Abstract 153: IL-10 Accelerates Re-Endothelialization and Inhibits Post-injury Intimal Hyperplasia following Carotid Artery Denudation by Attenuating TNF-alpha-induced Endothelial Cell Dysfunction

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Suresh K Verma ◽  
Prasanna Krishnamurthy ◽  
Alexander R Mackie ◽  
Erin E Vaughan ◽  
Mohsin Khan ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Inflammatory Responses ◽  
Thrombus Formation ◽  
Specific Effect ◽  
Mononuclear Phagocytes ◽  
Tnf Alpha ◽  
Post Injury ◽  
Knock Out ◽  
Cytokines And Chemokines

The association of inflammation with atherosclerosis and restenosis is now fairly well established. Restenosis, a persistent complication of percutaneous vascular interventions, is thought to be a complex response to injury, which includes early thrombus formation, neointimal growth and acute inflammation. Mononuclear phagocytes are likely participants in the host response to vascular injury, via the secretion of cytokines and chemokines, including TNF-alpha (TNF). Others and we have previously shown that IL-10 inhibits TNF and other inflammatory mediators produced in response to cardiovascular injuries. The specific effect of IL-10 on endothelial cell (EC) biology is not well elucidated. Here we report that in a mouse model of carotid denudation, IL-10 knock-out mice (IL10KO) displayed significantly delayed ReEndothelialization and enhanced neointimal growth compared to their WT counterparts. Exogenous treatment of recombinant IL-10 dramatically blunted the inflammatory cell infiltration and neointimal thickening while significantly accelerating the recovery of the injured endothelium both WT and IL10KO mice. In vitro, IL10 co-treatment reversed TNF-mediated growth arrest, EC cell cycle inhibition, EC-monocyte adhesion and EC apoptosis. At signaling level, IL-10 reduced TNF-induced activation of JNK MAP kinase while simultaneously activating PI3K/Akt pathway. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.

scholarly journals HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Irma Colombo ◽  
Enrico Sangiovanni ◽  
Roberta Maggio ◽  
Carlo Mattozzi ◽  
Stefania Zava ◽  
...  
Keyword(s):  
Cell Density ◽  
Skin Diseases ◽  
Inflammatory Responses ◽  
Human Keratinocytes ◽  
Suitable Model ◽  
Hacat Cells ◽  
Primary Human Keratinocytes ◽  
Proinflammatory Mediators ◽  
Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

scholarly journals Vascular endothelial cells and granulopoiesis: interleukin-1 stimulates release of G-CSF and GM-CSF

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 99-103 ◽  
Author(s):  
KM Zsebo ◽  
VN Yuschenkoff ◽  
S Schiffer ◽  
D Chang ◽  
E McCall ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Interleukin 1 ◽  
Colony Growth ◽  
Mononuclear Phagocytes ◽  
Conditioned Media ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Growth Assay

Abstract Cultured mononuclear phagocytes produce soluble factors that stimulate endothelial cells to release GM-colony-stimulating activity (GM-CSA). One such factor was recently identified as interleukin 1 (IL 1). Studies were designed to determine which types of granulopoietic factors are released by IL 1-stimulated endothelial cells. Supernatants from endothelial cells cultured for 3 days in medium containing IL 1 alpha and beta were tested in both murine and human CFU-GM colony growth assays. The effect of conditioned media on differentiation of WEHI-3B myelomonocytic leukemic cells was also examined. Control media containing IL 1 alone or unstimulated endothelial cell-conditioned media contained no detectable CSA in any bioassay. Medium conditioned by IL 1-stimulated endothelial cells stimulated the clonal growth of both human and murine CFU-GM and induced macrophage differentiation of WEHI-3B cells. Treatment of these conditioned media with a highly specific neutralizing monoclonal G-CSF antibody completely inhibited their activity in the murine CFU-GM assay, but only partially inhibited GM colony growth by human marrow. Treatment of the active conditioned media with a neutralizing rabbit anti-human GM-CSF antibody partially reduced the activity of the media in the human GM-colony growth assay. G-CSF radioimmunoassay of endothelial cell culture supernatants and Northern blot analysis of endothelial cell cytoplasmic RNA for GM-CSF gene transcripts confirmed that IL 1 induced expression of both G-CSF and GM-CSF genes. Because treatment of media with both antibodies abrogated all activity in the human GM colony growth assay, we conclude that IL 1-stimulated endothelial cells release both G and GM-CSF and that these are the only granulopoietic factors detectable in clonogenic assays released by these cells in vitro.

scholarly journals Microglial mitogens are produced in the developing and injured mammalian brain.

1991 ◽  
Vol 112 (2) ◽  
pp. 323-333 ◽  
Author(s):  
D Giulian ◽  
B Johnson ◽  
J F Krebs ◽  
J K George ◽  
M Tapscott
Keyword(s):  
Cerebral Cortex ◽  
Growth Factors ◽  
Molecular Mass ◽  
Inflammatory Responses ◽  
Mononuclear Phagocytes ◽  
Mammalian Brain ◽  
Blood Monocytes ◽  
Adult Rats

The central nervous system produces growth factors that stimulate proliferation of ameboid microglia during embryogenesis and after traumatic injury. Two microglial mitogens (MMs) are recovered from the brain of newborn rat. MM1 has an approximate molecular mass of 50 kD and a pI of approximately 6.8; MM2 has a molecular mass of 22 kD and a pI of approximately 5.2. These trypsin-sensitive proteins show specificity of action upon glia in vitro serving as growth factors for ameboid microglia but not astroglia or oligodendroglia. Although the MMs did not stimulate proliferation of blood monocytes or resident peritoneal macrophage, MM1 shows granulocyte macrophage colony-stimulating activity when tested upon bone marrow progenitor cells. Microglial mitogens may help to control brain mononuclear phagocytes in vivo. The MMs first appear in the cerebral cortex of rat during early development with peak levels around embryonic day E-20, a period of microglial proliferation. Microglial mitogens are also produced by traumatized brain of adult rats within 2 d after injury. When infused into the cerebral cortex, MM1 and MM2 elicit large numbers of mononuclear phagocytes at the site of injection. In vitro study shows that astroglia from newborn brain secrete MM2. These observations point to the existence of a regulatory system whereby secretion of proteins from brain glia helps to control neighboring inflammatory responses.

scholarly journals Insights into Endogenous Vs Exogenous Cargo-Containing Platelet Alpha-Granules

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1028-1028
Author(s):  
Sergey Zaytsev ◽  
Hyunsook Ahn ◽  
Victoria Stepanova ◽  
M. Anna Kowalska ◽  
Khalil Bdeir ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Ectopic Expression ◽  
Induced Pluripotent Stem Cell ◽  
Data Safety Monitoring Board ◽  
Factor V ◽  
Single Chain ◽  
Post Injury ◽  
Nsg Mice ◽  
The Media

Abstract Alpha granules in megakaryocytes contain a mixture of endogenously expressed proteins as well as proteins taken up from the intramedullar fluid. Both pools are thought to be found in all alpha granules in the megakaryocytes and released platelets. We have been studying the ectopic expression of urokinase (uPA) in platelets as a targeting strategy for fibrinolysis of nascent thrombi without causing fibrinolysis of established thrombi. These studies also demonstrated that there are two distinct pools of alpha granules, an endogenous cargo pool of granules and an exogenous uptake cargo pool of granules. Using in vitro grown megakaryocytes from two sources (1) CD34+-hematopoietic progenitor cells and (2) induced-pluripotent stem cell derived line imMKCL kindly provided by Dr. Koji Eto at Kyoto University, we demonstrated that urokinase can be localized within alpha granules in the megakaryocytes by either adding urokinase to the media or by ectopically expressing the protein using a lentiviral strategy. We observed that both a human single-chain uPA (scuPA) or a plasmin-insensitive but thrombin-activatable truncated human uPA mutant (uPA-T) in the media were internalized into granules distinct from granules containing ectopically expressed mouse scuPA following lentiviral transduction. Endocytosed uPA showed no co-localization with endogenous von Willebrand Factor (vWF), but significant colocalization with endocytosed Factor V or plasminogen (PLG) on confocal immunofluorescent microscopy. Further, Factor V competed with both uPA variants for uptake from the media. Uptake of these proteins was inhibited by the LRP1 antagonist receptor-associated protein (RAP) and by anti-LRP1 antibodies. This suggests that both proteins use the same endocytic receptor pathway and share this pathway with other proteins taken up from the media, including Factor V. We found that in vitro-generated CD34+ megakaryocytes pre-loaded with exogenously added PLG and co-incubated thereafter with recombinant scuPA and FV significantly degraded FV; however, no vWF degradation was observed in CD34+-derived megakaryocytes that had endocytosed or ectopically expressed scuPA with exogenously added PLG, suggesting that only the proteins which are endocytosed by in vitro-generated megakaryocytes are degraded by uPA-generated plasmin, whereas endogenous alpha-granular proteins remain intact. We then asked whether uPA localized in these two distinct pools can be released at sites of nascent thrombus formation and be effective in preventing nascent thrombus growth. We infused CD34+-derived MKs into NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human GPIb/IX). NSG/VWF R1326H mice have impaired clotting after vascular injury compared to NSG mice unless infused with human platelets or MKs . Significantly less post-injury clotting was seen upon infusion of either endogenous or exogenous scuPA-containing MK infusion. Further studies to define relative efficacy at the same levels of scuPA are being pursued. These studies show that there are two sets of alpha granules that remain separate during megakaryopoiesis in vitro: granules with endogenously expressed cargo and granules with endocytosed cargo with limited mixing between the two pools by confocal microscopy studies and following PLG uptake studies. The extent of mixing that occurs subsequently in released platelets was not studied nor has these finding been done with primary MKs not grown in culture; however, we believe that these studies extend our understanding of the nature of alpha granules and offer new insights into how to manipulate their cargo. Disclosures Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

scholarly journals Transcriptome Analysis of The Inflammatory Responses of Bovine Mammary Epithelial Cells: Exploring Immunomodulatory Target Genes for Bovine Mastitis

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 200 ◽  
Author(s):  
Md. Aminul Islam ◽  
Michihiro Takagi ◽  
Kohtaro Fukuyama ◽  
Ryoya Komatsu ◽  
Leonardo Albarracin ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Bacterial Infections ◽  
Target Genes ◽  
Inflammatory Responses ◽  
Bovine Mastitis ◽  
Immune Defense ◽  
Mammary Epithelial ◽  
Inflammatory Factors ◽  
Cytokines And Chemokines

Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1α and IL1β, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1α and IL1β expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

scholarly journals Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2755-2764 ◽  
Author(s):  
NC van de Kar ◽  
LA Monnens ◽  
MA Karmali ◽  
VW van Hinsbergh
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Human Endothelial Cells ◽  
Uremic Syndrome ◽  
Necrosis Factor

Abstract The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin- producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha- treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.

Enhanced Endothelial Cell Apoptosis in Splenic Tissues of Patients With Thrombotic Thrombocytopenic Purpura

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1264-1270 ◽  
Author(s):  
Chau T. Dang ◽  
Margret S. Magid ◽  
Babette Weksler ◽  
Amy Chadburn ◽  
Jeffrey Laurence
Keyword(s):  
Endothelial Cell ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Thrombus Formation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Endothelial Cell Proliferation ◽  
Endothelial Cell Apoptosis ◽  
Thrombocytopenic Purpura ◽  
Related Disorder ◽  
Uremic Syndrome

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy of obscure etiology. The fundamental pathologic lesion is a hyaline thrombus composed of platelets and some fibrin accompanied by endothelial cell proliferation and detachment, in the absence of an inflammatory response. We have previously demonstrated that plasmas from patients with both idiopathic TTP and a related disorder, sporadic hemolytic-uremic syndrome (HUS), induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) in vitro in those lineages of microvascular endothelial cells (MVECs) that are affected pathologically. We now demonstrate the presence of enhanced MVEC apoptosis in splenic tissues from patients with TTP, documented by terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL) and morphology. This is accompanied by elevated Fas expression. It contrasts with the absence of apoptosis in splenic tissues obtained after splenectomy for trauma or immune thrombocytopenic purpura. TUNEL-positive cells, identified by immunohistochemistry as MVECs or macrophages, presumably engulfing apoptotic ECs, are noted in numerous areas, including those apart from microthrombi. Thus, it is unlikely that EC apoptosis is simply a sequela of thrombus formation. Based on these data, we propose that MVEC apoptosis is of pathophysiologic significance in idiopathic TTP/sporadic HUS.

scholarly journals The Production of Nitric Oxide, IL-6, and TNF-Alpha in Palmitate-Stimulated PBMNCs Is Enhanced through Hyperglycemia in Diabetes

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Caroline Maria Oliveira Volpe ◽  
Luana Farnese Machado Abreu ◽  
Pollyanna Stephanie Gomes ◽  
Raquel Miranda Gonzaga ◽  
Clara Araújo Veloso ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Responses ◽  
Combined Therapy ◽  
Tnf Alpha ◽  
Low Grade ◽  
Simultaneous Control ◽  
Inflammatory Processes ◽  
Low Grade Inflammation

We examined nitric oxide (NO), IL-6, and TNF-αsecretion from cultured palmitate-stimulated PBMNCs or in the plasma from type 2 diabetes mellitus (T2MD) patients or nondiabetic (ND) controls. Free fatty acids (FFA) have been suggested to induce chronic low-grade inflammation, activate the innate immune system, and cause deleterious effects on vascular cells and other tissues through inflammatory processes. The levels of NO, IL-6, TNF-α, and MDA were higher in supernatant of palmitate stimulated blood cells (PBMNC) or from plasma from patients. The results obtained in the present study demonstrated that hyperglycemia in diabetes exacerbatesin vitroinflammatory responses in PBMNCs stimulated with high levels of SFA (palmitate). These results suggest that hyperglycemia primes PBMNCs for NO, IL-6, and TNF-alpha secretion underin vitroFFA stimulation are associated with the secretion of inflammatory biomarkers in diabetes. A combined therapy targeting signaling pathways activated by hyperglycemia in conjunction with simultaneous control of hyperglycemia and hypertriglyceridemia would be suggested for controlling the progress of diabetic complications.

scholarly journals Quantitative Proteomic Analysis of Mouse Sciatic Nerve Reveals Post-injury Upregulation of ADP-Dependent Glucokinase Promoting Macrophage Phagocytosis

2021 ◽  
Vol 14 ◽  
Author(s):  
Kai Zhang ◽  
Qingyao Wang ◽  
Yiyao Liang ◽  
Yu Yan ◽  
Haiqiong Wang ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Nerve Injury ◽  
Proteomic Analysis ◽  
Inflammatory Responses ◽  
Post Injury ◽  
Injured Nerve ◽  
Protein Levels ◽  
Macrophage Phagocytosis

Nerve injury induces profound and complex changes at molecular and cellular levels, leading to axonal self-destruction as well as immune and inflammatory responses that may further promote neurodegeneration. To better understand how neural injury changes the proteome within the injured nerve, we set up a mouse model of sciatic nerve injury (SNI) and conducted an unbiased, quantitative proteomic study followed by biochemical assays to confirm some of the changed proteins. Among them, the protein levels of ADP-dependent glucokinase (ADPGK) were significantly increased in the injured sciatic nerve. Further examination indicated that ADPGK was specifically expressed and upregulated in macrophages but not neurons or Schwann cells upon injury. Furthermore, culturing immortalized bone marrow-derived macrophages (iBMDMs) in vitro with the conditioned media from transected axons of mouse dorsal root ganglion (DRG) neurons induced ADPGK upregulation in iBMDMs, suggesting that injured axons could promote ADPGK expression in macrophages non-cell autonomously. Finally, we showed that overexpression of ADPGK per se did not activate macrophages but promoted the phagocytotic activity of lipopolysaccharides (LPS)-treated macrophages. Together, this proteomic analysis reveals interesting changes of many proteins within the injured nerve and our data identify ADPGK as an important in vivo booster of injury-induced macrophage phagocytosis.

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