Subject(s)
Adenosine Triphosphate , Pneumonia , Humans , Bleomycin , Apyrase/pharmacology , Hydrolysis , PhagocytesABSTRACT
INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. METHODS: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFß1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions. RESULTS: Tonabersat partially negated glucose/TGFß1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression. CONCLUSION: Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFß1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.
Subject(s)
Diabetes Mellitus , Diabetic Nephropathies , Humans , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Apyrase/metabolism , Apyrase/pharmacology , Communication , Connexin 43/metabolism , Diabetes Mellitus/metabolism , Diabetic Nephropathies/metabolism , Epithelial Cells/metabolism , Fibroblasts/metabolism , Fibrosis , Glucose/pharmacologyABSTRACT
Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) cause gastrointestinal infection and, in severe cases, hemolytic uremic syndrome which may lead to death. There is, to-date, no therapy for this infection. Stx induces ATP release from host cells and ATP signaling mediates its cytotoxic effects. Apyrase cleaves and neutralizes ATP and its effect on Stx and EHEC infection was therefore investigated. Apyrase decreased bacterial RecA and dose-dependently decreased toxin release from E. coli O157:H7 in vitro, demonstrated by reduced phage DNA and protein levels. The effect was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice infected with Stx2-producing E. coli O157:H7 were treated with apyrase intraperitoneally, on days 0 and 2 post-infection, and monitored for 11 days. Apyrase-treated mice developed disease two days later than untreated mice. Untreated infected mice lost significantly more weight than those treated with apyrase. Apyrase-treated mice exhibited less colonic goblet cell depletion and apoptotic cells, as well as lower fecal ATP and Stx2, compared to untreated mice. Apyrase also decreased platelet aggregation induced by co-incubation of human platelet-rich-plasma with Stx2 and E. coli O157 lipopolysaccharide in the presence of collagen. Thus, apyrase had multiple protective effects, reducing RecA levels, stx2 and toxin release from EHEC, reducing fecal Stx2 and protecting mouse intestinal cells, as well as decreasing platelet activation, and could thereby delay the development of disease.
Subject(s)
Bacteriophages , Escherichia coli Infections , Escherichia coli O157 , Gastrointestinal Microbiome , Adenosine Triphosphate/metabolism , Animals , Apyrase/metabolism , Apyrase/pharmacology , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Escherichia coli O157/genetics , Humans , Lipopolysaccharides/metabolism , Mice , Mice, Inbred BALB C , Shiga Toxin/metabolism , Shiga Toxin/pharmacology , Shiga Toxin 2/genetics , Shiga Toxin 2/metabolism , Shiga Toxin 2/pharmacologyABSTRACT
Emerging evidences have linked the air pollution particulate matters, especially the fine particulate matter PM2.5, to the disease development of chronic obstructive pulmonary disease (COPD). Our previous studies reported that biofuel PM2.5 can induce devastated damage of human bronchial epithelial cells, this study aims to further investigate the underlying molecular mechanisms how biofuel PM2.5 induces bronchial epithelial cell death and dysfunction. In this study, biofuel PM2.5 extracted from wood smoke (WSPM2.5) was used according to our previous publication. A 16-HBE cell line was used as the cell model. Results showed that: Firstly, WSPM2.5 induced significant pyroptosis in 16-HBE cells, reflected by the typical changes including elevated release of lactate dehydrogenase release (LDH) and activated activity and expression of Caspase-1/IL-1ß/IL-18 signaling pathway. Then, specific inhibitors for both Caspases (Z-VAD-FMK) and Caspase-1 (VX-765), as well as specific siRNA knockdown of IL-1ß all effectively attenuated the WSPM2.5-induced upregulation of downstream inflammatory cytokines and chemokines (IL-6, IL-8, CXCL-1, CXCL-2, etc), respectively. Notably, WSPM2.5 caused a novel increase of intracellular-to-extracellular ATP secretion, which could also contribute to the WSPM2.5-induced pyroptosis and inflammation by activating the Caspase-1/IL-1ß/IL-18 signaling pathway through possible autocrine and/or paracrine mechanisms. Antagonism of ATP (Apyrase) or specific siRNA knockdown against ATP receptors (P2Y2 and P2Y7) both significantly inhibited the WSPM2.5-induced pyroptosis and inflammation. These results add up to the current knowledge and bring up novel insights that WSPM2.5 could induce significant pyroptosis and inflammation of human bronchial epithelial cells, through both a classic NLRP3/Caspase-1/IL-1ß-dependent and a novel ATP/P2Y-dependent mechanisms.
Subject(s)
Pyroptosis , Smoke , Adenosine Triphosphate/metabolism , Apyrase/metabolism , Apyrase/pharmacology , Biofuels , Caspase 1/genetics , Caspase 1/metabolism , Caspase 1/pharmacology , Epithelial Cells , Humans , Inflammation/metabolism , Interleukin-18/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Lactate Dehydrogenases/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Particulate Matter/pharmacology , Pyroptosis/genetics , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Smoke/adverse effects , Nicotiana/metabolism , Wood/metabolismABSTRACT
Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.
Subject(s)
Platelet Aggregation , Platelet Membrane Glycoproteins , Agammaglobulinaemia Tyrosine Kinase/metabolism , Apyrase/pharmacology , Blood Platelets/metabolism , Collagen/pharmacology , Eptifibatide/pharmacology , Fluorescein-5-isothiocyanate/metabolism , Fluorescein-5-isothiocyanate/pharmacology , Humans , Indomethacin/metabolism , Indomethacin/pharmacology , Intracellular Signaling Peptides and Proteins , Lectins, C-Type/metabolism , Ligands , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein-Tyrosine Kinases , Syk Kinase/metabolism , Tyrosine/metabolism , Tyrosine/pharmacology , src-Family Kinases/metabolismABSTRACT
Pannexin1 (Panx-1) is a gap junction channel protein that mediates the release of intracellular ATP during autophagy, and thus plays an important role in tumor cell apoptosis and chemo-resistance. However, the role of Panx-1 in cisplatin-resistance of testicular cancer cells remains unclear. We found that cisplatin-resistant I-10 testicular cancer cell lines (I-10/CDDP) autophagy-associated proteins (p62, p-mTOR, mTOR and LC3) exhibited high levels of autophagy in their expression, while LC3-II expression was more significantly in the presence of lysosomal degradation blocked by chloroquine (CQ). Xenograft models using I-10/CDDP cells with knockdown ATG5 and ATG7 were established in mouse models and showed blockade of autophagic flux and inhibition of tumor growth. In addition, inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN), as well as shRNA-mediated knockdown promoted autophagy in the I-10/CDDP cells, which was accompanied by a decrease in the levels of extracellular ATP. In contrast, overexpression of Panx-1 decreased autophagy of I-10/CDDP cells and increased extracellular ATP levels. To further determine the effect of panx-1-mediated ATP release on the autophagy of I-10/CDDP cells, apyrase was used to hydrolyze the extracellular ATP. Apyrase promoted autophagy in I-10/CDDP cells city by decreasing extracellular ATP, regardless of Panx-1 expression. This study demonstrated for the first time that Panx-1-mediated ATP release inhibits autophagy of I-10/CDDP cells, which provides a potential therapeutic strategy for cisplatin-resistant testicular cancer.
Subject(s)
Antineoplastic Agents , Cisplatin , Connexins , Nerve Tissue Proteins , Testicular Neoplasms , Adenosine Triphosphate/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Apyrase/pharmacology , Autophagy , Cell Line, Tumor , Cisplatin/pharmacology , Connexins/genetics , Drug Resistance, Neoplasm , Humans , Male , Mice , Neoplasms, Germ Cell and Embryonal , Nerve Tissue Proteins/genetics , TOR Serine-Threonine Kinases/metabolism , Testicular Neoplasms/drug therapy , Testicular Neoplasms/geneticsABSTRACT
The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.
Subject(s)
Biocatalysis , Carbon/chemistry , Carbon/pharmacology , Nitrogen/chemistry , Nitrogen/pharmacology , Oxidative Stress/drug effects , Protein Corona/metabolism , Quantum Dots/chemistry , Quantum Dots/metabolism , A549 Cells , Animals , Apyrase/chemistry , Apyrase/pharmacology , Catalase/chemistry , Catalase/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cellular Microenvironment/drug effects , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/pharmacology , HeLa Cells , Humans , Rats , Reactive Oxygen Species/metabolism , beta-Galactosidase/chemistry , beta-Galactosidase/pharmacologyABSTRACT
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.
Subject(s)
5'-Nucleotidase/pharmacology , Anti-Inflammatory Agents/pharmacology , Apyrase/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Protein Engineering , 5'-Nucleotidase/chemistry , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , Adenine Nucleotides/metabolism , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Apyrase/chemistry , Apyrase/genetics , Apyrase/metabolism , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , GPI-Linked Proteins/pharmacology , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/metabolism , Protein Conformation , Recombinant Fusion Proteins/metabolism , Signal Transduction , Structure-Activity Relationship , Substrate SpecificityABSTRACT
In the current study, we explored the role of extracellular ATP (eATP) in promoting systemic inflammation during development of acute pancreatitis (AP). Release of extracellular (e)ATP was evaluated in plasma and bronchoalveolar lavage fluid (BALF) of mice with experimental acute pancreatitis (AP). Prophylactic intervention using apyrase or suramin was used to understand the role and contribution of eATP in pancreatitis-associated systemic injury. AP of varying severity was induced in C57BL/6 mice using 1-day or 2-day caerulein, caerulein + LPS and l-arginine models. eATP was measured in plasma and BALF. Mice were treated with suramin or apyrase in the caerulein and l-arginine models of AP. Plasma cytokines, lung, and pancreatic myeloperoxidase, and morphometric analysis of pancreatic and lung histology, were used to assess the severity of pancreatitis. Plasma eATP and purinergic 2 (P2) receptors in the pancreas and lungs were significantly elevated in the experimental models of AP. Blocking the effect of eATP by suramin led to reduced levels of plasma IL-6 and TNFα as well as reduced lung, and pancreatic injury. Neutralizing eATP with apyrase reduced systemic injury but did not ameliorate local injury. The results of this study support the role of eATP and P2 receptors in promoting systemic inflammation during AP. Modulating purinergic signaling during AP can be an important therapeutic strategy in controlling systemic inflammation and, thus, systemic inflammatory response syndrome during AP.NEW & NOTEWORTHY Released ATP from injured cells promotes systemic inflammation in acute pancreatitis.
Subject(s)
Adenosine Triphosphate/metabolism , Inflammation/metabolism , Pancreatitis/metabolism , Acute Disease , Adenosine Triphosphate/blood , Animals , Apyrase/pharmacology , Arginine , Bronchoalveolar Lavage Fluid/chemistry , Ceruletide , Cytokines/blood , Cytokines/metabolism , Inflammation/chemically induced , Inflammation/prevention & control , Lung/metabolism , Mice , Mice, Inbred C57BL , Pancreas/metabolism , Pancreatitis/chemically induced , Pancreatitis/prevention & control , Peroxidase/metabolism , Receptors, Purinergic/metabolism , Signal Transduction , Suramin/pharmacologyABSTRACT
BACKGROUND: Dogs with Babesia rossi infection display a normocoagulable thromboelastogram, despite being markedly thrombocytopenic, which is purportedly due to large-scale platelet activation. Thromboelastographic platelet mapping (TEG-PM) evaluates individual contributions of thrombin, fibrinogen, and platelets to clot formation, and may elucidate some of the pathomechanisms of thrombocytopenia-associated hemostatic alterations. OBJECTIVE: This study investigated potential differences in TEG-PM variables in dogs with complicated B rossi infection compared with healthy controls, and whether these variables correlated with platelet activation indices. METHODS: The maximum amplitude (MA) following thrombin generation (MAThrombin ) was determined using kaolin-activated TEG. The TEG-PM variables included MA following the addition of platelet agonists arachidonic acid (MAAA ) and adenosine diphosphate (MAADP ), and MA due to fibrin alone (MAFibrin ). In addition, platelet indices and fibrinogen concentrations were determined. RESULTS: Thirteen dogs with complicated B rossi infection and five healthy controls were included. The median MAFibrin and fibrinogen concentrations were significantly higher (P < 0.01 for both) and median platelet count was significantly lower (P < 0.01) in the babesiosis group vs the control group. No significant differences were found for MAThrombin and MAAA/ADP . maximum amplitude due to fibrin alone was positively correlated with fibrinogen concentration (r = 0.735), mean platelet volume (r = 0.517), and mean platelet mass (r = 0.498), and negatively correlated with hematocrit (r = -0.685), platelet count (r = -0.476), and plateletcrit (r = -0.479) (P < 0.05 for all). CONCLUSIONS: This study suggests that the presence of hyperfibrinogenemia offsets the severe thrombocytopenia associated with B rossi to result in normal thromboelastograms and lack of overt clinical bleeding.
Subject(s)
Babesia , Babesiosis/blood , Blood Platelets/physiology , Dog Diseases/blood , Thrombelastography/veterinary , Animals , Apyrase/pharmacology , Arachidonic Acid/pharmacology , Blood Platelets/chemistry , Blood Platelets/drug effects , Case-Control Studies , Dog Diseases/parasitology , Dogs , Female , Fibrinogen/analysis , MaleSubject(s)
Adenosine Diphosphate/metabolism , Adenylyl Cyclases/metabolism , Blood Platelets/drug effects , Platelet Glycoprotein GPIb-IX Complex/metabolism , Receptor, PAR-1/agonists , Apyrase/pharmacology , Blood Platelets/cytology , Blood Platelets/metabolism , Humans , Peptide Fragments/pharmacology , Receptor, PAR-1/metabolism , Receptors, Purinergic P2Y12/metabolism , Thrombin/metabolismABSTRACT
BACKGROUND: Whole blood impedance platelet aggregometry (Multiplate-) can be performed with different agonists to evaluate platelet function. Although the manufacturer recommends disposal of stored reagents after 1 month at -20°C or 24 hours at 4°C, reagent integrity after reconstitution under different storage conditions is unknown. If reagent integrity is stable for longer periods, assay costs could decrease dramatically. OBJECTIVES: This study aimed to determine the stability of reconstituted arachidonic acid (AA) and adenosine diphosphate (ADP) platelet agonists stored at -20°C and -80°C for up to 6 months. METHODS: Aliquots of reconstituted AA and ADP were stored at -20°C and -80°C each month for a total of 6 months. Six healthy staff-owned dogs were enrolled in the study. A physical examination, CBC, diagnostic panel, urinalysis, and baseline platelet aggregometry assessment was performed on all of the dogs. Platelet aggregometry was performed using fresh and stored aliquots of AA and ADP reagents on the same day. The area under the curve (AUC) was recorded from each platelet aggregometry analysis. Repeated measures (RM) analysis (one-way ANOVA) was performed and subsequent time points (1, 2, 3, 4, 5, and 6 months) were compared with fresh AUC results. RESULTS: All dogs were clinically healthy, and all diagnostic tests were normal. There were no differences in AUC obtained from fresh samples at any time point or either temperature for AA or ADP. CONCLUSIONS: Whole blood impedance platelet aggregometry reagents, AA and ADP, were stable for up to 6 months when stored at -20°C or -80°C, obviating the need to discard viable reagents, and decreasing assay costs.
Subject(s)
Apyrase/pharmacology , Arachidonic Acid/pharmacology , Blood Specimen Collection/veterinary , Platelet Aggregation , Platelet Function Tests/veterinary , Animals , Blood Platelets/drug effects , Blood Specimen Collection/methods , Dogs/blood , Electric Impedance , Platelet Aggregation/drug effectsABSTRACT
INTRODUCTION: MSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology. METHODS: Bone marrow derived MSCs were exposed to ATP for 4 days, in the presence of 100 ng IFNγ/mL. Intracellular expression of IDO in MSCs was assessed by FACS. Conditioned media from thus stimulated MSCs was analyzed for kynurenine content and its suppressive effect on lymphocyte proliferation. Apyrase or P2 × 7-receptor antagonist (AZ 11645373) were applied in order to inhibit ATP induced effect on MSCs. RESULTS: We demonstrate, that ATP at concentrations between 0.062 and 0.5 mM increases dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentrations within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2 × 7-receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP, inhibited lymphocyte proliferation from 65% to 16%. CONCLUSIONS: We characterized ATP as a DAMP family member responsible for necrosis-induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment.
Subject(s)
Adenosine Triphosphate/pharmacology , Immune Tolerance , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Mesenchymal Stem Cells/immunology , Apyrase/pharmacology , Cell Proliferation , Cells, Cultured , Culture Media, Conditioned/chemistry , Humans , Immunomodulation , Kynurenine/metabolism , Lymphocyte Activation , Tryptophan/metabolismABSTRACT
OBJECTIVE: The objective of this study was to investigate the effect of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) during acute antibody-mediated rejection (AMR). METHODS: NTPDase1 overexpression, NTPDase1 knockout, and wild-type nude mice skin graft models were used to induce acute AMR. NTPDase1 expression in B cells, NTPDase1 messenger RNA expression in skin grafts, extracellular adenosine diphosphate (ADP) concentration, B-cell volume and surface antigens expression, average platelet transport rate, and ultrastructure and apoptosis of skin graft cells were investigated. RESULTS: During acute AMR in nude mice, higher NTPDase1 expression caused lower extracellular ADP concentration, smaller increase in B-cell volume, and major histocompatibility complex II surface antigen expression, suggesting a negative correlation between them; higher NTPDase1 expression also caused slower average platelet transport rate and less severe skin graft injury, suggesting a negative correlation between them. Pretreatment with high-dose exogenous NTPDase1 inhibited platelet activation and protected skin grafts, but it resulted in prolonged bleeding time (by 51.4%) and prolonged coagulation time (by 44.1%). CONCLUSION: An NTPDase1-associated imbalance in extracellular ADP degradation may contribute to B-cell activation, platelet activation, and more severe skin graft injury in nude mice. Pretreatment with high-dose exogenous NTPDase1 effectively protected skin grafts in nude mice at 1 week, but it increased the risk of bleeding.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/physiology , Antigens, CD/metabolism , Apyrase/metabolism , B-Lymphocytes/physiology , Graft Rejection/enzymology , Platelet Activation/physiology , Skin Transplantation , Animals , Antigens, CD/genetics , Antigens, CD/pharmacology , Apyrase/genetics , Apyrase/pharmacology , Cell Size , Graft Rejection/immunology , Lymphocyte Activation/physiology , Male , Mice , Mice, Nude , RNA, Messenger/metabolismABSTRACT
Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis.
Subject(s)
Adenosine Triphosphate/metabolism , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/metabolism , Cell Movement/drug effects , S100 Calcium-Binding Protein A4/metabolism , Animals , Apyrase/pharmacology , Cancer-Associated Fibroblasts/drug effects , Female , Gene Expression Profiling , Gene Knockdown Techniques , Humans , MCF-7 Cells , Mice , Mice, Nude , Niclosamide/pharmacology , Oligonucleotide Array Sequence Analysis , Primary Cell Culture , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , S100 Calcium-Binding Protein A4/genetics , Up-RegulationABSTRACT
A novel apyrase from Russell's viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell's viper apyrase). It is a high molecular weight (79.4â¯kDa) monomeric glycoprotein that contains 2.4% neutral sugars and 58.4% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (pâ¯<â¯.05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5'-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54⯵M and 615⯵M of Piâ¯releasedâ¯min-1, respectively with a turnover number (Kcat) of 24,600â¯min-1. Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92â¯nM and 1.25â¯nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom.
Subject(s)
Antivenins/chemistry , Apyrase/chemistry , Daboia , Viper Venoms/enzymology , Animals , Anticoagulants , Antivenins/pharmacology , Apyrase/isolation & purification , Apyrase/pharmacology , Blood Platelets/drug effects , Humans , Platelet Aggregation/drug effectsABSTRACT
In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Microvessels/drug effects , Polyphosphates/pharmacology , Adenosine Triphosphate/metabolism , Alkaline Phosphatase/metabolism , Apyrase/pharmacology , Cell Line, Tumor , Cells, Cultured , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Microvessels/metabolism , Microvessels/ultrastructureABSTRACT
Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro-inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1-methyl-4-phenylpyridinium (MPP+ ) treatment increased the level of UDP/P2Y6R on neuronal SH-SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP+ -induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down-regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP+ , which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and p38 (p-p38) caused by MPP+ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP+ , suggesting that P2Y6R may play an important role in the pathogenesis of PD.
Subject(s)
Cell Death/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Herbicides/pharmacology , Oxidative Stress/drug effects , Receptors, Purinergic P2/metabolism , 1-Methyl-4-phenylpyridinium/pharmacology , Apyrase/pharmacology , Cell Line, Tumor , Humans , Isothiocyanates/pharmacology , Malondialdehyde/metabolism , Neuroblastoma/pathology , Nitric Oxide Synthase Type II/metabolism , Purinergic P2 Receptor Agonists/pharmacology , Purinergic P2 Receptor Antagonists/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Receptors, Purinergic P2/genetics , Superoxide Dismutase-1/metabolism , Thiourea/analogs & derivatives , Thiourea/pharmacology , Transfection , Uridine Diphosphate/pharmacologyABSTRACT
Cisplatin (DDP) is the most commonly used drug in testicular cancer. However, drug resistance severely limits its clinical use and the underlying mechanisms need to be further clarified. The aim of present study was to investigate the role of ATP/IP3 pathway mediated by pannexin1 (Panx-1) channels on DDP-induced apoptosis and to reveal the potential mechanisms of DDP-resistance in testicular cancer. We found that the expression of Panx-1 in I-10/DDP cells (DDP-resistance) was decreased compared with parental I-10 cells determined by western blotting and immunofluorescence assay. To further clarify the role of Panx-1 in DDP resistance, Panx-1 function was modulated by overexpression and knockdown of Panx-1 expression. Panx-1 overexpression increased DDP-induced apoptosis, ATP release and IP3 levels. On the contrary, Panx-1 silencing decreased DDP-induced apoptosis, ATP release and IP3 levels. Apyrase (hydrolyzing extracellular ATP) or xestospongin C (antagonizing IP3 receptor) also decreased DDP-induced apoptosis. Our findings demonstrate that Panx-1 is involved in DDP-resistance and ATP/IP3 pathway mediated by Panx-1 channels participates in DDP-induced apoptosis in testicular cancer. Panx-1 modulation may be interesting to amplify the clinical effect of DDP and reverse the resistance of testicular cancer cells to DDP.
Subject(s)
Adenosine Triphosphate/metabolism , Cisplatin/pharmacology , Connexins/metabolism , Drug Resistance, Neoplasm/drug effects , Inositol 1,4,5-Trisphosphate/metabolism , Nerve Tissue Proteins/metabolism , Testicular Neoplasms/metabolism , Testicular Neoplasms/pathology , Animals , Apoptosis/drug effects , Apyrase/pharmacology , Cell Line, Tumor , Gene Knockdown Techniques , Gene Silencing/drug effects , Humans , Macrocyclic Compounds/pharmacology , Male , Mice , Oxazoles/pharmacology , Signal Transduction/drug effectsABSTRACT
Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 µM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.