38 results on '"Azcutia V"'
Search Results
2. Pro-inflammatory effects of early non-enzymatic glycated proteins in human mesothelial cells vary with cell donorʼs age
- Author
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Rodríguez-Mañas, L, Sánchez-Rodríguez, C, Vallejo, S, El-Assar, M, Peiró, C, Azcutia, V, Matesanz, N, Sánchez-Ferrer, C F, and Nevado, J
- Published
- 2006
- Full Text
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3. 345 Endothelial dysfunction by genetic deletion or inhibition of the G prteoin-coupled receptor Mas — a new target to improve endothelial function
- Author
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Walther, T., Vallejo, S., Sanchez-Ferrer, C., Heringer-Walther, S., Azcutia, V., Rodriguez-Manas, L., Gembardt, F., and Peiro, C.
- Subjects
G proteins ,MEMBRANE proteins - Abstract
An abstract of the study "Endothelial dysfunction by genetic deletion or inhibition of the G protein-coupled receptor Mas--a new target to improve endothelial function," by T. Walther and colleagues, is presented.
- Published
- 2006
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4. The human CD47 checkpoint is targeted by an immunosuppressive Aedes aegypti salivary factor to enhance arboviral skin infectivity.
- Author
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Marin-Lopez A, Huck JD, Esterly AT, Azcutia V, Rosen C, Garcia-Milian R, Sefik E, Vidal-Pedrola G, Raduwan H, Chen TY, Arora G, Halene S, Shaw AC, Palm NW, Flavell RA, Parkos CA, Thangamani S, Ring AM, and Fikrig E
- Subjects
- Animals, Humans, Female, Insect Proteins immunology, Zika Virus Infection immunology, Salivary Proteins and Peptides immunology, Mosquito Vectors immunology, Mosquito Vectors virology, CD47 Antigen, Aedes immunology, Aedes virology, Skin immunology, Skin virology, Zika Virus immunology, Zika Virus physiology
- Abstract
The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.
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- 2024
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5. Inhibition of Soluble Stem Cell Factor Promotes Intestinal Mucosal Repair.
- Author
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Garcia-Hernandez V, Raya-Sandino A, Azcutia V, Miranda J, Kelm M, Flemming S, Birkl D, Quiros M, Brazil JC, Parkos CA, and Nusrat A
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- Animals, Mice, Dextran Sulfate, Disease Models, Animal, Inflammation drug therapy, Inflammation pathology, Intestinal Mucosa pathology, Stem Cell Factor antagonists & inhibitors, Stem Cell Factor metabolism, Colitis drug therapy, Colitis pathology, Colitis, Ulcerative drug therapy, Colitis, Ulcerative pathology, Inflammatory Bowel Diseases drug therapy, Inflammatory Bowel Diseases pathology
- Abstract
Background: Incidences of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are escalating worldwide and can be considered a global public health problem. Given that the gold standard approach to IBD therapeutics focuses on reducing the severity of symptoms, there is an urgent unmet need to develop alternative therapies that halt not only inflammatory processes but also promote mucosal repair. Previous studies have identified increased stem cell factor (SCF) expression in inflamed intestinal mucosal tissues. However, the role that SCF plays in mediating intestinal inflammation and repair has not been explored., Methods: Changes in the expression of SCF were evaluated in the colonic tissue of healthy mice and during dextran sodium sulfate (DSS)-induced colitis. Furthermore, mucosal wound healing and colitis severity were analyzed in mice subjected to either mechanical biopsy or DSS treatment, respectively, following intestinal epithelial cell-specific deletion of SCF or anti-SCF antibody administration., Results: We report robust expression of SCF by intestinal epithelial cells during intestinal homeostasis with a switch to immune cell-produced SCF during colitis. Data from mice with intestinal epithelial cell-specific deletion of SCF highlight the importance of immune cell-produced SCF in driving the pathogenesis of colitis. Importantly, antibody-mediated neutralization of total SCF or the specific SCF248 isoform decreased immune cell infiltration and enhanced mucosal wound repair following biopsy-induced colonic injury or DSS-induced colitis., Conclusions: These data demonstrate that SCF functions as a pro-inflammatory mediator in mucosal tissues and that specific neutralization of SCF248 could be a viable therapeutic option to reduce intestinal inflammation and promote mucosal wound repair in individuals with IBD., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2023
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6. Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function.
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Azcutia V, Kelm M, Fink D, Cummings RD, Nusrat A, Parkos CA, and Brazil JC
- Subjects
- Intestinal Mucosa, Neuraminidase, Polysaccharides, CD11b Antigen immunology, CD18 Antigens immunology, Neutrophils physiology, Transendothelial and Transepithelial Migration
- Abstract
Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.
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- 2023
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7. Distinct stimulus-dependent neutrophil dynamics revealed by real-time imaging of intestinal mucosa after acute injury.
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Azcutia V, Kelm M, Kim S, Luissint AC, Flemming S, Abernathy-Close L, Young VB, Nusrat A, Miller MJ, and Parkos CA
- Abstract
Clinical symptoms in many inflammatory diseases of the intestine are directly related to neutrophil (PMN) migration across colonic mucosa and into the intestinal lumen, yet in-vivo studies detailing this process are lacking. Using real-time intravital microscopy and a new distal colon loop model, we report distinct PMN migratory dynamics in response to several models of acute colonic injury. PMNs exhibited rapid swarming responses after mechanically induced intestinal wounds. Similar numbers of PMNs infiltrated colonic mucosa after wounding in germ-free mice, suggesting microbiota-independent mechanisms. By contrast, acute mucosal injury secondary to either a treatment of mice with dextran sodium sulfate or an IL-10 receptor blockade model of colitis resulted in lamina propria infiltration with PMNs that were largely immotile. Biopsy wounding of colonic mucosa in DSS-treated mice did not result in enhanced PMN swarming however, intraluminal application of the neutrophil chemoattractant LTB
4 under such conditions resulted in enhanced transepithelial migration of PMNs. Analyses of PMNs that had migrated into the colonic lumen revealed that the majority of PMNs were directly recruited from the circulation and not from the immotile pool in the mucosa. Decreased PMN motility parallels upregulation of the receptor CXCR4 and apoptosis. Similarly, increased expression of CXCR4 on human PMNs was observed in colonic biopsies from people with active ulcerative colitis. This new approach adds an important tool to investigate mechanisms regulating PMN migration across mucosa within the distal intestine and will provide new insights for developing future anti-inflammatory and pro-repair therapies., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)- Published
- 2022
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8. Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo.
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Azcutia V, Kelm M, Luissint AC, Boerner K, Flemming S, Quiros M, Newton G, Nusrat A, Luscinskas FW, and Parkos CA
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- Animals, CD11b Antigen metabolism, CD18 Antigens metabolism, CD47 Antigen genetics, Cells, Cultured, Chemotaxis, Mice, Mice, Inbred C57BL, Mice, Knockout, Neutrophil Activation, Organ Specificity, Transendothelial and Transepithelial Migration, CD47 Antigen metabolism, Inflammation immunology, Intestines immunology, Neutrophils immunology
- Abstract
Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.
- Published
- 2021
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9. Targeting epithelium-expressed sialyl Lewis glycans improves colonic mucosal wound healing and protects against colitis.
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Kelm M, Quiros M, Azcutia V, Boerner K, Cummings RD, Nusrat A, Brazil JC, and Parkos CA
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- Animals, Cell Movement physiology, Cell Proliferation physiology, Colitis chemically induced, Colitis drug therapy, Colon metabolism, Disease Models, Animal, Epithelial Cells pathology, Inflammatory Bowel Diseases drug therapy, Inflammatory Bowel Diseases metabolism, Intestinal Mucosa metabolism, Mice, Inbred C57BL, Colitis metabolism, Colon pathology, Epithelial Cells metabolism, Intestinal Mucosa pathology, Wound Healing physiology
- Abstract
Dysregulated healing of injured mucosa is a hallmark of many pathological conditions, including inflammatory bowel disease. Mucosal injury and chronic intestinal inflammation are also associated with alterations in epithelial glycosylation. Previous studies have revealed that inflammation-induced glycan sialyl Lewis A on epithelial CD44v6 acts as a ligand for transmigrating PMNs. Here we report that robust sialylated Lewis glycan expression was induced in colonic mucosa from individuals with ulcerative colitis and Crohn disease as well as in the colonic epithelium of mice with colitis induced by dextran sodium sulfate (DSS). Targeting of sialylated epithelial Lewis glycans with mAb GM35 reduced disease activity and improved mucosal integrity during DSS-induced colitis in mice. Wound healing studies revealed increased epithelial proliferation and migration responses as well as improved mucosal repair after ligation of epithelial sialyl Lewis glycans. Finally, we showed that GM35-mediated increases in epithelial proliferation and migration were mediated through activation of kinases that signal downstream of CD44v6 (Src, FAK, Akt). These findings suggest that sialylated Lewis glycans on CD44v6 represent epithelial targets for improved recovery of intestinal barrier function and restitution of mucosal homeostasis after inflammation or injury.
- Published
- 2020
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10. Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.
- Author
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Kelm M, Lehoux S, Azcutia V, Cummings RD, Nusrat A, Parkos CA, and Brazil JC
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- CD11b Antigen chemistry, CD18 Antigens chemistry, Epitopes chemistry, Humans, Mannose-Binding Lectins chemistry, Mannose-Binding Lectins pharmacology, Neutrophils chemistry, Phagocytosis, Phytohemagglutinins chemistry, Phytohemagglutinins pharmacology, Plant Lectins chemistry, Plant Lectins pharmacology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Superoxides chemistry, Superoxides immunology, Transendothelial and Transepithelial Migration, CD11b Antigen immunology, CD18 Antigens immunology, Epitopes immunology, Neutrophils immunology
- Abstract
Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases., (© 2019 Federation of American Societies for Experimental Biology.)
- Published
- 2020
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11. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.
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Birkl D, O'Leary MN, Quiros M, Azcutia V, Schaller M, Reed M, Nishio H, Keeney J, Neish AS, Lukacs NW, Parkos CA, and Nusrat A
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- Animals, Bone Marrow Transplantation, Chemokine CCL20 genetics, Chemokine CCL20 metabolism, Colitis chemically induced, Colitis metabolism, Colitis pathology, Dextran Sulfate toxicity, Inflammation etiology, Inflammation pathology, Intestinal Mucosa injuries, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Monocytes cytology, Receptors, CCR6 genetics, Receptors, CCR6 metabolism, Cell Movement, Inflammation therapy, Intestinal Mucosa physiology, Monocytes metabolism, Receptors, Formyl Peptide physiology, Wound Healing
- Abstract
Mucosal wound repair is coordinated by dynamic crosstalk between endogenous and exogenous mediators and specific receptors on epithelial cells and infiltrating immune cells. One class of such receptor-ligand pairs involves formyl peptide receptors (FPRs) that have been shown to influence inflammatory response and repair. Here we explored the role of murine Fpr2/3, an ortholog of human FPR2/receptor for lipoxin A4 (ALX), in orchestrating intestinal mucosal repair. Compared with wild-type (WT) mice, Fpr2/3
-/- mice exhibited delayed recovery from acute experimental colitis and perturbed repair after biopsy-induced colonic mucosal injury. Decreased numbers of infiltrating monocytes were observed in healing wounds from Fpr2/3-/- mice compared with WT animals. Bone marrow transplant experiments revealed that Fpr2/3-/- monocytes showed a competitive disadvantage when infiltrating colonic wounds. Moreover, Fpr2/3-/- monocytes were defective in chemotactic responses to the chemokine CC chemokine ligand (CCL)20, which is up-regulated during early phases of inflammation. Analysis of Fpr2/3-/- monocytes revealed altered expression of the CCL20 receptor CC chemokine receptor (CCR)6, suggesting that Fpr2/3 regulates CCL20-CCR6-mediated monocyte chemotaxis to sites of mucosal injury in the gut. These findings demonstrate an important contribution of Fpr2/3 in facilitating monocyte recruitment to sites of mucosal injury to influence wound repair.-Birkl, D., O'Leary, M. N., Quiros, M., Azcutia, V., Schaller, M., Reed, M., Nishio, H., Keeney, J., Neish, A. S., Lukacs, N. W., Parkos, C. A., Nusrat, A. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.- Published
- 2019
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12. Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo.
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Reed M, Luissint AC, Azcutia V, Fan S, O'Leary MN, Quiros M, Brazil J, Nusrat A, and Parkos CA
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- Animals, CD47 Antigen genetics, Colitis chemically induced, Colitis genetics, Colitis metabolism, Dextran Sulfate, Disease Models, Animal, Humans, Intestinal Mucosa pathology, Intestines pathology, Male, Mice, Inbred C57BL, Mice, Knockout, Signal Transduction genetics, Thrombospondin 1 metabolism, Transforming Growth Factor beta1 metabolism, Wound Healing genetics, CD47 Antigen metabolism, Epithelial Cells metabolism, Intestinal Mucosa metabolism, Intestines physiopathology, Wound Healing physiology
- Abstract
CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial β1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-β1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.
- Published
- 2019
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13. Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice.
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Luissint AC, Williams HC, Kim W, Flemming S, Azcutia V, Hilgarth RS, Leary MNO, Denning TL, Nusrat A, and Parkos CA
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- Animals, Cell Adhesion Molecules genetics, Cells, Cultured, Chemokine CXCL1 metabolism, Disease Models, Animal, Gastrointestinal Microbiome, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, NF-kappa B metabolism, Neutrophil Infiltration, Peritonitis chemically induced, Permeability, Receptors, Cell Surface genetics, Zymosan, Cell Adhesion Molecules metabolism, Intestinal Mucosa immunology, Macrophages immunology, Neutrophils immunology, Peritonitis immunology, Receptors, Cell Surface metabolism, Tight Junctions pathology
- Abstract
Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-a
fl/fl ). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.- Published
- 2019
- Full Text
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14. Role of negative regulation of immune signaling pathways in neutrophil function.
- Author
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Azcutia V, Parkos CA, and Brazil JC
- Abstract
Polymorphonuclear neutrophils (PMNs) play a critical role in host defense against infection and in the resolution of inflammation. However, immune responses mediated by PMN must be tightly regulated to facilitate elimination of invading pathogens without inducing detrimental inflammation and host tissue damage. Specific engagement of cell surface immunoreceptors by a diverse range of extracellular signals regulates PMN effector functions through differential activation of intracellular signaling cascades. Although mechanisms of PMN activation mediated via cell signaling pathways have been well described, less is known about negative regulation of PMN function by immune signaling cascades. Here, we provide an overview of immunoreceptor-mediated negative regulation of key PMN effector functions including maturation, migration, phagocytosis, reactive oxygen species release, degranulation, apoptosis, and NET formation. Increased understanding of mechanisms of suppression of PMN effector functions may point to possible future therapeutic targets for the amelioration of PMN-mediated autoimmune and inflammatory diseases., (©2017 Society for Leukocyte Biology.)
- Published
- 2017
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15. Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.
- Author
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Quiros M, Nishio H, Neumann PA, Siuda D, Brazil JC, Azcutia V, Hilgarth R, O'Leary MN, Garcia-Hernandez V, Leoni G, Feng M, Bernal G, Williams H, Dedhia PH, Gerner-Smidt C, Spence J, Parkos CA, Denning TL, and Nusrat A
- Subjects
- Animals, CD11 Antigens metabolism, Cell Proliferation, Colon pathology, Cyclic AMP Response Element-Binding Protein metabolism, Epithelial Cells cytology, Epithelial Cells metabolism, Gene Deletion, Gene Expression Regulation, Humans, Inflammation, Intestinal Mucosa metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Signal Transduction, Wound Healing, CCN Intercellular Signaling Proteins metabolism, Interleukin-10 metabolism, Macrophages metabolism, Proto-Oncogene Proteins metabolism
- Abstract
In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.
- Published
- 2017
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16. Erratum to: Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway.
- Author
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Peiró C, Romacho T, Azcutia V, Villalobos L, Fernández E, Bolaños JP, Moncada S, and Sánchez-Ferrer CF
- Published
- 2017
- Full Text
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17. Defects in CD4+ T cell LFA-1 integrin-dependent adhesion and proliferation protect Cd47-/- mice from EAE.
- Author
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Azcutia V, Bassil R, Herter JM, Engelbertsen D, Newton G, Autio A, Mayadas T, Lichtman AH, Khoury SJ, Parkos CA, Elyaman W, and Luscinskas FW
- Subjects
- Adoptive Transfer, Animals, Antibodies, Monoclonal pharmacology, Antigen Presentation drug effects, Antigen Presentation immunology, CD28 Antigens metabolism, CD4-Positive T-Lymphocytes drug effects, Cell Adhesion drug effects, Cell Proliferation drug effects, Chemokines pharmacology, Encephalomyelitis, Autoimmune, Experimental pathology, Female, Humans, Immunization, Integrin alpha4beta1 metabolism, Intercellular Adhesion Molecule-1 metabolism, Lymph Nodes drug effects, Lymph Nodes pathology, Lymphocyte Activation drug effects, Lymphocyte Activation immunology, Mice, Inbred C57BL, Myelin-Oligodendrocyte Glycoprotein immunology, Receptors, Antigen, T-Cell metabolism, Vascular Cell Adhesion Molecule-1 metabolism, CD4-Positive T-Lymphocytes immunology, CD47 Antigen metabolism, Encephalomyelitis, Autoimmune, Experimental immunology, Encephalomyelitis, Autoimmune, Experimental prevention & control, Lymphocyte Function-Associated Antigen-1 metabolism
- Abstract
CD47 is known to play an important role in CD4
+ T cell homeostasis. We recently reported a reduction in mice deficient in the Cd47 gene (Cd47-/- ) CD4+ T cell adhesion and transendothelial migration (TEM) in vivo and in vitro as a result of impaired expression of high-affinity forms of LFA-1 and VLA-4 integrins. A prior study concluded that Cd47-/- mice were resistant to experimental autoimmune encephalomyelitis (EAE) as a result of complete failure in CD4+ T cell activation after myelin oligodendrocyte glycoprotein peptide 35-55 aa (MOG35-55 ) immunization. As the prior EAE study was published before our report, authors could not have accounted for defects in T cell integrin function as a mechanism to protect Cd47-/- in EAE. Thus, we hypothesized that failure of T cell activation involved defects in LFA-1 and VLA-4 integrins. We confirmed that Cd47-/- mice were resistant to MOG35-55 -induced EAE. Our data, however, supported a different mechanism that was not a result of failure of CD4+ T cell activation. Instead, we found that CD4+ T cells in MOG35-55 -immunized Cd47-/- mice were activated, but clonal expansion contracted within 72 h after immunization. We used TCR crosslinking and mitogen activation in vitro to investigate the underlying mechanism. We found that naïve Cd47-/- CD4+ T cells exhibited a premature block in proliferation and survival because of impaired activation of LFA-1, despite effective TCR-induced activation. These results identify CD47 as an important regulator of LFA-1 and VLA-4 integrin-adhesive functions in T cell proliferation, as well as recruitment, and clarify the roles played by CD47 in MOG35-55 -induced EAE., (© Society for Leukocyte Biology.)- Published
- 2017
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18. A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton.
- Author
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Janssen E, Tohme M, Hedayat M, Leick M, Kumari S, Ramesh N, Massaad MJ, Ullas S, Azcutia V, Goodnow CC, Randall KL, Qiao Q, Wu H, Al-Herz W, Cox D, Hartwig J, Irvine DJ, Luscinskas FW, and Geha RS
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Animals, Cell Movement, Cytoskeletal Proteins, HEK293 Cells, Humans, Immunological Synapses metabolism, Lymph Nodes cytology, Mechanotransduction, Cellular, Mice, Inbred C57BL, Mice, Knockout, Protein Binding, Protein Interaction Maps, Protein Multimerization, Protein Transport, T-Lymphocytes physiology, Actin Cytoskeleton metabolism, Carrier Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Receptors, Antigen, T-Cell metabolism, Wiskott-Aldrich Syndrome Protein metabolism
- Abstract
Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.
- Published
- 2016
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19. Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway.
- Author
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Peiró C, Romacho T, Azcutia V, Villalobos L, Fernández E, Bolaños JP, Moncada S, and Sánchez-Ferrer CF
- Subjects
- Animals, Cells, Cultured, Endothelium, Vascular drug effects, Endothelium, Vascular metabolism, Glutathione, Humans, Hyperglycemia metabolism, Interleukin-1beta pharmacology, Male, Myocytes, Smooth Muscle drug effects, NADPH Oxidases metabolism, Oxidation-Reduction drug effects, Rats, Sprague-Dawley, Reactive Oxygen Species metabolism, Glucose metabolism, Inflammation metabolism, Myocytes, Smooth Muscle metabolism, Pentose Phosphate Pathway drug effects
- Abstract
Background: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1β. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1β., Methods: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1β in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-κB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined., Results: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1β. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1β activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1β by a mechanism which was abrogated by the inhibition of the PPP., Conclusions: A pro-inflammatory stimulus like IL1β transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia.
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- 2016
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20. AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation.
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Herter JM, Grabie N, Cullere X, Azcutia V, Rosetti F, Bennett P, Herter-Sprie GS, Elyaman W, Luscinskas FW, Lichtman AH, and Mayadas TN
- Subjects
- A Kinase Anchor Proteins immunology, Animals, Antigen-Presenting Cells immunology, Cell Adhesion immunology, Cell Migration Assays, Leukocyte, Cells, Cultured, Endosomes, Gene Knockout Techniques, Glomerular Basement Membrane immunology, In Vitro Techniques, Inflammation, Kidney blood supply, Kidney immunology, Mice, Microtubule-Associated Proteins immunology, Receptors, Antigen, T-Cell, Transendothelial and Transepithelial Migration immunology, A Kinase Anchor Proteins genetics, Cell Movement immunology, Encephalomyelitis, Autoimmune, Experimental immunology, Lymph Nodes immunology, Lymphocyte Activation immunology, Microtubule-Associated Proteins genetics, Nephritis immunology, Reperfusion Injury immunology, T-Lymphocytes immunology
- Abstract
The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis.
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- 2015
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21. Migration of myeloid cells during inflammation is differentially regulated by the cell surface receptors Slamf1 and Slamf8.
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Wang G, van Driel BJ, Liao G, O'Keeffe MS, Halibozek PJ, Flipse J, Yigit B, Azcutia V, Luscinskas FW, Wang N, and Terhorst C
- Subjects
- Animals, Antigens, CD genetics, Antigens, CD metabolism, Cell Movement genetics, Cell Movement physiology, Chemotaxis drug effects, Dendritic Cells cytology, Dendritic Cells metabolism, Macrophages cytology, Macrophages metabolism, Membrane Proteins, Mice, Mice, Inbred BALB C, Myeloid Cells cytology, Neutrophils cytology, Neutrophils metabolism, Peritonitis metabolism, Peritonitis pathology, Reactive Oxygen Species metabolism, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Signaling Lymphocytic Activation Molecule Family Member 1, Antigens, CD physiology, Myeloid Cells metabolism, Receptors, Cell Surface physiology
- Abstract
Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.
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- 2015
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22. A Lupus-Associated Mac-1 Variant Has Defects in Integrin Allostery and Interaction with Ligands under Force.
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Rosetti F, Chen Y, Sen M, Thayer E, Azcutia V, Herter JM, Luscinskas FW, Cullere X, Zhu C, and Mayadas TN
- Abstract
Leukocyte CD18 integrins increase their affinity for ligand by transmitting allosteric signals to and from their ligand-binding αI domain. Mechanical forces induce allosteric changes that paradoxically slow dissociation by increasing the integrin/ligand bond lifetimes, referred to as catch bonds. Mac-1 formed catch bonds with its ligands. However, a Mac-1 gene (ITGAM) coding variant (rs1143679, R77H), which is located in the β-propeller domain and is significantly associated with systemic lupus erythematosus risk, exhibits a marked impairment in 2D ligand affinity and affinity maturation under mechanical force. Targeted mutations and activating antibodies reveal that the failure in Mac-1 R77H allostery is rescued by induction of cytoplasmic tail separation and full integrin extension. These findings demonstrate roles for R77, and the β-propeller in which it resides, in force-induced allostery relay and integrin bond stabilization. Defects in these processes may have pathological consequences, as the Mac-1 R77H variant is associated with increased susceptibility to lupus., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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23. Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies.
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Leick M, Azcutia V, Newton G, and Luscinskas FW
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- Animals, Humans, Image Processing, Computer-Assisted methods, Inflammation immunology, Leukocytes immunology, Microscopy, Fluorescence methods
- Abstract
The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article reviews basic mechanisms in the recruitment of leukocytes to tissues and then selectively reviews new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. Investigators have also begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise.
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- 2014
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24. CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions.
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Azcutia V, Routledge M, Williams MR, Newton G, Frazier WA, Manica A, Croce KJ, Parkos CA, Schmider AB, Turman MV, Soberman RJ, and Luscinskas FW
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- Animals, CD47 Antigen genetics, CD47 Antigen metabolism, Cell Adhesion immunology, Cells, Cultured, Endothelial Cells drug effects, Endothelial Cells immunology, Endothelial Cells metabolism, Humans, Immunoblotting, Integrin alpha4beta1 metabolism, Jurkat Cells, Lymphocyte Function-Associated Antigen-1 metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Microscopy, Fluorescence, Protein Binding immunology, T-Lymphocytes metabolism, Transendothelial and Transepithelial Migration immunology, Tumor Necrosis Factor-alpha immunology, Tumor Necrosis Factor-alpha pharmacology, CD47 Antigen immunology, Integrin alpha4beta1 immunology, Lymphocyte Function-Associated Antigen-1 immunology, T-Lymphocytes immunology
- Abstract
CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.
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- 2013
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25. Complete blockade of the vasorelaxant effects of angiotensin-(1-7) and bradykinin in murine microvessels by antagonists of the receptor Mas.
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Peiró C, Vallejo S, Gembardt F, Palacios E, Novella S, Azcutia V, Rodríguez-Mañas L, Hermenegildo C, Sánchez-Ferrer CF, and Walther T
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- Angiotensin I pharmacology, Angiotensin II pharmacology, Animals, Bradykinin pharmacology, Human Umbilical Vein Endothelial Cells drug effects, Human Umbilical Vein Endothelial Cells metabolism, Humans, Male, Mesenteric Arteries drug effects, Mesenteric Arteries physiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Microvessels physiology, Nitric Oxide metabolism, Nitric Oxide Synthase Type III metabolism, Proto-Oncogene Mas, Proto-Oncogene Proteins physiology, Receptors, G-Protein-Coupled physiology, Vasodilation drug effects, Vasodilator Agents pharmacology, Angiotensin II analogs & derivatives, Microvessels drug effects, Peptide Fragments pharmacology, Proto-Oncogene Proteins antagonists & inhibitors, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
The heptapeptide angiotensin-(1-7) is a biologically active metabolite of angiotensin II, the predominant peptide of the renin-angiotensin system. Recently, we have shown that the receptor Mas is associated with angiotensin-(1-7)-induced signalling and mediates, at least in part, the vasodilatory properties of angiotensin-(1-7). However, it remained controversial whether an additional receptor could account for angiotensin-(1-7)-induced vasorelaxation. Here, we used two different angiotensin-(1-7) antagonists, A779 and d-Pro-angiotensin-(1-7), to address this question and also to study their influence on the vasodilatation induced by bradykinin. Isolated mesenteric microvessels from both wild-type and Mas-deficient C57Bl/6 mice were precontracted with noradrenaline, and vascular reactivity to angiotensin-(1-7) and bradykinin was subsequently studied using a small-vessel myograph. Furthermore, mechanisms for Mas effects were investigated in primary human umbilical vein endothelial cells. Both angiotensin-(1-7) and bradykinin triggered a concentration-dependent vasodilatation in wild-type microvessels, which was absent in the presence of a nitric oxide synthase inhibitor. In these vessels, the pre-incubation with the Mas antagonists A779 or d-Pro-angiotensin-(1-7) totally abolished the vasodilatory capacity of both angiotensin-(1-7) and bradykinin, which was nitric oxide mediated. Accordingly, Mas-deficient microvessels lacked the capacity to relax in response to either angiotensin-(1-7) or bradykinin. Pre-incubation of human umbilical vein endothelial cells with A779 prevented bradykinin-mediated NO generation and NO synthase phosphorylation at serine 1177. The angiotensin-(1-7) antagonists A779 and d-Pro-angiotensin-(1-7) equally block Mas, which completely controls the angiotensin-(1-7)-induced vasodilatation in mesenteric microvessels. Importantly, Mas also appears to be a critical player in NO-mediated vasodilatation induced by renin-angiotensin system-independent agonists by altering phosphorylation of NO synthase.
- Published
- 2013
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26. Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.
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Zheng C, Azcutia V, Aikawa E, Figueiredo JL, Croce K, Sonoki H, Sacks FM, Luscinskas FW, and Aikawa M
- Subjects
- Animals, Aorta, Cell Adhesion physiology, Cells, Cultured, Humans, Leukocytes, Mononuclear physiology, Mice, Mice, Inbred C57BL, NF-kappa B metabolism, Saphenous Vein, Vascular Cell Adhesion Molecule-1 metabolism, Apolipoprotein C-III antagonists & inhibitors, Endothelial Cells physiology, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Leukocytes, Mononuclear drug effects, Quinolines pharmacology
- Abstract
Aims: Activation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo., Methods and Results: Physiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr(-/-) mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect., Conclusions: These findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.
- Published
- 2013
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27. Signaling lymphocyte activation molecule regulates development of colitis in mice.
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van Driel B, Liao G, Romero X, O'Keeffe MS, Wang G, Faubion WA, Berger SB, Magelky EM, Manocha M, Azcutia V, Grisham M, Luscinskas FW, Mizoguchi E, de Waal Malefyt R, Reinecker HC, Bhan AK, Wang N, and Terhorst C
- Subjects
- Animals, Antigens, CD genetics, CD40 Antigens adverse effects, Cell Movement, Chemokine CCL2 blood, Chemokine CCL7 blood, Colitis blood, Colitis chemically induced, Disease Models, Animal, Intestines pathology, Macrophages pathology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, Receptors, Cell Surface deficiency, Receptors, Cell Surface genetics, Signaling Lymphocytic Activation Molecule Family Member 1, Antigens, CD physiology, Colitis physiopathology, Receptors, Cell Surface physiology
- Abstract
Background & Aims: Signaling lymphocyte activation molecule (Slamf)1 is a co-stimulatory receptor on T cells and regulates cytokine production by macrophages and dendritic cells. Slamf1 regulates microbicidal mechanisms in macrophages, therefore we investigated whether the receptor affects development of colitis in mice., Methods: We transferred CD45RB(hi) CD4(+) T cells into Rag(-/-) or Slamf1(-/-)Rag(-/-) mice to induce colitis. We also induced colitis by injecting mice with an antibody that activates CD40. We determined the severity of enterocolitis based on disease activity index, histology scores, and levels of cytokine production, and assessed the effects of antibodies against Slamf1 on colitis induction. We quantified migration of monocytes and macrophage to inflamed tissues upon induction of colitis or thioglycollate-induced peritonitis and in response to tumor necrosis factor-α in an air-pouch model of leukocyte migration., Results: Colitis was reduced in Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after transfer of CD45RB(hi) CD4(+) T cells or administration of the CD40 agonist. The numbers of monocytes and macrophages were reduced in inflamed tissues of Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after induction of colitis and other inflammatory disorders. An antibody that inhibited Slamf1 reduced the level of enterocolitis in Rag(-/-) mice., Conclusions: Slamf1 contributes to the development of colitis in mice. It appears to indirectly regulate the appearance of monocytes and macrophages in inflamed intestinal tissues. Antibodies that inhibit Slamf1 reduce colitis in mice, so human SLAMF1 might be a therapeutic target for inflammatory bowel disease., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
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28. Endothelial CD47 promotes vascular endothelial-cadherin tyrosine phosphorylation and participates in T cell recruitment at sites of inflammation in vivo.
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Azcutia V, Stefanidakis M, Tsuboi N, Mayadas T, Croce KJ, Fukuda D, Aikawa M, Newton G, and Luscinskas FW
- Subjects
- Animals, CD47 Antigen genetics, CD47 Antigen metabolism, Disease Models, Animal, Human Umbilical Vein Endothelial Cells, Humans, Inflammation blood, Ligands, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorylation immunology, Recombinant Proteins toxicity, Signal Transduction genetics, Signal Transduction immunology, T-Lymphocyte Subsets metabolism, T-Lymphocyte Subsets pathology, Tumor Necrosis Factor-alpha toxicity, CD47 Antigen physiology, Cadherins blood, Chemotaxis, Leukocyte immunology, Endothelium, Vascular metabolism, Inflammation immunology, Inflammation pathology, T-Lymphocyte Subsets immunology, Tyrosine blood
- Abstract
At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47⁻/⁻ mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α-induced inflammation. Reconstitution of CD47⁻/⁻ mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47⁻/⁻ endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(αi)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(αi), had no inhibitory effect, whereas G(αi) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(αi). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.
- Published
- 2012
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29. IL-17 and TNF-α sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation.
- Author
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Griffin GK, Newton G, Tarrio ML, Bu DX, Maganto-Garcia E, Azcutia V, Alcaide P, Grabie N, Luscinskas FW, Croce KJ, and Lichtman AH
- Subjects
- Animals, Chemokines biosynthesis, Endothelial Cells immunology, Flow Cytometry, Inflammation immunology, Interleukin-17 immunology, Leukocyte Rolling immunology, Mice, Mice, Inbred C57BL, Mice, Knockout, Reverse Transcriptase Polymerase Chain Reaction, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tumor Necrosis Factor-alpha immunology, Endothelial Cells metabolism, Inflammation metabolism, Interleukin-17 metabolism, Neutrophil Infiltration immunology, Tumor Necrosis Factor-alpha metabolism
- Abstract
IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.
- Published
- 2012
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30. Emerging mechanisms of neutrophil recruitment across endothelium.
- Author
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Williams MR, Azcutia V, Newton G, Alcaide P, and Luscinskas FW
- Subjects
- Animals, Cell Adhesion Molecules immunology, Cell Adhesion Molecules metabolism, Chemokines metabolism, Chemotaxis, Leukocyte immunology, Endothelium, Vascular immunology, Endothelium, Vascular metabolism, Humans, Inflammation metabolism, Inflammation pathology, Mice, Neutrophil Infiltration immunology, Receptors, Chemokine metabolism, Cell Adhesion immunology, Chemokines immunology, Immunity, Innate, Inflammation immunology, Neutrophils immunology, Neutrophils metabolism, Receptors, Chemokine immunology, Signal Transduction immunology, Transendothelial and Transepithelial Migration immunology
- Abstract
Neutrophils are the all-terrain vehicle of the innate immune system because of their ability to gain entry into tissues and organs, and thus, play an essential role in host defense. Exactly how this marvel of nature works is still incompletely understood. In the past 2-3 years, new players and processes have been identified in the endothelial-leukocyte adhesion cascade. Novel signaling pathways have been discovered in both the endothelium and the neutrophils that regulate various steps in the recruitment process. This review focuses on these emerging pathways and the mechanisms that regulate neutrophil recruitment across endothelium., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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31. Pathways responsible for apoptosis resulting from amadori-induced oxidative and nitrosative stress in human mesothelial cells.
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Sánchez-Rodríguez C, Peiró C, Vallejo S, Matesanz N, El-Assar M, Azcutia V, Romacho T, Sánchez-Ferrer CF, Rodríguez-Mañas L, and Nevado J
- Subjects
- Animals, Cattle, Cell Death, Cytochromes c metabolism, Humans, Hyperglycemia metabolism, Inflammation, L-Lactate Dehydrogenase metabolism, MAP Kinase Signaling System, Proto-Oncogene Proteins c-jun metabolism, Signal Transduction, Apoptosis, Epithelium pathology, Glycolipids pharmacology, Nitrogen metabolism, Oxidative Stress, Phosphatidylethanolamines pharmacology
- Abstract
Background: Apoptosis and inflammatory/oxidative stress have been associated with hyperglycemia in human peritoneal mesothelial cells (HPMCs) and other cell types. We and others have highlighted the role of early products of non-enzymatic protein glycation in inducing proinflammatory conditions and increasing apoptotic rates in HPMCs. Loss of HPMCs seems to be a hallmark of complications associated with peritoneal membrane dysfunction. The aim of this work is to elucidate the mechanisms by which Amadori adducts may act upon HPMC apoptosis., Methods: HPMCs isolated from different patients were exposed to different Amadori adducts, i.e. highly glycated hemoglobin (10 nM) and glycated bovine serum albumin (250 μg/ml), to study cell death and several proapoptotic markers by different experimental approaches., Results: Amadori adducts, but not their respective controls, impaired cell proliferation and cell viability by means of apoptosis in a time-dependent manner. They regulated the intrinsic mitochondrial cell death signaling pathway and modulated activation of caspases, Bax, iNOS, p53, NF-κB, and mitogen-activated protein kinases (p38 and JNK) through different reactive oxygen and nitrosative species., Conclusions: Our data strongly support the idea that long-term hyperglycemia could act as an inducer of apoptosis in HPMCs through Amadori adducts, involving different oxidative and nitrosative reactive species., (Copyright © 2011 S. Karger AG, Basel.)
- Published
- 2011
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32. Inflammation determines the pro-adhesive properties of high extracellular d-glucose in human endothelial cells in vitro and rat microvessels in vivo.
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Azcutia V, Abu-Taha M, Romacho T, Vázquez-Bella M, Matesanz N, Luscinskas FW, Rodríguez-Mañas L, Sanz MJ, Sánchez-Ferrer CF, and Peiró C
- Subjects
- Adhesiveness, Animals, Cell Adhesion, Chemotaxis, Leukocyte, Dose-Response Relationship, Drug, Endothelial Cells metabolism, Endothelium, Vascular pathology, Glucose adverse effects, Humans, Inflammation chemically induced, Leukocyte Rolling, Rats, Umbilical Veins, Endothelium, Vascular metabolism, Glucose metabolism, Hyperglycemia pathology, Inflammation pathology, Microvessels metabolism
- Abstract
Background: Hyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early pro-atherosclerotic events., Methods and Findings: In the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1beta (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-kappaB (NF-kappaB) elicited by IL-1beta, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-kappaB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1beta was co-administered., Conclusions: These results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia.
- Published
- 2010
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33. The deleterious effect of high concentrations of D-glucose requires pro-inflammatory preconditioning.
- Author
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Lafuente N, Matesanz N, Azcutia V, Romacho T, Nevado J, Rodríguez-Mañas L, Moncada S, Peiró C, and Sánchez-Ferrer CF
- Subjects
- Aorta, Cells, Cultured, Gene Expression, Humans, Interleukin-1beta metabolism, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular immunology, Myocytes, Smooth Muscle drug effects, Glucose pharmacology, Myocytes, Smooth Muscle metabolism, NF-kappa B biosynthesis, Nitric Oxide Synthase biosynthesis, Phosphotransferases biosynthesis
- Abstract
Objectives: The present study investigated whether high concentrations of D-glucose can trigger pro-inflammatory mechanisms in human aortic smooth muscle cells., Methods: The expression and/or the activity of inducible nitric oxide synthase (iNOS), the extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF)-kappaB were studied in cultured human aortic smooth muscle cells (HASMC) in response to increasing concentrations of D-glucose and/or the inflammatory cytokine interleukin (IL)-1beta., Results: Increasing D-glucose in the medium from 5.5 to 22 mmol/l had no effect on any of these parameters. However, the high concentration of D-glucose did increase iNOS expression in response to low concentrations of IL-1beta (2.5 and 5 ng/ml), as well as the IL-1beta-induced activation of both ERK 1/2 and NF-kappaB. D-glucose also enhanced, concentration-dependently, the expression and activity of iNOS induced by co-incubation with IL-1beta (10 ng/ml). Pretreatment with IL-1beta sensitized the cells to the subsequent effects of high D-glucose., Conclusions: The results indicate that high concentrations of D-glucose exacerbate the pro-inflammatory effects of IL-1beta. We suggest that the observed association between inflammation and diabetes is the result of elevated D-glucose enhancing a pre-existing inflammatory condition, rather than a direct effect of D-glucose on the production of inflammatory mediators.
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- 2008
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34. Endothelial dysfunction through genetic deletion or inhibition of the G protein-coupled receptor Mas: a new target to improve endothelial function.
- Author
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Peiró C, Vallejo S, Gembardt F, Azcutia V, Heringer-Walther S, Rodríguez-Mañas L, Schultheiss HP, Sánchez-Ferrer CF, and Walther T
- Subjects
- Acetylcholine pharmacology, Angiotensin I pharmacology, Angiotensin II analogs & derivatives, Angiotensin II pharmacology, Animals, Bradykinin pharmacology, Cells, Cultured, Endothelium, Vascular drug effects, Humans, In Vitro Techniques, Mesenteric Arteries drug effects, Mesenteric Arteries physiopathology, Mice, Mice, Inbred C57BL, Mice, Knockout, Nitric Oxide physiology, Peptide Fragments pharmacology, Proto-Oncogene Mas, Proto-Oncogene Proteins agonists, Proto-Oncogene Proteins deficiency, Proto-Oncogene Proteins physiology, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled deficiency, Receptors, G-Protein-Coupled physiology, Vasodilation drug effects, Vasodilation physiology, Endothelium, Vascular physiopathology, Proto-Oncogene Proteins antagonists & inhibitors, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
Background: Endothelial dysfunction is an initial step in the pathogenesis of cardiovascular diseases. Since we previously identified the G protein-coupled receptor Mas as a receptor for angiotensin (Ang)-(1-7), a heptapeptide with endothelium-dependent vasorelaxant properties, we investigated whether alterations on the Ang-(1-7)/Mas axis alter endothelial function., Results: Ang-(1-7)-mediated relaxation of murine wild-type mesenteric arteries was equally impaired in both wild-type arteries pretreated with the Ang-(1-7) receptor blocker, A779, and arteries isolated from Mas-deficient mice. Importantly, the response to the endothelium-dependent vasorelaxant, bradykinin (BK), and acetylcholine (ACh) effects were comparably inhibited, while endothelium-independent vessel relaxation by sodium nitroprusside was unaltered in these vessels. Hypothesizing endothelial dysfunction, we proved the in-vivo relevance of the ex-vivo findings investigating mesenteric properties after 1 week of minipump infusion of A779 in wild-type mice. Both BK- and ACh-induced relaxation were significantly impaired in wild-type vessels of pretreated animals. A779-induced impairment of endothelial function was confirmed in vitro, since BK-mediated nitric oxide (NO) release was increased by Ang-(1-7) and blunted by A779 pretreatment in primary human endothelial cell cultures., Conclusions: Our data highlight a pivotal role for the receptor Mas in preserving normal vascular relaxation. Consequently, Mas agonists arise as a promising tool in the treatment of cardiovascular diseases characterized by endothelial dysfunction.
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- 2007
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35. Xanthine oxidase-derived extracellular superoxide anions stimulate activator protein 1 activity and hypertrophy in human vascular smooth muscle via c-Jun N-terminal kinase and p38 mitogen-activated protein kinases.
- Author
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Matesanz N, Lafuente N, Azcutia V, Martín D, Cuadrado A, Nevado J, Rodríguez-Mañas L, Sánchez-Ferrer CF, and Peiró C
- Subjects
- Allopurinol pharmacology, Aorta cytology, Aorta drug effects, Cell Enlargement drug effects, Cell Proliferation drug effects, Cells, Cultured, Humans, Hypertrophy, JNK Mitogen-Activated Protein Kinases metabolism, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular enzymology, Muscle, Smooth, Vascular pathology, Myocytes, Smooth Muscle cytology, Myocytes, Smooth Muscle metabolism, Xanthine Oxidase antagonists & inhibitors, Xanthine Oxidase metabolism, Myocytes, Smooth Muscle drug effects, Replication Protein C metabolism, Superoxides pharmacology, Xanthine Oxidase pharmacology, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
Objective: Vascular xanthine oxidase (XO) activity has been found to be elevated in chronic vascular disease. Although a role for XO in endothelial dysfunction has been proposed, little is known about its influence on vascular smooth muscle maladaptive growth., Methods: The proliferative and hypertrophic response of human aortic smooth muscle cells (HASMC) stimulated with xanthine/xanthine oxidase (X/XO) was quantified by determining cell number, cell size and protein synthesis. The levels and activity of the growth-related transcription factor activator protein 1 (AP-1) and the activation of mitogen-activated protein kinase (MAPK) by X/XO were determined by either Western blot or transient transfection experiments., Results: X/XO did not affect HASMC proliferation, but led to enhanced planar cell surface area and protein synthesis. In addition, X/XO enhanced c-jun levels and AP-1 transcriptional activity. Although X/XO did not modify extracellular signal-regulated protein kinases 1/2 MAPK or Akt/PKB activity, it promoted the activation of c-Jun N-terminal kinase and p38 MAPK, which were both necessary for X/XO to increase AP-1 activity and cell size in HASMC cultures. Finally, the effects of X/XO on MAPK activation, AP-1 activity and cell size were dependent on the extracellular release of superoxide anions through the enzymatic activity of XO, as they were prevented by both superoxide dismutase and allopurinol., Conclusion: X/XO exhibits hypertrophic properties for human vascular smooth muscle, which are mediated by redox-sensitive pathways involving MAPK activation. XO can therefore participate in the maladaptive vascular remodeling observed in chronic cardiovascular diseases exhibiting elevated vascular XO activity.
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- 2007
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36. Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.
- Author
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Nevado J, Peiró C, Vallejo S, El-Assar M, Lafuente N, Matesanz N, Azcutia V, Cercas E, Sánchez-Ferrer CF, and Rodríguez-Mañas L
- Subjects
- Blotting, Western, Cells, Cultured, Cytokines biosynthesis, Cytokines genetics, Electrophoretic Mobility Shift Assay, Epithelium drug effects, Gene Expression drug effects, Glycated Hemoglobin pharmacology, Humans, Luciferases metabolism, NF-kappa B physiology, Nitric Oxide physiology, Nitrites metabolism, Peritoneum cytology, Peritoneum drug effects, Plasmids genetics, Reactive Oxygen Species metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Epithelial Cells drug effects, Glycation End Products, Advanced pharmacology, Inflammation genetics, NF-kappa B genetics
- Abstract
Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7+/-16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml(-1)), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-kappaB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF-kappaB-related proinflammatory molecules, including cytokines, such as TNF-alpha, IL-1beta or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-kappaB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-kappaB-related proinflammatory signals in human mesothelial cells.
- Published
- 2005
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37. Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein.
- Author
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Lafuente N, Azcutia V, Matesanz N, Cercas E, Rodríguez-Mañas L, Sánchez-Ferrer CF, and Peiró C
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- Blotting, Western, Cells, Cultured, Dialysis, Humans, Indicators and Reagents, Interleukin-1 pharmacology, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular enzymology, Nitric Oxide biosynthesis, Nitric Oxide Synthase Type II, Nitrites metabolism, Recombinant Proteins pharmacology, Risk Assessment, Artifacts, C-Reactive Protein pharmacology, Nitric Oxide Synthase biosynthesis, Sodium Azide adverse effects
- Abstract
C-reactive protein (CRP) is an acute-phase protein identified as a cardiovascular risk marker. In recent years, an increasing number of studies have investigated the possible direct effects of CRP on the vasculature, using mainly commercial CRP. In the present work, a potential role for CRP as a modulator of inducible nitric oxide synthase (iNOS) induction was explored. Cultured human aortic vascular smooth muscle cells (HASMC) were stimulated for 18 hours with 10 ng/mL interleukin-1beta (IL-1beta), resulting in a marked increase of iNOS levels and NO production, as determined by Western blotting and nitrite measurement, respectively. Commercial CRP (1 to 100 microg/mL) concentration-dependently inhibited the effects elicited by IL-1beta. Unexpectedly, similar results were observed when the commercial CRP solution was replaced by the corresponding vehicle medium containing growing concentrations of sodium azide. The inhibitory effects of commercial CRP or vehicle medium were lost on sodium azide removal by dialysis. In conclusion, sodium azide from the commercial CRP solution, but not CRP itself, mainly accounts for the inhibitory effect on IL-1beta-evoked iNOS induction and NO release. Care should be taken before attributing any biologic role to commercial CRP containing sodium azide.
- Published
- 2005
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38. Glycosylated human oxyhaemoglobin activates nuclear factor-kappaB and activator protein-1 in cultured human aortic smooth muscle.
- Author
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Peiro C, Matesanz N, Nevado J, Lafuente N, Cercas E, Azcutia V, Vallejo S, Rodriguez-Manas L, and Sanchez-Ferrer CF
- Subjects
- Aorta cytology, Cell Division drug effects, Cell Division physiology, Electrophoretic Mobility Shift Assay methods, Fluorescent Antibody Technique methods, Gene Expression drug effects, Humans, Muscle, Smooth, Vascular cytology, NF-kappa B genetics, Oxidative Stress drug effects, Oxyhemoglobins chemistry, Reactive Oxygen Species chemistry, Reactive Oxygen Species metabolism, Signal Transduction, Superoxide Dismutase chemistry, Superoxide Dismutase metabolism, Thiourea metabolism, Transcription Factor AP-1 genetics, Transcription Factors chemistry, Transcription Factors metabolism, Aorta metabolism, Cells, Cultured, Glycosylation, Muscle, Smooth, Vascular metabolism, NF-kappa B metabolism, Oxyhemoglobins metabolism, Thiourea analogs & derivatives, Transcription Factor AP-1 metabolism
- Abstract
Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox-regulated transcription factors, like activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. In this study, we analysed whether Amadori-modified human oxyhaemoglobin, glycosylated at either normal (N-Hb) or elevated (E-Hb) levels, can induce cell growth and activate AP-1 and NF-kappaB in cultured human aortic smooth muscle cells (HASMC). E-Hb (1 nm-1 x microm), but not N-Hb, promoted a concentration-dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nm, E-Hb stimulated both AP-1 and NF-kappaB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E-Hb resembled those of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). E-Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. In conclusion, E-Hb stimulates growth and activates AP-1 and NF-kappaB in human vascular smooth muscle by redox-sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy.
- Published
- 2003
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