DEL-1 promotes macrophage efferocytosis and clearance of inflammation.

Resolution of inflammation is essential for tissue homeostasis and represents a promising approach to inflammatory disorders. Here we found that developmental endothelial locus-1 (DEL-1), a secreted protein that inhibits leukocyte-endothelial adhesion and inflammation initiation, also functions as a non-redundant downstream effector in inflammation clearance. In human and mouse periodontitis, waning of inflammation was correlated with DEL-1 upregulation, whereas resolution of experimental periodontitis failed in DEL-1 deficiency. This concept was mechanistically substantiated in acute monosodium-urate-crystal-induced inflammation, where the pro-resolution function of DEL-1 was attributed to effective apoptotic neutrophil clearance (efferocytosis). DEL-1-mediated efferocytosis induced liver X receptor-dependent macrophage reprogramming to a pro-resolving phenotype and was required for optimal production of at least certain specific pro-resolving mediators. Experiments in transgenic mice with cell-specific overexpression of DEL-1 linked its anti-leukocyte-recruitment action to endothelial cell-derived DEL-1 and its efferocytic/pro-resolving action to macrophage-derived DEL-1. Thus, the compartmentalized expression of DEL-1 facilitates distinct homeostatic functions in an appropriate context that can be harnessed therapeutically.

Glycolysis is integral to histamine-induced endothelial hyperpermeability.

Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis.

DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1.

Leukocytes are rapidly recruited to sites of inflammation via interactions with the vascular endothelium. The steroid hormone dehydroepiandrosterone (DHEA) exerts anti-inflammatory properties; however, the underlying mechanisms are poorly understood. In this study, we show that an anti-inflammatory mechanism of DHEA involves the regulation of developmental endothelial locus 1 (DEL-1) expression. DEL-1 is a secreted homeostatic factor that inhibits ß2-integrin-dependent leukocyte adhesion, and the subsequent leukocyte recruitment and its expression is downregulated upon inflammation. Similarly, DHEA inhibited leukocyte adhesion to the endothelium in venules of the inflamed mouse cremaster muscle. Importantly, in a model of lung inflammation, DHEA limited neutrophil recruitment in a DEL-1-dependent manner. Mechanistically, DHEA counteracted the inhibitory effect of inflammation on DEL-1 expression. Indeed, whereas TNF reduced DEL-1 expression and secretion in endothelial cells by diminishing C/EBPß binding to the DEL-1 gene promoter, DHEA counteracted the inhibitory effect of TNF via activation of tropomyosin receptor kinase A (TRKA) and downstream PI3K/AKT signaling that restored C/EBPß binding to the DEL-1 promoter. In conclusion, DHEA restrains neutrophil recruitment by reversing inflammation-induced downregulation of DEL-1 expression. Therefore, the anti-inflammatory DHEA/DEL-1 axis could be harnessed therapeutically in the context of inflammatory diseases.

Hypothalamo-pituitary and immune-dependent adrenal regulation during systemic inflammation.

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immune-adrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPA-axis activation mediated by LPS were found in Mx1(Cre+)-MyD88(fl/fl) mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Impact of Hey2 and COUP-TFII on genes involved in arteriovenous differentiation in primary human arterial and venous endothelial cells.

Arteries and veins show marked differences in their anatomy, physiology and genetic expression pattern. In this study, we analyzed impact of overexpression or downregulation of arterial marker gene Hey2 and venous marker gene COUP-TFII in human venous and arterial endothelial cells on genes involved in arteriovenous differentiation. Lentiviral overexpression of venous marker gene COUP-TFII in arterial endothelial cells led to downregulation of NICD4, arterial marker gene Hey2 and EphrinB2. Downregulation of Hey2 could be mediated by direct binding of COUP-TFII to Hey2 promoter as shown by ChIP, EMSA and promoter analysis. Downregulation of Hey2 by shRNA causes downregulation of EphrinB2 expression. Overexpression of arterial marker Hey2 in venous endothelial cells did not change expression pattern of COUP-TFII. Downregulation of venous marker gene COUP-TFII in venous endothelial cells resulted in upregulation of VEGF-A, Dll4 and EphrinB2 expression. Our data support an important role of Hey2 and COUP-TFII in arteriovenous differentiation of human endothelial cells.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

53BP1 Deficiency Promotes Pathological Neovascularization in Proliferative Retinopathy.

The replication stress inflicted on retinal endothelial cells (ECs) in the context of hypoxia-induced pathological neovascularization during proliferative retinopathy is linked with activation of the deoxyribonucleic acid (DNA) repair response. Here, we studied the effect of deficiency of the DNA damage response adaptor 53BP1, which is an antagonist of homologous recombination (HR), in the context of proliferative retinopathy. In the model of retinopathy of prematurity (ROP), 53BP1-deficient mice displayed increased hypoxia-driven pathological neovascularization and tuft formation, accompanied by increased EC proliferation and reduced EC apoptosis, as compared with 53BP1-sufficient mice. In contrast, physiological retina angiogenesis was not affected by 53BP1 deficiency. Knockdown of 53BP1 in ECs in vitro also resulted in enhanced proliferation and reduced apoptosis of the cells under hypoxic conditions. Additionally, upon 53BP1 knockdown, ECs displayed increased HR rate in hypoxia. Consistently, treatment with an HR inhibitor reversed the hyper-proliferative angiogenic phenotype associated with 53BP1 deficiency in ROP. Thus, by unleashing HR, 53BP1 deletion increases pathological EC proliferation and neovascularization in the context of ROP. Our data shed light to a previously unknown interaction between the DNA repair response and pathological neovascularization in the retina.

Hes3 expression in the adult mouse brain is regulated during demyelination and remyelination.

Hes3 is a component of the STAT3-Ser/Hes3 Signaling Axis controlling the growth and survival of neural stem cells and other plastic cells. Pharmacological activation of this pathway promotes neuronal rescue and behavioral recovery in models of ischemic stroke and Parkinson's disease. Here we provide initial observations implicating Hes3 in the cuprizone model of demyelination and remyelination. We focus on the subpial motor cortex of mice because we detected high Hes3 expression. This area is of interest as it is impacted both in human demyelinating diseases and in the cuprizone model. We report that Hes3 expression is reduced at peak demyelination and is partially restored within 1 week after cuprizone withdrawal. This raises the possibility of Hes3 involvement in demyelination/remyelination that may warrant additional research. Supporting a possible role of Hes3 in the maintenance of oligodendrocyte markers, a Hes3 null mouse strain shows lower levels of myelin basic protein in undamaged adult mice, compared to wild-type controls. We also present a novel method for culturing the established oligodendrocyte progenitor cell line oli-neu in a manner that maintains Hes3 expression as well as its self-renewal and differentiation potential, offering an experimental tool to study Hes3. Based upon this approach, we identify a Janus kinase inhibitor and dbcAMP as powerful inducers of Hes3 gene expression. We provide a new biomarker and cell culture method that may be of interest in demyelination/remyelination research.

Milk Fat Globule-Epidermal Growth Factor 8 (MFG-E8) Is a Novel Anti-inflammatory Factor in Rheumatoid Arthritis in Mice and Humans.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells and is implicated in the pathogenesis of autoimmune and inflammatory diseases. Because MFG-E8 also controls bone metabolism, we investigated its role in rheumatoid arthritis (RA), focusing on inflammation and joint destruction. The regulation of MFG-E8 by inflammation was assessed in vitro using osteoblasts, in arthritic mice and in patients with RA. K/BxN serum transfer arthritis (STA) was applied to MFG-E8 knock-out mice to assess its role in the pathogenesis of arthritis. Stimulation of osteoblasts with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α downregulated the expression of MFG-E8 by 30% to 35%. MFG-E8-deficient osteoblasts responded to LPS with a stronger production of pro-inflammatory cytokines. In vivo, MFG-E8 mRNA levels were 52% lower in the paws of collagen-induced arthritic (CIA) mice and 24% to 42% lower in the serum of arthritic mice using two different arthritis models (CIA and STA). Similarly, patients with RA (n = 93) had lower serum concentrations of MFG-E8 (-17%) compared with healthy controls (n = 140). In a subgroup of patients who had a moderate to high disease activity (n = 21), serum concentrations of MFG-E8 rose after complete or partial remission had been achieved (+67%). Finally, MFG-E8-deficient mice subjected to STA exhibited a stronger disease burden, an increased number of neutrophils in the joints, and a more extensive local and systemic bone loss. This was accompanied by an increased activation of osteoclasts and a suppression of osteoblast function in MFG-E8-deficient mice. Thus, MFG-E8 is a protective factor in the pathogenesis of RA and subsequent bone loss. Whether MFG-E8 qualifies as a novel biomarker or therapeutic target for the treatment of RA is worth addressing in further studies.

Brief Report: Endothelial-Specific X-Box Binding Protein 1 Deficiency Limits Tumor Necrosis Factor-Induced Leukocyte Recruitment and Vasculitis.

OBJECTIVE: Endothelial cell activation by tumor necrosis factor (TNF) and associated leukocyte infiltration are hallmarks of vasculitis. The aim of this study was to investigate the potential role of the cellular stress-associated endothelial X-box binding protein 1 (XBP-1) transcription factor in TNF-induced endothelial cell inflammation and vasculitis. METHODS: Mice with an endothelial cell-specific XBP-1 deficiency were used in a modified local Shwartzman reaction (LSR) model of TNF-induced small vessel vasculitis. To address the contribution of XBP-1 to the TNF-mediated inflammatory response in endothelial cells, we examined the activation of XBP-1 expression by TNF as well as the effect of XBP-1 knockdown in endothelial cells on TNF-induced signaling, proinflammatory gene expression, and leukocyte-endothelial cell adhesion. RESULTS: The active spliced form of XBP-1 in endothelial cells was triggered by TNF. In addition, endothelial XBP-1 contributed to the sustained TNF-triggered NF-κB-dependent transcriptional activation of proinflammatory molecules, which was associated with leukocyte-endothelial cell adhesion. In the LSR model, endothelial cell-specific XBP-1-deficient mice displayed significantly less vascular damage, accompanied by reduced perivascular neutrophil infiltration, as compared with wild-type mice. CONCLUSION: Endothelial XBP-1 is activated by TNF and regulates leukocyte-endothelial cell adhesion in vitro as well as neutrophil infiltration and vascular damage in murine vasculitis.

Loss of milk fat globule-epidermal growth factor 8 (MFG-E8) in mice leads to low bone mass and accelerates ovariectomy-associated bone loss by increasing osteoclastogenesis.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein that controls the engulfment of apoptotic cells and exerts inflammation-modulatory effects. Recently, it has been implicated in osteoclastogenesis and the pathogenesis of inflammatory periodontal bone loss, but its role in physiological bone homeostasis is still not well defined. Here, we evaluated the influence of MFG-E8 on osteoblasts and osteoclasts and its impact on bone remodeling in healthy and ovariectomized mice as a model for post-menopausal osteoporosis. Total and trabecular bone mineral densities at the lumbar spine in 6-week-old MFG-E8 KO mice were reduced by 11% (p < 0.05) and 17% (p < 0.01), respectively, as compared to wild-type (WT) mice. Accordingly, serum levels of the bone formation marker P1NP were decreased by 37% (p < 0.01) in MFG-E8 KO mice as were the ex vivo mineralization capacity and expression of osteoblast genes (Runx2, alkaline phosphatase, osteocalcin) in MFG-E8 KO osteoblasts. In contrast, serum bone resorption markers CTX1 and TRAP5b were increased by 30% and 60% (p < 0.05), respectively, in MFG-E8 KO mice. Furthermore, bone marrow macrophages from MFG-E8-KO mice differentiated more effectively into osteoclasts, as compared to WT cells. MFG-E8-deficient osteoclasts displayed increased bone resorption ex vivo, which could be reversed by the presence of recombinant MFG-E8. To determine the significance of the enhanced osteoclastogenesis in MFG-E8 KO mice, we performed an ovariectomy, which is associated with bone loss due to increased osteoclast activity. Indeed, MFG-E8 KO mice lost 12% more trabecular bone density than WT mice after ovariectomy. Together, these data indicate that MFG-E8 controls steady-state and pathological bone turnover and may therefore represent a new target gene in the treatment of bone diseases.

Endothelial-specific deficiency of Junctional Adhesion Molecule-C promotes vessel normalisation in proliferative retinopathy.

In proliferative retinopathies, like proliferative diabetic retinopathy and retinopathy of prematurity (ROP), the hypoxia response is sustained by the failure of the retina to revascularise its ischaemic areas. Non-resolving retina ischaemia/hypoxia results in upregulation of pro-angiogenic factors and pathologic neovascularisation with ectopic, fragile neovessels. Promoting revascularisation of the retinal avascular area could interfere with this vicious cycle and lead to vessel normalisation. Here, we examined the function of endothelial junctional adhesion molecule-C (JAM-C) in the context of ROP. Endothelial-specific JAM-C-deficient (EC-JAM-C KO) mice and littermate JAM-C-proficient (EC-JAM-C WT) mice were subjected to the ROP model. An increase in total retinal vascularisation was found at p17 owing to endothelial JAM-C deficiency, which was the result of enhanced revascularisation and vessel normalisation, thereby leading to significantly reduced avascular area in EC-JAM-C KO mice. In contrast, pathologic neovessel formation was not affected by endothelial JAM-C deficiency. Consistent with improved vessel normalisation, tip cell formation at the interface between vascular and avascular area was higher in EC-JAM-C KO mice, as compared to their littermate controls. Consistently, JAM-C inactivation in endothelial cells resulted in increased spreading on fibronectin and enhanced sprouting in vitro in a manner dependent on ß1-integrin and on the activation of the small GTPase RAP1. Together, endothelial deletion of JAM-C promoted endothelial cell sprouting, and consequently vessel normalisation and revascularisation of the hypoxic retina without altering pathologic neovascularisation. Thus, targeting endothelial JAM-C may provide a novel therapeutic strategy for promoting revascularisation and vessel normalisation in the treatment of proliferative retinopathies.

Role of the endothelial-derived endogenous anti-inflammatory factor Del-1 in inflammation-mediated adrenal gland dysfunction.

Inflammation in the course of systemic inflammatory response syndrome (SIRS) or sepsis often results in dysregulation of the hypothalamic-pituitary-adrenal axis; however, the underlying mechanisms are not well understood. The adrenal gland is highly vascularized; thus, we hypothesized that endothelial dysfunction may actively participate in inflammation-related adrenal insufficiency. To address this hypothesis, we used the properties of developmental endothelial locus-1 (Del-1), which is an endothelial-derived anti-inflammatory factor that antagonizes integrin-dependent leukocyte adhesion. Here we identified that Del-1 is expressed in the adrenal gland and that its expression was down-regulated upon SIRS induction by systemic lipopolysaccharide administration. Furthermore, we observed increased leukocyte accumulation, inflammation, and higher apoptosis in the adrenal glands of Del-1-deficient mice as compared with wild-type mice. Strikingly, Del-1 deficiency was also associated with reduced corticosterone and ACTH levels 24 hours after lipopolysaccharide administration. Together, these data suggest that Del-1 may act as a gatekeeper of adrenal gland inflammation and may regulate the integrity of the hypothalamic-pituitary-adrenal axis stress response, thereby modulating adrenal (dys)function in the course of SIRS.

Caveolin-1 opens endothelial cell junctions by targeting catenins.

AIMS: A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown. METHODS AND RESULTS: Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with ß- and γ-catenin. Consequently, the association of ß- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1. CONCLUSION: Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.

Hematogenous dissemination of melanoma is a life-threatening complication of this malignant tumor. Here, we identified junctional adhesion molecule-C (JAM-C) as a novel player in melanoma metastasis to the lung. JAM-C expression was identified in human and murine melanoma cell lines, in human malignant melanoma, as well as in metastatic melanoma including melanoma lung metastasis. JAM-C expressed on both murine B16 melanoma cells as well as on endothelial cells promoted the transendothelial migration of the melanoma cells. We generated mice with inactivation of JAM-C. JAM-C(-/-) mice as well as endothelial-specific JAM-C-deficient mice displayed significantly decreased B16 melanoma cell metastasis to the lung, whereas treatment of mice with soluble JAM-C prevented melanoma lung metastasis. Together, JAM-C represents a novel therapeutic target for melanoma metastasis.