples compared to peritumoral liver tissue samples (p = 0.0105). Loss of endothelial GPR182 expression was also seen in fibrotic and cirrhotic liver tissues. In vitro, GPR182 differentially regulated canonical GPCR signaling pathways as shown using reporter luciferase assays in HEK293T cells. Whereas ERK and RhoA signaling were inhibited, CREB and Calcium signaling were activated by ectopic GPR182 overexpression. Our data demonstrate that GPR182 is an endothelial subtype-specific marker for human sinusoidal EC of the liver, spleen, lymph node and bone marrow. In addition, we provide evidence for GPR182-dependent downstream signaling via ERK and SRF pathways that may be involved in regulating endothelial subtype-specific sinusoidal differentiation and sinusoidal functions such as permeability.

Angiocrine Wnt signaling controls liver growth and metabolic maturation in mice.

Postnatal liver development is characterized by hepatocyte growth, proliferation, and functional maturation. Notably, canonical Wnt signaling in hepatocytes has been identified as an important regulator of final adult liver size and metabolic liver zonation. The cellular origin of Wnt ligands responsible for homeostatic liver/body weight ratio (LW/BW) remained unclear, which was also attributable to a lack of suitable endothelial Cre driver mice. To comprehensively analyze the effects of hepatic angiocrine Wnt signaling on liver development and metabolic functions, we used endothelial subtype-specific Stab2-Cre driver mice to delete Wls from hepatic endothelial cells (HECs). The resultant Stab2-Cretg/wt ;Wlsfl/fl (Wls-HECKO) mice were viable, but showed a significantly reduced LW/BW. Specifically, ablation of angiocrine Wnt signaling impaired metabolic zonation in the liver, as shown by loss of pericentral, ß-catenin-dependent target genes such as glutamine synthase (Glul), RhBg, Axin2, and cytochrome P450 2E1, as well as by extended expression of periportal genes such as arginase 1. Furthermore, endothelial subtype-specific expression of a c-terminally YFP-tagged Wls fusion protein in Wls-HECKO mice (Stab2-Cretg/wt ;Wlsfl/fl ;Rosa26:Wls-YFPfl/wt [Wls-rescue]) restored metabolic liver zonation. Interestingly, lipid metabolism was altered in Wls-HECKO mice exhibiting significantly reduced plasma cholesterol levels, while maintaining normal plasma triglyceride and blood glucose concentrations. On the contrary, zonal expression of Endomucin, LYVE1, and other markers of HEC heterogeneity were not altered in Wls-HECKO livers. CONCLUSION: Angiocrine Wnt signaling controls liver growth as well as development of metabolic liver zonation in mice, whereas intrahepatic HEC zonation is not affected. (Hepatology 2017).