Melanomas contain subsets of cancer stem-like cells with tumor-initiating capacity. The frequency of these cells in the tumor is still a topic of debate. We investigated the phenotypic plasticity of cancer cells grown as melanospheres to elucidate the influence of the microenvironment on some features of melanoma stem-like cells. Cells from surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in a stem cell medium and as adherent monolayer cultures in the presence of serum. Proliferation and viability were measured by cell counting and an acid phosphatase assay; surface marker expression was evaluated by flow cytometry, and the clonogenic potential of single cells was assessed by growth in soft agar. Patient-derived melanoma cells could be maintained in cell culture for more than 16 months when grown as melanospheres. In the presence of serum, melanospheres completely changed their growth characteristics and formed adherent monolayers. The transition from melanospheres to monolayers was accompanied by an apparent loss of clonogenic potential, an increased proliferation rate, and altered expressions of cell surface markers ABCB5, CD133, and CD49f. These changes, however, were reversible. Compared with adherent monolayer cultures, melanospheres are enriched in cells with clonogenic potential, reflecting the self-renewing capacity of cancer stem-like cells. This clonogenic potential can be lost and regained depending on the growth conditions. Our results demonstrate how easily melanoma cells change their function upon exposure to external stimuli and suggest that the frequency of melanoma stem-like cells strongly depends on the microenvironment.

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7 2/2 and Cxcl4 2/2 knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7 2/2 mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4 2/2 mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

Background: Cancer cell invasion requires integrins for adhesion/de-adhesion and MMPs for focalized proteolysis. Results: MMP-2 is up-regulated in invasive colorectal tumors and degrades ␤1 integrins. Conclusion: Shedding of the I-like domain from ␤1 integrins results in decreased adhesion and enhanced cell motility. Significance: MMP-2 amplifies the motility of cancer cells, not only degrading extracellular matrix but also reducing the ␤1 integrin expression.

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Background: PDI regulates cytoskeleton reorganization by the thiol-disulfide exchange in ␤-actin. Results: PDI directly binds to Cys 374 of ␤-actin during cell adhesion and spreading. Conclusion: Interaction of PDI with ␤-actin is induced by integrin-mediated cell adhesion and promotes cytoskeleton reorganization. Significance: PDI is a new regulator of the intramolecular disulfide-thiol rearrangement of ␤-actin in response to ␣IIb␤3 integrin engagement.

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of ␣ IIb ␤ 3 in blood platelets. The aim of this study was to explain whether Ero1␣ can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1␣ can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and ␣ IIb ␤ 3 , as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1␣, anti-␣ IIb ␤ 3 immunoprecipitates showed the presence of several Ero1␣-positive bands that corresponded to the complexes ␣ IIb ␤ 3 -PDIEro1␣, PDI-Ero1␣, and Ero1␣-Ero1␣ dimers. It binds more efficiently to the activated ␣ IIb ␤ 3 conformer, and its interaction is inhibited by RGD peptides. Ero1␣ appears to be involved in the regulation of ␣ IIb ␤ 3 receptor activity because of the following: (a) blocking the cell surface Ero1␣ by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1␣ increases ␣ IIb ␤ 3 receptor activity, as indicated by increased binding of fibrinogen.

Background Clinical complexity is increasingly prevalent among patients with atrial fibrillation (AF). The ‘Atrial fibrillation Better Care’ (ABC) pathway approach has been proposed to streamline a more holistic and integrated approach to AF care; however, there are limited data on its usefulness among clinically complex patients. We aim to determine the impact of ABC pathway in a contemporary cohort of clinically complex AF patients. Methods From the ESC-EHRA EORP-AF General Long-Term Registry, we analysed clinically complex AF patients, defined as the presence of frailty, multimorbidity and/or polypharmacy. A K-medoids cluster analysis was performed to identify different groups of clinical complexity. The impact of an ABC-adherent approach on major outcomes was analysed through Cox-regression analyses and delay of event (DoE) analyses. Results Among 9966 AF patients included, 8289 (83.1%) were clinically complex. Adherence to the ABC pathway in the clinically complex group reduced the risk of all-cause death (adjusted HR [aHR]: 0.72, 95%CI 0.58–0.91), major adverse cardiovascular events (MACEs; aHR: 0.68, 95%CI 0.52–0.87) and composite outcome (aHR: 0.70, 95%CI: 0.58–0.85). Adherence to the ABC pathway was associated with a significant reduction in the risk of death (aHR: 0.74, 95%CI 0.56–0.98) and composite outcome (aHR: 0.76, 95%CI 0.60–0.96) also in the high-complexity cluster; similar trends were observed for MACEs. In DoE analyses, an ABC-adherent approach resulted in significant gains in event-free survival for all the outcomes investigated in clinically complex patients. Based on absolute risk reduction at 1 year of follow-up, the number needed to treat for ABC pathway adherence was 24 for all-cause death, 31 for MACEs and 20 for the composite outcome. Conclusions An ABC-adherent approach reduces the risk of major outcomes in clinically complex AF patients. Ensuring adherence to the ABC pathway is essential to improve clinical outcomes among clinically complex AF patients.