Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

EGF-Receptor against Amphiregulin (AREG) Influences Costimulatory Molecules on Monocytes and T Cells and Modulates T-Cell Responses.

Amphiregulin (AREG) is a ligand of the epidermal growth factor receptor (EGFR) and has been shown to regulate the phagocytosis-induced cell death of monocytes in peripheral blood. AREG-dependent apoptotic signaling engages factors of the intrinsic and extrinsic apoptotic pathway, such as BCL-2, BCL-XL, and death ligand/receptor CD95/CD95L. Here, we tested the hypothesis that AREG influences costimulatory monocyte functions, which are crucial for T-cell responses. We found a stronger expression of AREG and EGFR in monocytes compared to lymphocytes. As a novel function of AREG, we observed reduced T-cell proliferation following polyclonal T-cell stimulation with OKT3. This reduction of proliferation occurred in the presence of monocytes as well as in their absence, monocyte signaling being replaced by crosslinking of OKT3. Increasing concentrations of AREG down-modulated the concentration of costimulatory B7 molecules (CD80/CD86) and HLA-DR on monocytes. In proliferation assays, CD28 expression on T cells was down-modulated on the application of OKT3 but unaltered by AREG. LcK activation, following OKT3-stimulation, was reduced in T cells that had been coincubated with AREG. The effects of AREG on T-cell phenotypes were also present when monocytes were depleted and OKT3 was crosslinked. The rearranged expression of immunological synapse proteins was accompanied by an alteration of T-cell polarization. Although the proportion of regulatory T cells was not shifted by AREG, IL-17-expressing T cells were significantly enhanced, with a bias toward TH1-polarization. Taken together, these results suggest that AREG acts as an immunoregulatory molecule at the interface between antigen-presenting cells and T cells.

Analysis of flow-induced transcriptional response and cell alignment of different sources of endothelial cells used in vascular tissue engineering.

Endothelialization of tissue-engineered vascular grafts has proven crucial for implant functionality and thus clinical outcome, however, the choice of endothelial cells (ECs) is often driven by availability rather than by the type of vessel to be replaced. In this work we studied the response to flow of different human ECs with the aim of examining whether their response in vitro is dictated by their original in vivo conditions. Arterial, venous, and microvascular ECs were cultured under shear stress (SS) of 0, 0.3, 3, 1, 10, and 30 dyne/cm2 for 24 h. Regulation of flow-induced marker KLF2 was similar across the different ECs. Upregulation of anti-thrombotic markers, TM and TPA, was mainly seen at higher SS. Cell elongation and alignment was observed for the different ECs at 10 and 30 dyne/cm2 while at lower SS cells maintained a random orientation. Downregulation of pro-inflammatory factors SELE, IL8, and VCAM1 and up-regulation of anti-oxidant markers NQO1 and HO1 was present even at SS for which cell alignment was not observed. Our results evidenced similarities in the response to flow among the different ECs, suggesting that the maintenance of the resting state in vitro is not dictated by the SS typical of the tissue of origin and that absence of flow-induced cell orientation does not necessarily correlate with a pro-inflammatory state of the ECs. These results support the use of ECs from easily accessible sources for in vitro vascular tissue engineering independently from the target vessel.

Mechanical activation of lung epithelial cells through the ion channel Piezo1 activates the metalloproteinases ADAM10 and ADAM17 and promotes growth factor and adhesion molecule release.

In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

Influence of Aerosolization on Endothelial Cells for Efficient Cell Deposition in Biohybrid and Regenerative Applications.

The endothelialization of gas exchange membranes can increase the hemocompatibility of extracorporeal membrane oxygenators and thus become a long-term lung replacement option. Cell seeding on large or uneven surfaces of oxygenator membranes is challenging, with cell aerosolization being a possible solution. In this study, we evaluated the endothelial cell aerosolization for biohybrid lung application. A Vivostat® system was used for the aerosolization of human umbilical vein endothelial cells with non-sprayed cells serving as a control. The general suitability was evaluated using various flow velocities, substrate distances and cell concentrations. Cells were analyzed for survival, apoptosis and necrosis levels. In addition, aerosolized and non-sprayed cells were cultured either static or under flow conditions in a dynamic microfluidic model. Evaluation included immunocytochemistry and gene expression via quantitative PCR. Cell survival for all tested parameters was higher than 90%. No increase in apoptosis and necrosis levels was seen 24 h after aerosolization. Spraying did not influence the ability of the endothelial cells to form a confluent cell layer and withstand shear stresses in a dynamic microfluidic model. Immunocytochemistry revealed typical expression of CD31 and von Willebrand factor with cobble-stone cell morphology. No change in shear stress-induced factors after aerosolization was reported by quantitative PCR analysis. With this study, we have shown the feasibility of endothelial cell aerosolization with no significant changes in cell behavior. Thus, this technique could be used for efficient the endothelialization of gas exchange membranes in biohybrid lung applications.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Functional changes in long-term incubated rat precision-cut lung slices.

BACKGROUND: Respiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days. METHODS: Rat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation. RESULTS: Cultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin. CONCLUSIONS: Overall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

Differential maturation and chaperone dependence of the paralogous protein kinases DYRK1A and DYRK1B.

The HSP90/CDC37 chaperone system not only assists the maturation of many protein kinases but also maintains their structural integrity after folding. The interaction of mature kinases with the HSP90/CDC37 complex is governed by the conformational stability of the catalytic domain, while the initial folding of the protein kinase domain is mechanistically less well characterized. DYRK1A (Dual-specificity tyrosine (Y)-phosphorylation Regulated protein Kinase 1A) and DYRK1B are closely related protein kinases with discordant HSP90 client status. DYRK kinases stoichiometrically autophosphorylate on a tyrosine residue immediately after folding, which served us as a traceable marker of successful maturation. In the present study, we used bacterial expression systems to compare the capacity of autonomous maturation of DYRK1A and DYRK1B in the absence of eukaryotic cofactors or chaperones. Under these conditions, autophosphorylation of human DYRK1B was severely compromised when compared with DYRK1A or DYRK1B orthologs from zebrafish and Xenopus. Maturation of human DYRK1B could be restored by bacterial expression at lower temperatures, suggesting that folding was not absolutely dependent on eukaryotic chaperones. The differential folding properties of DYRK1A and DYRK1B were largely due to divergent sequences of the C-terminal lobes of the catalytic domain. Furthermore, the mature kinase domain of DYRK1B featured lower thermal stability than that of DYRK1A when exposed to heat challenge in vitro or in living cells. In summary, our study enhances the mechanistic understanding of the differential thermodynamic properties of two closely related protein kinases during initial folding and as mature kinases.

Inflammatory activation of surface molecule shedding by upregulation of the pseudoprotease iRhom2 in colon epithelial cells.

The metalloproteinase ADAM17 contributes to inflammatory and proliferative responses by shedding of cell-surface molecules. By this ADAM17 is implicated in inflammation, regeneration, and permeability regulation of epithelial cells in the colon. ADAM17 maturation and surface expression requires the adapter proteins iRhom1 or iRhom2. Here we report that expression of iRhom2 but not iRhom1 is upregulated in intestinal tissue of mice with acute colitis. Our analysis of public databases indicates elevated iRhom2 expression in mucosal tissue and epithelial cells from patients with inflammatory bowel disease (IBD). Consistently, expression of iRhom2 but not iRhom1 is upregulated in colon or intestinal epithelial cell lines after co-stimulation with tumor necrosis factor (TNF) and interferon gamma (IFNgamma). This upregulation can be reduced by inhibition of Janus kinases or transcription factors NF-kappaB or AP-1. Upregulation of iRhom2 can be mimicked by iRhom2 overexpression and is associated with enhanced maturation and surface expression of ADAM17 which then results in increased cleavage of transforming growth factor (TGF) alpha and junctional adhesion molecule (JAM)-A. Finally, the induction of these responses is suppressed by inhibition of iRhom2 transcription. Thus, inflammatory induction of iRhom2 may contribute to upregulated ADAM17-dependent mediator and adhesion molecule release in IBD. The development of iRhom2-dependent inhibitors may allow selective targeting of inflammatory ADAM17 activities.

Never Change a Flowing System? The Effects of Retrograde Flow on Isolated Perfused Lungs and Vessels.

Retrograde perfusion may occur during disease, surgery or extracorporeal circulation. While it is clear that endothelial cells sense and respond to changes in blood flow, the consequences of retrograde perfusion are only poorly defined. Similar to shear stress or disturbed flow, retrograde perfusion might result in vasomotor responses, edema formation or inflammation in and around vessels. In this study we investigated in rats the effects of retrograde perfusion in isolated systemic vessels (IPV) and in pulmonary vessels of isolated perfused lungs (IPL). Anterograde and retrograde perfusion was performed for 480 min in IPV and for 180 min in the IPL. Perfusion pressure, cytokine levels in perfusate and bronchoalveolar lavage fluid (BALF), edema formation and mRNA expression were studied. In IPV, an increased perfusion pressure and initially also increased cytokine levels were observed during retrograde perfusion. In the IPL, increased edema formation occurred, while cytokine levels were not increased, though dilution of cytokines in BALF due to pulmonary edema cannot be excluded. In conclusion, effects of flow reversal were visible immediately after initiation of retrograde perfusion. Pulmonary edema formation was the only effect of the 3 h retrograde perfusion. Therefore, further research should focus on identification of possible long-term complications of flow reversal.

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro.

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1ß, TNF-α, IFN-γ, and TGF-ß. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation, migration and malignant signalling events in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of ß1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.

Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells.

Background: Endothelial function significantly depends on the proteolytic release of surface expressed signal molecules, their receptors and adhesion molecules via the metalloproteinase ADAM17. The pseudoproteases iRhom1 and 2 independently function as adapter proteins for ADAM17 and are essential for the maturation, trafficking, and activity regulation of ADAM17. Bioinformatic data confirmed that immune cells predominantly express iRhom2 while endothelial cells preferentially express iRhom1. Objective: Here, we investigate possible reasons for higher iRhom1 expression and potential inflammatory regulation of iRhom2 in endothelial cells and analyze the consequences for ADAM17 maturation and function. Methods: Primary endothelial cells were cultured in absence and presence of flow with and without inflammatory cytokines (TNFα and INFγ). Regulation of iRhoms was studied by qPCR, involved signaling pathways were studied with transcriptional inhibitors and consequences were analyzed by assessment of ADAM17 maturation, surface expression and cleavage of the ADAM17 substrate junctional adhesion molecule JAM-A. Results: Endothelial iRhom1 is profoundly upregulated by physiological shear stress. This is accompanied by a homeostatic phenotype driven by the transcription factor KLF2 which is, however, only partially responsible for regulation of iRhom1. By contrast, iRhom2 is most prominently upregulated by inflammatory cytokines. This correlates with an inflammatory phenotype driven by the transcription factors NFκB and AP-1 of which AP-1 is most relevant for iRhom2 regulation. Finally, shear stress exposure and inflammatory stimulation have independent and no synergistic effects on ADAM17 maturation, surface expression and JAM-A shedding. Conclusion: Conditions of shear stress and inflammation differentially upregulate iRhom1 and 2 in primary endothelial cells which then results in independent regulation of ADAM17.

The metalloproteinase ADAM15 is upregulated by shear stress and promotes survival of endothelial cells.

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.

Status update on iRhom and ADAM17: It's still complicated.

Several membrane-bound proteins with a single transmembrane domain are subjected to limited proteolysis at the cell surface. This cleavage leads to the release of their biologically active ectodomains, which can trigger different signalling pathways. In many cases, this ectodomain shedding is mediated by members of the family of a disintegrins and metalloproteinases (ADAMs). ADAM17 in particular is responsible for the cleavage of several proinflammatory mediators, growth factors, receptors and adhesion molecules. Due to its direct involvement in the release of these signalling molecules, ADAM17 can be positively and negatively involved in various physiological processes as well as in inflammatory, fibrotic and malignant pathologies. This central role of ADAM17 in a variety of processes requires strict multi-level regulation, including phosphorylation, various conformational changes and endogenous inhibitors. Recent research has shown that an early, crucial control mechanism is interaction with certain adapter proteins identified as iRhom1 and iRhom2, which are pseudoproteases of the rhomboid superfamily. Thus, iRhoms have also a decisive influence on physiological and pathophysiological signalling processes regulated by ADAM17. Their characteristic gene expression profiles, the specific consequences of gene knockouts and finally the occurrence of disease-associated mutations suggest that iRhom1 and iRhom2 undergo different gene regulation in order to fulfil their function in different cell types and are therefore only partially redundant. Therefore, there is not only interest in ADAM17, but also in iRhoms as therapeutic targets. However, to exploit the therapeutic potential, the regulation of ADAM17 activity and in particular its interaction with iRhoms must be well understood.

Elevated expression of the metalloproteinase ADAM8 associates with vascular diseases in mice and humans.

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.

Argon reduces the pulmonary vascular tone in rats and humans by GABA-receptor activation.

Argon exerts neuroprotection. Thus, it might improve patients' neurological outcome after cerebral disorders or cardiopulmonary resuscitation. However, limited data are available concerning its effect on pulmonary vessel and airways. We used rat isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of rats and humans to assess this topic. IPL: Airway and perfusion parameters, oedema formation and the pulmonary capillary pressure (Pcap) were measured and the precapillary and postcapillary resistance (Rpost) was calculated. In IPLs and PCLS, the pulmonary vessel tone was enhanced with ET-1 or remained unchanged. IPLs were ventilated and PCLS were gassed with argon-mixture or room-air. IPL: Argon reduced the ET-1-induced increase of Pcap, Rpost and oedema formation (p < 0.05). PCLS (rat): Argon relaxed naïve pulmonary arteries (PAs) (p < 0.05). PCLS (rat/human): Argon attenuated the ET-1-induced contraction in PAs (p < 0.05). Inhibition of GABAB-receptors abolished argon-induced relaxation (p < 0.05) in naïve or ET-1-pre-contracted PAs; whereas inhibition of GABAA-receptors only affected ET-1-pre-contracted PAs (p < 0.01). GABAA/B-receptor agonists attenuated ET-1-induced contraction in PAs and baclofen (GABAB-agonist) even in pulmonary veins (p < 0.001). PLCS (rat): Argon did not affect the airways. Finally, argon decreases the pulmonary vessel tone by activation of GABA-receptors. Hence, argon might be applicable in patients with pulmonary hypertension and right ventricular failure.

The metalloproteinase ADAM8 promotes leukocyte recruitment in vitro and in acute lung inflammation.

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.