Targeted JAM-C deletion in germ cells by Spo11-controlled Cre recombinase

Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26(loxP) and EYFP-R26(loxP) reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11(Cre) mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11(Cre) transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells

The complex function of macrophages and their subpopulations in metabolic injury associated fatty liver disease

Non-alcoholic fatty liver disease (NAFLD), recently also defined as metabolic dysfunction-associated fatty liver disease (MAFLD), is a major health problem, as it affects ∼25% of the population globally and is a major cause of hepatic cirrhosis and thereby liver failure, as well as hepatocellular carcinoma. MALFD comprises a broad range of pathological conditions in the liver, including simple fat accumulation (steatosis) and the more progressive non-alcoholic steatohepatitis (NASH) that can lead to fibrosis development. Cells of innate immunity, and particularly macrophages, comprising the liver resident Kupffer cells and the recruited monocyte-derived macrophages, play complex roles in NASH-related inflammation and disease progression to fibrosis. Here, we discuss the recent developments with regards to the function of liver macrophage subpopulations during MAFLD development and progression

Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia

BackgroundCritical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities.Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization.Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues.MethodsBalb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 mu l physiological serum (SC, n:8) or 5x10(5) human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related.ResultsAdministration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown.ConclusionsOur results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.Institute of Health Carlos III, ISCIII; Junta de Andaluci

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (SFB/TR23)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (Project A2)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre.(Project Z5)Else Kroner-Fresenius-Stiftung (2013_A2

Activation of proteinase 3 contributes to Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance

Contains fulltext : 169891.pdf (publisher's version ) (Open Access)Activation of inflammatory pathways is known to accompany development of obesity-induced non-alcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive pro-inflammatory mediators IL-1beta and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In the present study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human alpha-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3 deficient mice showed strongly reduced levels of lipids in the liver after fed a high fat diet. Moreover, these mice were resistant to high fat diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1(-/-) mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with alpha-1 antitrypsin during the last 10 days of a 16 week high fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential

Epithelial calcineurin controls microbiota-dependent intestinal tumor development.

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell-intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ZE814/5-1 (S.Z.), BA2863/5-1 (J.F.B.) and CH279/5-1 (T.C.), the European Research Council (ERC) starting grant 336528 (S.Z.), a Postdoctoral Fellowship Award from the Crohn's and Colitis Foundation of America (S.Z.), the European Commission (Marie Curie International Reintegration grant 256363; S.Z.), the DFG Excellence Cluster 'Inflammation at Interfaces' (S.Z. and J.F.B.), the DFG Excellence Cluster 'Center for Regenerative Therapies' (S.Z.); the US National Institutes of Health grants DK044319 (R.S.B.), DK051362 (R.S.B.), DK053056 (R.S.B.) and DK088199 (R.S.B.), the Harvard Digestive Diseases Center (HDDC) grant DK0034854 (R.S.B.), and the AIRC grant IG-14233 (M.E.B.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nm.407

Biological responses to spider silk - antibiotic fusion protein

The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous implants were studied to access the in vivo biological response to 6mer + hepcidin, which were compared with controls of silk alone (6mer), polylactic–glycolic acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer + hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28603/2006, Chimera project (No. PTDC/EBB-EBI/109093/2008), Proteo-Light (No. PTDC/FIS/68517/2006), NIH (Grant No. P41 EB002520) Tissue Engineering Resource Center; and the NIH (Grant Nos EB003210 and DE017207).Tissue Engineering Resource Center - Bolsa No. P41 EB002520, bolsa Nos EB003210 and DE017207European - Projeto EXPERTISSUES (No. NMP3- CT-2004-500283

Antimicrobial functionalized genetically engineered spider silk

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of b-sheets to lock in structures via physical interactions without the need for chemical crosslinking. These new functional silk proteins were assessed for antimicrobial activity against Gram e Escherichia coli and Gram þ Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutoramento (SFRH/BD/28603/2006); Chimera project (PTDC/EBB-EBI/109093/2008); NIH and Tissue Engineering Resource Center EB003210, P41 EB002520, DE017207