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.

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.

Stabilin-1 is expressed in human breast cancer and supports tumor growth in mammary adenocarcinoma mouse model.

Stabilin-1 is a multifunctional scavenger receptor expressed on alternatively-activated macrophages. Stabilin-1 mediates phagocytosis of "unwanted-self" components, intracellular sorting, and endocytic clearance of extracellular ligands including SPARC that modulates breast cancer growth. The expression of stabilin-1 was found on tumor-associated macrophages (TAM) in mouse and human cancers including melanoma, lymphoma, glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphoma the expression and functional role of stabilin-1 in breast cancer was unknown. Here, we demonstrate that stabilin-1 is expressed on TAM in human breast cancer, and its expression is most pronounced on stage I disease. Using stabilin-1 knockout (ko) mice we show that stabilin-1 facilitates growth of mouse TS/A mammary adenocarcinoma. Endocytosis assay on stabilin-1 ko TAM demonstrated impaired clearance of stabilin-1 ligands including SPARC that was capable of inducing cell death in TS/A cells. Affymetrix microarray analysis on purified TAM and reporter assays in stabilin-1 expressing cell lines demonstrated no influence of stabilin-1 expression on intracellular signalling. Our results suggest stabilin-1 mediated silent clearance of extracellular tumor growth-inhibiting factors (e.g. SPARC) as a mechanism of stabilin-1 induced tumor growth. Silent clearance function of stabilin-1 makes it an attractive candidate for delivery of immunomodulatory anti-cancer therapeutic drugs to TAM.

Deficiency of liver sinusoidal scavenger receptors stabilin-1 and -2 in mice causes glomerulofibrotic nephropathy via impaired hepatic clearance of noxious blood factors.

Tissue homeostasis and remodeling are processes that involve high turnover of biological macromolecules. Many of the waste molecules that are by-products or degradation intermediates of biological macromolecule turnover enter the circulation and are subsequently cleared by liver sinusoidal endothelial cells (LSEC). Besides the mannose receptor, stabilin-1 and stabilin-2 are the major scavenger receptors expressed by LSEC. To more clearly elucidate the functions of stabilin-1 and -2, we have generated mice lacking stabilin-1, stabilin-2, or both stabilin-1 and -2 (Stab1­/­ Stab2­/­ mice). Mice lacking either stabilin-1 or stabilin-2 were phenotypically normal; however, Stab1­/­ Stab2­/­ mice exhibited premature mortality and developed severe glomerular fibrosis, while the liver showed only mild perisinusoidal fibrosis without dysfunction. Upon kidney transplantation into WT mice, progression of glomerular fibrosis was halted, indicating the presence of profibrotic factors in the circulation of Stab1­/­ Stab2­/­ mice. While plasma levels of known profibrotic cytokines were unaltered, clearance of the TGF-ß family member growth differentiation factor 15 (GDF-15) was markedly impaired in Stab1­/­ Stab2­/­ mice but not in either Stab1­/­ or Stab2­/­ mice, indicating that it is a common ligand of both stabilin-1 and stabilin-2. These data lead us to conclude that stabilin-1 and -2 together guarantee proper hepatic clearance of potentially noxious agents in the blood and maintain tissue homeostasis not only in the liver but also distant organs.

Identification of HLA class I dependent immunogenic peptides from clonotypic TCRbeta expressed in cutaneous T-cell lymphoma.

The clonotypic T-cell receptor (TCR) is a potential target antigen for specific immunotherapy of cutaneous T-cell lymphoma (CTCL). We identified T-cell epitopes from the rearranged TCR beta chain of the malignant T-cell population by the "reverse immunology" approach. Peptide-specific T-cell lines were generated against predicted epitopes and tested for the recognition of tumor cells and cells transfected with the full-length DNA coding for TCRV beta chain. Two peptides derived from the clonotypic TCRVbeta of a HLA-A2 positive patient could induce peptide-specific T cells from peripheral blood mononuclear cells of healthy donors and the patient as assessed by IFN-gamma ELISpot assay. Furthermore, the reactive CTLs efficiently recognized autologous Sézary tumor cells, as well as HLA-A2 positive 293 cells transfected with recombinant plasmid expressing the corresponding TCRVbeta29 protein. Similar results were obtained in a HLA-A3+ patient for TCRVbeta7-Jbeta2.7. In conclusion, our experiments show that the TCR beta chain harbors epitopes suitable as targets for specific vaccination which might be a promising approach for the specific immunotherapy of cutaneous T-cell lymphoma patients.