Alexandra Demory

Wnt2 acts as a cell type–specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross‐stimulating the VEGF pathway

The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC‐specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse‐transcription polymerase chain reaction (RT‐PCR). As confirmed via quantitative RT‐PCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P < 0.05). The Wnt inhibitor secreted frizzled‐related protein 1 (sFRP1) (P < 0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP1 and WIF (P < 0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of β‐catenin. GST (glutathione transferase) pull‐down and co‐immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor‐2 (VEGFR‐2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down‐regulated VEGFR‐2 messenger RNA and protein. Wnt2 siRNA knock‐down confirmed Wnt2 specificity of VEGFR‐2 regulation in HSECs. Conclusion: Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects. (HEPATOLOGY 2008;47:1018–1031.)

Liver sinusoidal endothelium: A microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1†

Liver sinusoidal endothelium (LSEC) is a prime example of organ‐specific microvascular differentiation and functions. Disease‐associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC‐specific molecular differentiation program in the rat we used a two‐sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42 hours. The LSEC signature consisted of 48 genes both down‐regulated in LMEC and in LSEC upon culture (fold change >7 in at least one comparison); quantitative reverse‐transcription polymerase chain reaction confirmation of these genes included numerous family members and signaling pathway‐associated molecules. The LSEC differentiation program comprised distinct sets of growth (Wnt2, Fzd4, 5, 9, Wls, vascular endothelial growth factors [VEGFR] 1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosis‐related (Stabilin‐1/2, Lyve1, and Ehd3) and cytoskeleton‐associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm‐1, Aatf) and up‐regulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. In addition, our analysis identified a novel 26‐kDa single‐pass transmembrane protein, liver endothelial differentiation‐associated protein (Leda)‐1, that was selectively expressed in all liver endothelial cells and preferentially localized to the abluminal cell surface. Upon forced overexpression in MDCK cells, Leda‐1 was sorted basolaterally to E‐cadherin‐positive adherens junctions, suggesting functional involvement in cell adhesion and polarity. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironment‐dependent LSEC‐specific differentiation program including the novel junctional molecule Leda‐1. HEPATOLOGY 2010

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis.

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A

Tumor‐associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE‐1+ M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow‐derived macrophages (BMDMs) by combining M2 mediators (IL‐4, glucocorticoids) and tumor‐conditioned media (TCM). Admixture of MS4A8A+ TCM/IL‐4/GC‐treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage‐like cells displayed a special gene signature. Admixture of these MS4A8A+ Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF‐κB activation and of the MAPK JNK and ERK did not show relevant effects, the p38α/β MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A+ TAM may serve as a novel cellular target for selective cancer therapy.

Wnt2 acts as an angiogenic growth factor for non-sinusoidal endothelial cells and inhibits expression of stanniocalcin-1

Recently, we have shown that Wnt2 is an autocrine growth and differentiation factor for hepatic sinusoidal endothelial cells. As Wnt signaling has become increasingly important in vascular development and cancer, we analyzed Wnt signaling in non-sinusoidal endothelial cells of different vascular origin (HUVEC, HUAEC, HMVEC-LLy). Upon screening the multiple components of the Wnt pathway, we demonstrated lack of Wnt2 expression, but presence of Frizzled-4, one of its receptors, in cultured non-sinusoidal endothelial cells. Treatment of these cells by exogenous Wnt2 induced endothelial proliferation and sprouting angiogenesis in vitro. Upon analysis of Wnt2 tissue expression as a basis for paracrine Wnt2 effects on non-sinusoidal endothelial cells in vivo, Wnt2 was found to be expressed in densely vascularized murine malignant tumors and in wound healing tissues in close proximity to CD31+ endothelial cells. By gene profiling, stanniocalcin-1 (STC1), a known regulator of angiogenesis, was identified as a target gene of Wnt2 signaling in HUVEC down-regulated by Wnt2 treatment. Tumor-conditioned media counter-acted Wnt2 and up-regulated STC1 expression in HUVEC. In conclusion, we provide evidence that Wnt2 acts as an angiogenic factor for non-sinusoidal endothelium in vitro and in vivo whose target genes undergo complex regulation by the tissue microenvironment.

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.