Targeting of Scavenger Receptors Stabilin-1 and Stabilin-2 Ameliorates Atherosclerosis by a Plasma Proteome Switch Mediating Monocyte/Macrophage Suppression.

BACKGROUND: Scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are preferentially expressed by liver sinusoidal endothelial cells. They mediate the clearance of circulating plasma molecules controlling distant organ homeostasis. Studies suggest that Stab1 and Stab2 may affect atherosclerosis. Although subsets of tissue macrophages also express Stab1, hematopoietic Stab1 deficiency does not modulate atherogenesis. Here, we comprehensively studied how targeting Stab1 and Stab2 affects atherosclerosis. METHODS: ApoE-KO mice were interbred with Stab1-KO and Stab2-KO mice and fed a Western diet. For antibody targeting, Ldlr-KO mice were also used. Unbiased plasma proteomics were performed and independently confirmed. Ligand binding studies comprised glutathione-S-transferase-pulldown and endocytosis assays. Plasma proteome effects on monocytes were studied by single-cell RNA sequencing in vivo, and by gene expression analyses of Stabilin ligand-stimulated and plasma-stimulated bone marrow-derived monocytes/macrophages in vitro. RESULTS: Spontaneous and Western diet-associated atherogenesis was significantly reduced in ApoE-Stab1-KO and ApoE-Stab2-KO mice. Similarly, inhibition of Stab1 or Stab2 by monoclonal antibodies significantly reduced Western diet-associated atherosclerosis in ApoE-KO and Ldlr-KO mice. Although neither plasma lipid levels nor circulating immune cell numbers were decisively altered, plasma proteomics revealed a switch in the plasma proteome, consisting of 231 dysregulated proteins comparing wildtype with Stab1/2-single and Stab1/2-double KO, and of 41 proteins comparing ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO. Among this broad spectrum of common, but also disparate scavenger receptor ligand candidates, periostin, reelin, and TGFBi (transforming growth factor, ß-induced), known to modulate atherosclerosis, were independently confirmed as novel circulating ligands of Stab1/2. Single-cell RNA sequencing of circulating myeloid cells of ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO mice showed transcriptomic alterations in patrolling (Ccr2-/Cx3cr1++/Ly6Clo) and inflammatory (Ccr2+/Cx3cr1+/Ly6Chi) monocytes, including downregulation of proatherogenic transcription factor Egr1. In wildtype bone marrow-derived monocytes/macrophages, ligand exposure alone did not alter Egr1 expression in vitro. However, exposure to plasma from ApoE-Stab1-KO and ApoE-Stab2-KO mice showed a reverted proatherogenic macrophage activation compared with ApoE-KO plasma, including downregulation of Egr1 in vitro. CONCLUSIONS: Inhibition of Stab1/Stab2 mediates an anti-inflammatory switch in the plasma proteome, including direct Stabilin ligands. The altered plasma proteome suppresses both patrolling and inflammatory monocytes and, thus, systemically protects against atherogenesis. Altogether, anti-Stab1- and anti-Stab2-targeted therapies provide a novel approach for the future treatment of atherosclerosis.

Association of Differentially Altered Liver Fibrosis with Deposition of TGFBi in Stabilin-Deficient Mice.

Liver sinusoidal endothelial cells (LSECs) control clearance of Transforming growth factor, beta-induced, 68kDa (TGFBi) and Periostin (POSTN) through scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2). Stabilin inhibition can ameliorate atherosclerosis in mouse models, while Stabilin-double-knockout leads to glomerulofibrosis. Fibrotic organ damage may pose a limiting factor in future anti-Stabilin therapies. While Stab1-deficient (Stab1-/-) mice were shown to exhibit higher liver fibrosis levels upon challenges, fibrosis susceptibility has not been studied in Stab2-deficient (Stab2-/-) mice. Wildtype (WT), Stab1-/- and Stab2-/- mice were fed experimental diets, and local ligand abundance, hepatic fibrosis, and ligand plasma levels were measured. Hepatic fibrosis was increased in both Stab1-/- and Stab2-/- at baseline. A pro-fibrotic short Methionine-Choline-deficient (MCD) diet induced slightly increased liver fibrosis in Stab1-/- and Stab2-/- mice. A Choline-deficient L-amino acid-defined (CDAA) diet induced liver fibrosis of similar distribution and extent in all genotypes (WT, Stab1-/- and Stab2-/-). A hepatic abundance of Stabilin ligand TGFBi correlated very highly with liver fibrosis levels. In contrast, plasma levels of TGFBi were increased only in Stab2-/- mice after the CDAA diet but not the MCD diet, indicating the differential effects of these diets. Here we show that a single Stabilin deficiency of either Stab1 or Stab2 induces mildly increased collagen depositions under homeostatic conditions. Upon experimental dietary challenge, the local abundance of Stabilin ligand TGFBi was differentially altered in Stabilin-deficient mice, indicating differentially affected LSEC scavenger functions. Since anti-Stabilin-directed therapies are in clinical evaluation for the treatment of diseases, these findings bear relevance to treatment with novel anti-Stabilin agents.

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).

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.

Phenotypical and biochemical characterization of murine psoriasiform and fibrotic skin disease models in Stabilin-deficient mice.

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

Deficiency for scavenger receptors Stabilin-1 and Stabilin-2 leads to age-dependent renal and hepatic depositions of fasciclin domain proteins TGFBI and Periostin in mice.

Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two major scavenger receptors of liver sinusoidal endothelial cells that mediate removal of diverse molecules from the plasma. Double-knockout mice (Stab-DKO) develop impaired kidney function and a decreased lifespan, while single Stabilin deficiency or therapeutic inhibition ameliorates atherosclerosis and Stab1-inhibition is subject of clinical trials in immuno-oncology. Although POSTN and TFGBI have recently been described as novel Stabilin ligands, the dynamics and functional implications of these ligands have not been comprehensively studied. Immunofluorescence, Western Blotting and Simple Western™ as well as in situ hybridization (RNAScope™) and qRT-PCR were used to analyze transcription levels and tissue distribution of POSTN and TGFBI in Stab-KO mice. Stab-POSTN-Triple deficient mice were generated to assess kidney and liver fibrosis and function in young and aged mice. TGFBI and POSTN protein accumulated in liver tissue in Stab-DKO mice and age-dependent in glomeruli of Stabilin-deficient mice despite unchanged transcriptional levels. Stab-POSTN-Triple KO mice showed glomerulofibrosis and a reduced lifespan comparable to Stab-DKO mice. However, alterations of the glomerular diameter and vascular density were partially normalized in Stab-POSTN-Triple KO. TGFBI and POSTN are Stabilin-ligands that are deposited in an age-dependent manner in the kidneys and liver due to insufficient scavenging in the liver. Functionally, POSTN might partially contribute to the observed renal phenotype in Stab-DKO mice. This study provides details on downstream effects how Stabilin dysfunction affects organ function on a molecular and functional level.

Vascular Remodeling Is a Crucial Event in the Early Phase of Hepatocarcinogenesis in Rodent Models for Liver Tumorigenesis.

The investigation of hepatocarcinogenesis is a major field of interest in oncology research and rodent models are commonly used to unravel the pathophysiology of onset and progression of hepatocellular carcinoma. HCC is a highly vascularized tumor and vascular remodeling is one of the hallmarks of tumor progression. To date, only a few detailed data exist about the vasculature and vascular remodeling in rodent models used for hepatocarcinogenesis. In this study, the vasculature of HCC and the preneoplastic foci of alteration (FCA) of different mouse models with varying genetic backgrounds were comprehensively characterized by using immunohistochemistry (CD31, Collagen IV, αSMA, Desmin and LYVE1) and RNA in situ hybridization (VEGF-A). Computational image analysis was performed to evaluate selected parameters including microvessel density, pericyte coverage, vessel size, intratumoral vessel distribution and architecture using the Aperio ImageScope and Definiens software programs. HCC presented with a significantly lower number of vessels, but larger vessel size and increased coverage, leading to a higher degree of maturation, whereas FCA lesions presented with a higher microvessel density and a higher amount of smaller but more immature vessels. Our results clearly demonstrate that vascular remodeling is present and crucial in early stages of experimental hepatocarcinogenesis. In addition, our detailed characterization provides a strong basis for further angiogenesis studies in these experimental models.

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 Notch signaling controls insulin transport in muscle.

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

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.

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.