(Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the 38 histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory 39 lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical 40 and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation 41 of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. 42 Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent 43 protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain 44 inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-45 intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10 , Ccl2 , and 46 Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic 47 disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and 48 repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell 49 recruitment and, in consequence, MS lesion formation. 50

Reduced shear stress resulting from disturbed blood flow can impair endothelial integrity and drive the development of vascular inflammatory lesions. Metalloproteinases of the ADAM family have been implicated in the regulation of cell survival and inflammatory responses. Here we investigate the mechanism and function of ADAM15 upregulation in primary flow cultured endothelial cells. Transcriptomic analysis indicated that within the ADAM family ADAM15 mRNA is most prominently upregulated (4-fold) when endothelial cells are exposed to physiologic shear stress. This induction was confirmed in venous, arterial and microvascular endothelial cells and is associated with increased presence of ADAM15 protein in the cell lysates (5.6-fold) and on the surface (3.1-fold). The ADAM15 promoter contains several consensus sites for the transcription factor KLF2 which is also upregulated by shear stress. Induction of endothelial KLF2 by simvastatin treatment is associated with ADAM15 upregulation (1.8-fold) which is suppressed by counteracting simvastatin with geranylgeranyl pyrophosphate. KLF2 overexpression promotes ADAM15 expression (2.1-fold) under static conditions whereas KLF2 siRNA knockdown prevents ADAM15 induction by shear stress. Functionally, ADAM15 promotes survival of endothelial cells challenged by growth factor depletion or TNF stimulation as shown by ADAM15 shRNA knockdown (1.6-fold). Exposure to shear stress increases endothelial survival while additional knockdown of ADAM15 reduces survival (6.7-fold) under flow conditions. Thus, physiologic shear stress resulting from laminar flow promotes KLF2 induced ADAM15 expression which contributes to endothelial survival. The absence of ADAM15 at low shear stress or static conditions may therefore lead to increased endothelial damage and promote vascular inflammation.

BACKGROUND AND AIMS:Members of the family of a disintegrin and metalloproteinases (ADAMs) and their substrates have been previously shown to modulate the inflammatory response in cardiac diseases, but studies investigating the relevance of ADAM8 are still rare. Our aim is to provide evidence for the inflammatory dysregulation of ADAM8 in vascular diseases and its association with disease severity.METHODS:Western-type diet fed Apoe-/- and Ldlr-/- mice and artery ligation served as murine model for atherosclerosis and myocardial infarction, respectively. Human bypass grafts were used to study the association with coronary artery disease (CAD), with the simplified acute physiology score II (SAPS II) as a measure of postoperative organ dysfunction. Human primary vascular and blood cells were analyzed under basal and inflammatory conditions. mRNA levels were determined by RT-qPCR, ADAM8 protein levels by ELISA, immunohistochemistry or flow cytometry.RESULTS:ADAM8/ADAM8 expression is associated with atherosclerosis and CAD such as myocardial infarction in both mice and humans, especially in endothelial cells and leukocytes. We observed a strong in vivo and in vitro correlation of ADAM8 with the vascular disease markers VCAM-1, ICAM-1, TNF, IL-6, and CCL-2. Serum analysis revealed a significant elevation of soluble ADAM8 serum levels correlating with soluble CXCL16 levels and SAPS II.CONCLUSIONS:We demonstrate a general association of ADAM8 with cardiovascular diseases in mice and humans predominantly acting in endothelial cells and leukocytes. The correlation with postoperative organ dysfunctions in CAD patients highlights the value of further studies investigating the specific function of ADAM8 in cardiovascular diseases.

Background and Aims: Through shedding of various membrane molecules, including adhesion molecules and chemokines, A Disintegrin And Metalloproteinase 10 (ADAM10) could regulate endothelial functions e.g. permeability and leukocyte recruitment, critical processes in inflammatory diseases like atherosclerosis. Indeed, proteomic analysis on mouse endothelial cell sheddome revealed ±300 differentially regulated proteins upon ADAM10 inhibition, of which 10% appeared involved in permeability and leukocyte transmigration. Accordingly, in vitro inhibition of endothelial ADAM10 decreased neutrophil transmigration under flow.

Estrogens modulate the immune system and possess anti-inflammatory properties. In line, immune cells express a variety of estrogen receptors (ER) including ER-alpha and -beta. In the present study, we examined the influence of 17beta-estradiol (E2) serum concentrations on blood leukocyte composition and their ex vivo polarization/activation status by FACS analysis in sub-fertile human females under controlled ovarian stimulation (COS). Using a set of cell-type and polarization-specific markers, we demonstrate that increased 17ß-estradiol (E2) serum concentrations yield an overall increase in leukocytes, neutrophils and monocytes but decreased lymphocytes. There was a clear ratio shift towards an increase in M2 monocytes with a protective quality and an increase in T-helper cells compared to a decrease in cytotoxic T-cells. These data support experimental findings and clinical trials, i.e. related to multiple sclerosis and other autoimmune-related diseases, that have shown a down-regulation of CD8(+) T cells and up-regulation of T-regulatory cells. Further studies have to pinpoint to which extent the immune system/-responsiveness of otherwise healthy female patients is affected by medium-term systemic E2 variations.

Proteases of the disintegrin and metalloproteinase (ADAM) family mediate the proteolytic shedding of various surface molecules including cytokine precursors, adhesion molecules, growth factors, and receptors. Within the vasculature ADAM10 and ADAM17 regulate endothelial permeability, transendothelial leukocyte migration, and the adhesion of leukocytes and platelets. In vivo studies show that both proteases are implicated in several inflammatory pathologies, for example, edema formation, leukocyte infiltration, and thrombosis. However, both proteases also contribute to developmental and regenerative processes. Thus, although ADAMs can be regarded as valuable drug targets in many aspects, the danger of severe side effects is clearly visible. To circumvent these side effects, traditional inhibition approaches have to be improved to target ADAMs at the right time in the right place. Moreover, the inhibitors need to be more selective for the target protease and if possible also for the substrate. Antibodies recognizing the active conformation of ADAMs or small molecules blocking exosites of ADAM proteases may represent inhibitors with the desired selectivities.

Recently, the IMPRES study revealed that systemic imatinib improves exercise capacity in patients with advanced pulmonary arterial hypertension. Imatinib blocks the tyrosine kinase activity of the platelet-derived growth factor (PDGF)-receptor (PDGFR), acts antiproliferative and relaxes pulmonary arteries. However so far, the relaxant effects of imatinib on pulmonary veins (PVs) and on the postcapillary resistance are unknown, although pulmonary hypertension (PH) due to left heart disease (LHD) is most common and primarily affects PVs. Next, it is unknown whether activation of PDGFR alters the pulmonary venous tone. Due to the reported adverse effects of systemic imatinib, we evaluated the effects of nebulized imatinib on the postcapillary resistance.

Alveolar leukocyte recruitment is a hallmark of acute lung inflammation and involves transmigration of leukocytes through endothelial and epithelial layers. The disintegrin and metalloproteinase (ADAM) 8 is expressed on human isolated leukocytic cells and can be further upregulated on cultured endothelial and epithelial cells by proinflammatory cytokines. By shRNA-mediated knockdown we show that leukocytic ADAM8 is required on monocytic THP-1 cells for chemokine-induced chemotaxis as well as transendothelial and transepithelial migration. Furthermore, ADAM8 promotes αL-integrin upregulation and THP-1 cell adhesion to endothelial cells. On endothelial cells ADAM8 enhances transendothelial migration and increases cytokine-induced permeability. On epithelial cells the protease facilitates migration in a wound closure assay but does not affect transepithelial leukocyte migration. Blood leukocytes and bone marrow-derived macrophages (BMDM) from ADAM8-deficient mice show suppressed chemotactic response. Intranasal application of LPS to mice is accompanied with ADAM8 upregulation in the lung. In this model of acute lung inflammation ADAM8-deficient mice are protected against leukocyte infiltration. Finally, transfer experiments of BMDM in mice indicate that ADAM8 exerts a promigratory function predominantly on leukocytes. Our study provides in vitro and in vivo evidence that ADAM8 on leukocytes holds a proinflammatory function in acute lung inflammation by promoting alveolar leukocyte recruitment.

The CXC-chemokine receptor 6 (CXCR6) is a class A GTP-binding protein-coupled receptor (GPCRs) that mediates adhesion of leukocytes by interacting with the transmembrane cell surface-expressed chemokine ligand 16 (CXCL16), and also regulates leukocyte migration by interacting with the soluble shed variant of CXCL16. In contrast to virtually all other chemokine receptors with chemotactic activity, CXCR6 carries a DRF motif instead of the typical DRY motif as a key element in receptor activation and G protein coupling. In this work, modeling analyses revealed that the phenylalanine F3.51 in CXCR6 might have impact on intramolecular interactions including hydrogen bonds by this possibly changing receptor function. Initial investigations with embryonic kidney HEK293 cells and further studies with monocytic THP-1 cells showed that mutation of DRF into DRY does not influence ligand binding, receptor internalization, receptor recycling, and protein kinase B (AKT) signaling. Adhesion was slightly decreased in a time-dependent manner. However, CXCL16-induced calcium signaling and migration were increased. Vice versa, when the DRY motif of the related receptor CX3CR1 was mutated into DRF the migratory response towards CX3CL1 was diminished, indicating that the presence of a DRF motif generally impairs chemotaxis in chemokine receptors. Transmembrane and soluble CXCL16 play divergent roles in homeostasis, inflammation, and cancer, which can be beneficial or detrimental. Therefore, the DRF motif of CXCR6 may display a receptor adaptation allowing adhesion and cell retention by transmembrane CXCL16 but reducing the chemotactic response to soluble CXCL16. This adaptation may avoid permanent or uncontrolled recruitment of inflammatory cells as well as cancer metastasis.

Flow conditions critically regulate endothelial cell functions in the vasculature. Reduced shear stress resulting from disturbed blood flow can drive the development of vascular inflammatory lesions. On endothelial cells, the transmembrane chemokine CX3CL1/fractalkine promotes vascular inflammation by functioning as a surface-expressed adhesion molecule and by becoming released as soluble chemoattractant for monocytic cells expressing the receptor CX3CR1. Here, we report that endothelial cells from human artery, vein, or microvasculature constitutively express CX3CL1 when cultured under static conditions. Stimulation with TNFα under static or very low shear stress conditions strongly upregulates CX3CL1 expression. By contrast, CX3CL1 induction is profoundly reduced when cells are exposed to higher shear stress. When endothelial cells were grown and subsequently stimulated with TNFα under low shear stress, strong adhesion of monocytic THP-1 cells to endothelial cells was observed. This adhesion was in part mediated by transmembrane CX3CL1 as demonstrated with a neutralizing antibody. By contrast, no CX3CL1-dependent adhesion to stimulated endothelium was observed at high shear stress. Thus, during early stages of vascular inflammation, low shear stress typically seen at atherosclerosis-prone regions promotes the induction of endothelial CX3CL1 and monocytic cell recruitment, whereas physiological shear stress counteracts this inflammatory activation of endothelial cells.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity.

Proteases of the disintegrin and metalloproteinase (ADAM) family mediate the proteolytic shedding of various surface molecules including cytokine precursors, adhesion molecules, growth factors, and receptors. Within the vasculature ADAM10 and ADAM17 regulate endothelial permeability, transendothelial leukocyte migration, and the adhesion of leukocytes and platelets. In vivo studies show that both proteases are implicated in several inflammatory pathologies, for example, edema formation, leukocyte infiltration, and thrombosis. However, both proteases also contribute to developmental and regenerative processes. Thus, although ADAMs can be regarded as valuable drug targets in many aspects, the danger of severe side effects is clearly visible. To circumvent these side effects, traditional inhibition approaches have to be improved to target ADAMs at the right time in the right place. Moreover, the inhibitors need to be more selective for the target protease and if possible also for the substrate. Antibodies recognizing the active conformation of ADAMs or small molecules blocking exosites of ADAM proteases may represent inhibitors with the desired selectivities.

The metalloproteinase ADAM8 promotes leukocyte recruitment in vitro and in acute 1 lung inflammation 2 3 Daniela Dreymueller, Jessica Pruessmeyer, Julian Schumacher, Sandra Fellendorf, Franz 4 Martin Hess, Anke Seifert, Aaron Babendreyer, Jörg W. Bartsch, Andreas Ludwig 5 6 1 Institute of Pharmacology and Toxicology, RWTH Aachen University, Aachen, Germany 7 2 Department of Neurosurgery, Philipps University Marburg, University Hospital Marburg, 8 Marburg, Germany 9 10 11 Running head: ADAM8 promotes leukocyte recruitment 12 13 Corresponding author: 14 Andreas Ludwig 15 Institute of Pharmacology and Toxicology, RWTH Aachen University 16 Wendlingweg 2, 52074 Aachen, Germany; Phone: +492418035771; Fax: +492418082433 17 Email: aludwig@ukaachen.de 18 19 Articles in PresS. Am J Physiol Lung Cell Mol Physiol (June 8, 2017). doi:10.1152/ajplung.00444.2016

A disintegrin and metalloproteinase 8 (ADAM8) has been identified as a signature gene associated with moderate and severe asthma. Studies in mice have demonstrated that the severity of asthma can be reduced by either transgenic knock-out or by antibodies blocking ADAM8 function, highlighting ADAM8 as potential drug target for asthma therapy. Here, we examined the therapeutic effect of an ADAM8 inhibitor peptide (BK-1361) that specifically blocks cellular ADAM8 activity in ovalbumin-sensitized and challenged Balb/c mice. We found that BK-1361 (25 μg/g body weight) attenuated airway responsiveness to methacholine stimulation by up to 42%, concomitantly reduced tissue remodeling by 50% and decreased inflammatory cells (e.g. eosinophils down by 54%)/inflammatory factors (e.g. sCD23 down by 50%)/T H 2 cytokines (e.g. IL-5 down by 70%)/ADAM8-positive eosinophils (down by 60%) in the lung. We further verified that BK-1361 specifically targets ADAM8 in vivo as the peptide caused significantly reduced levels of soluble CD23 in wild-type but not in ADAM8-deficient mice. These findings suggest that BK-1361 blocks ADAM8-dependent asthma effects in vivo by inhibiting infiltration of eosinophils and T H 2 lymphocytes, thus leading to reduction of T H 2-mediated inflammation, tissue remodeling and bronchial hyperresponsiveness. Taken together, pharmacological ADAM8 inhibition appears as promising novel therapeutic strategy for the treatment of asthma.

Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35–55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells.SIGNIFICANCE STATEMENTWe identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.

Proteases of the disintegrin and metalloproteinase (ADAM) family mediate the proteolytic shedding of various surface molecules including cytokine precursors, adhesion molecules, growth factors, and receptors. Within the vasculature ADAM10 and ADAM17 regulate endothelial permeability, transendothelial leukocyte migration, and the adhesion of leukocytes and platelets. In vivo studies show that both proteases are implicated in several inflammatory pathologies, for example, edema formation, leukocyte infiltration, and thrombosis. However, both proteases also contribute to developmental and regenerative processes. Thus, although ADAMs can be regarded as valuable drug targets in many aspects, the danger of severe side effects is clearly visible. To circumvent these side effects, traditional inhibition approaches have to be improved to target ADAMs at the right time in the right place. Moreover, the inhibitors need to be more selective for the target protease and if possible also for the substrate. Antibodies recognizing the active conformation of ADAMs or small molecules blocking exosites of ADAM proteases may represent inhibitors with the desired selectivities.

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics.

The chemokine CXCL 16 and its receptor CXCR 6 have been linked to the pathogenesis of acute and chronic cardiovascular disease. However, data on the clinical significance of CXCL 16 in patients undergoing cardiac surgery with acute myocardial ischemia/reperfusion (I/R) are still lacking. Therefore, we determined CXCL 16 in the serum of cardiac surgery patients and investigated its kinetics and association with the extent of organ dysfunction. 48 patients underwent conventional cardiac surgery with myocardial I/R and the use of cardiopulmonary bypass ( CPB ) were consecutively enrolled in the present study. We investigated the peri‐ and post‐operative profile of CXCL 16. Clinical relevant data were assessed and documented throughout the entire observation period. To identify the influence of myocardial I/R and CPB on CXCL 16 release data were compared to those received from patients that underwent off‐pump procedure. Pre‐operative serum CXCL 16 levels were comparable to those obtained from healthy volunteers (1174 ± 55.64 pg/ml versus 1225 ± 70.94). However, CXCL 16 levels significantly increased during surgery (1174 ± 55.64 versus 1442 ± 75.42 pg/ml; P = 0.0057) and reached maximum levels 6 hrs after termination of surgery (1174 ± 55.64 versus 1648 ± 74.71 pg/ml; P < 0.001). We revealed a positive correlation between the intraoperative serum levels of CXCL 16 and the extent of organ dysfunction ( r 2 = 0.356; P = 0.031). Patients with high CXCL 16 release showed an increased extent of organ dysfunction compared to patients with low CXCL 16 release. Our study shows that CXCL 16 is released into the circulation as a result of cardiac surgery and that high post‐operative CXCL 16 levels are associated with an increased severity of post‐operative organ dysfunctions.

By mediating proteolytic shedding on the cell surface the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of growth factors, cytokines and adhesion molecules. We here report that stimulation of lung epithelial A549 tumor cells with phorbol-12-myristate-13-acetate (PMA) leads to the downregulation of the surface expressed mature form of ADAM17 without affecting ADAM10 expression. This reduction could not be sufficiently explained by metalloproteinase-mediated degradation, dynamin-mediated internalization or microdomain redistribution of ADAM17. Instead, surface downregulation of ADAM17 was correlated with the presence of its mature form in exosomes. Exosomal ADAM17 release was also observed in monocytic and primary endothelial cells where it could be induced by stimulation with lipopolysaccharide. Antibody-mediated surface labelling of ADAM17 revealed that at least part of exosomal ADAM17 was oriented with the metalloproteinase domain outside and had been expressed on the cell surface. Suppression of iRHOM2-mediated ADAM17 maturation prevented surface expression and exosomal release of ADAM17. Further, deletion of the protease's C-terminus or cell treatment with a calcium chelator diminished exosomal release as well as surface downregulation of ADAM17, underlining that both processes are closely associated. Co-incubation of ADAM17 containing exosomes with cells expressing the ADAM17 substrates TGFα or amphiregulin lead to increased shedding of both substrates. This was prevented when exosomes were prepared from cells with shRNA-mediated ADAM17 knockdown. These data indicate that cell stimulation can downregulate expression of mature ADAM17 from the cell surface and induce release of exosomal ADAM17, which can then distribute and contribute to substrate shedding on more distant cells.