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Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of beta-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that beta-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.

This study proposes an alternative technique to prevent heat degradation induced by classic procedures of bioactive compound extraction, comparing classical maceration/decoction in hot water of polyphenols from Mango (Mangifera indica L.) (MI) with ultrasound-assisted extraction (UAE) in a water solution of β-cyclodextrin (β-CD) at room temperature and testing their biological activity on TNFα-induced endothelial dysfunction. Both extracts counteracted TNFα effects on EAhy926 cells, down-modulating interleukin-6, interleukin-8, cyclooxygenase-2 and intracellular adhesion molecule-1, while increasing endothelial nitric oxide synthase levels. β-CD extract showed higher efficacy in improving endothelial function. These effects were abolished after pre-treatment with the oestrogen receptor inhibitor ICI1182,780. Moreover, the β-CD extract induced Akt activation and completely abolished the TNFα-induced p38MAPK phosphorylation. UAE and β-CD encapsulation provide an efficient extraction protocol that increases polyphenol bioavailability. Polyphenols from MI play a protective role on endothelial cells and may be further considered as oestrogen-like molecules with vascular protective properties.

Purpose One of the limitations emerged with both synthetic and degradable vascular grafts is the lack of endothelialization after implantation that is known to be the main reason leading to unfavourable outcomes. It emerges the need to find new strategies to promote a rapid endothelialization of the scaffold. Pleiotrophin is a growth/differentiation cytokine for various cell type. We here evaluated the effect of Pleiotrophin on endothelial cells (EC), monocytes and macrophages that have been shown as key cells promoting neovascularization. Material/methods EA.hy926 endothelial cells, THP-1 monocytes and PMA-differentiated macrophages were treated with Pleiotrophin (10 and 100 ng/ml). VEGF, Flk-1, Nrp-1, COX-2, ICAM-1 and TGFβ expression were detected by Western Blot, IL-10, MCP-1 and TNFα levels by ELISA. Chemotaxis was performed in Boyden chambers. Wound healing was performed by scratch wound assay. Results Pleiotrophin induces in EC the expression of VEGF and its receptors Flk-1 and Nrp-1 and improves the migratory capacity. In THP-1 monocytes, Pleiotrophin induces the expression of VEGF and its receptor Nrp-1 and decreases the levels of COX-2 and TNFα. In PMA-differentiated macrophages COX-2 expression was significantly reduced by Pleiotrophin, while IL-10 and TGFβ were increased. Conclusions Pleiotrophin acts as an angiogenesis ‘driver’ by promoting the creation of a pro-angiogenic environment, a migratory behaviour in EC and a pro-regenerative alternative phenotype in macrophages. Our results suggest that Pleiotrophin might be considered for vascular prosthesis engineering.

Nowadays, engineering studies are characterized by high mobility of students, lecturers and workforce and by the dynamics of multinational companies where “classes” or “students’ teams” composed of persons with different competencies and backgrounds, working together in projects to solve complex problems. Such an environment will become increasingly relevant in multinational universities and companies, and it has brought a number of challenges to existing e-learning technologies. COOPER is an ongoing project that focuses on developing and testing such a collaborative and project-centred leaning environment. This paper proposes a COOPER framework and shows its approaches to address the various research challenges. This work is partially supported by EU/IST FP6 STREP project COOPER (contract number IST-2005-027073).

MicroRNAs have been proposed as novel regulators of vascular inflammation and dysfunction. This study aimed to evaluate the role of miR-149 in regulating the expression of key molecules associated with TNFα-induced endothelial activation. miR-149 was selected by in silico analysis and microRNA target prediction. Endothelial dysfunction was induced by TNFα treatment in Eahy926 endothelial cells and HUVEC. miR-149 level was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR). Metalloproteinase-9 (MMP-9) was measured by zymography, Inducible Nitric Oxide Synthase (iNOS) by immunoblotting, Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by ELISA. miR-149 regulatory effect was evaluated by gain-of-function technique upon miR-149 mimics transfection. TNFα down-modulated miR-149 level in Eahy926 and HUVEC. This effect was significantly abolished in Eahy926 by treatment with p38MAPK inhibitor. miR-149 mimic transfection counteracted the TNFα-induced expression of MMP-9, iNOS and IL-6. No effect was detected on IL-8 expression. Our results suggest that miR-149 represents an important new regulator of endothelial function through negative regulation of molecules associated with TNFα-induced endothelial dysfunction.

Background The ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3′UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression. Results The ACVR1/Alk-2 3′UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3′UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3′UTR sequence thereby modulating its expression. Conclusion In this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3′UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3′UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.

Aims Congestive heart failure (CHF) can be thought of as a state of chronic immune activation. Polymorphonuclear neutrophil (PMN) apoptosis is one of the mechanisms responsible for the resolution of inflammation. A reduced PMN apoptotic rate in CHF patients may generate a persistent inflammatory response and hence mediate tissue damage in this group of patients. We aimed to measure levels of spontaneous apoptosis of circulating PMNs in CHF patients and in controls, and to examine whether NYHA class, left ventricular ejection fraction (LV-EF), and laboratory parameters of inflammation, endothelial damage, and of liver and renal function, could predict the rate of PMN apoptosis in CHF patients. Methods and results A total of 29 CHF patients and 26 controls were studied. Propidium iodide and flow cytometry were used to assess PMN apoptosis. Delay in PMN apoptosis was expressed as percentage (expressed as median, first and third quartiles) of surviving PMNs in the study subjects. We found an increased percentage of surviving PMNs [38(27.1–47.1)] in CHF patients compared with controls [19.4 (15.8–25.2)] (P < 0.05). The PMN survival rate in the CHF group was correlated to NYHA class, and plasma levels of C-reactive protein and alkaline phosphatase, while it was inversely correlated to LV-EF and protein levels. A positive relationship between PMN survival and increased ex vivo endothelial apoptosis was found. Conclusion Increased PMN lifespan in patients with worsening CHF could be used as a novel measurement of tissue and endothelial damage in this group of patients.

Background The ACVR1 gene encodes a type I receptor for bone morphogenetic proteins (BMPs). Mutations in the ACVR1 gene are associated with Fibrodysplasia Ossificans Progressiva (FOP), a rare and extremely disabling disorder characterized by congenital malformation of the great toes and progressive heterotopic endochondral ossification in muscles and other non-skeletal tissues. Several aspects of FOP pathophysiology are still poorly understood, including mechanisms regulating ACVR1 expression. This work aimed to identify regulatory elements that control ACVR1 gene transcription. Methods and results We first characterized the structure and composition of human ACVR1 gene transcripts by identifying the transcription start site, and then characterized a 2.9 kb upstream region. This region showed strong activating activity when tested by reporter gene assays in transfected cells. We identified specific elements within the 2.9 kb region that are important for transcription factor binding using deletion constructs, co-transfection experiments with plasmids expressing selected transcription factors, site-directed mutagenesis of consensus binding-site sequences, and by protein/DNA binding assays. We also characterized a GC-rich minimal promoter region containing binding sites for the Sp1 transcription factor. Conclusions Our results showed that several transcription factors such as Egr-1, Egr-2, ZBTB7A/LRF, and Hey1, regulate the ACVR1 promoter by binding to the -762/-308 region, which is essential to confer maximal transcriptional activity. The Sp1 transcription factor acts at the most proximal promoter segment upstream of the transcription start site. We observed significant differences in different cell types suggesting tissue specificity of transcriptional regulation. These findings provide novel insights into the molecular mechanisms that regulate expression of the ACVR1 gene and that could be targets of new strategies for future therapeutic treatments.