RESUMO
BACKGROUND: Ischemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate, triggering microglial activation and causing a thromboinflammatory response, culminating in endothelial activation and vascular disruption. This is further aggravated by ischemia-reperfusion injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates extracellular adenosine triphosphate by hydrolyzing it to adenosine, which has antithrombotic and anti-inflammatory properties and reverses ischemia-reperfusion injury. OBJECTIVES: The objective off the study was to determine the efficacy of our therapeutic, anti-VCAM-CD39 in ischaemic stroke. METHODS: We developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion and analyzed at 24 hours. Anti-VCAM-CD39 or control agents (saline, nontargeted CD39, or anti-VCAM-inactive CD39) were given at 3 hours after middle cerebral artery occlusion. RESULTS: Anti-VCAM-CD39 treatment reduced neurologic deficit; magnetic resonance imaging confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood-brain barrier integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tissue plasminogen activator [tPA]) further reduced infarct volumes and attenuated blood-brain barrier permeability with no associated increase in intracranial hemorrhage. CONCLUSION: Anti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 hours postischemia and may further synergize with thrombolytic therapy to improve stroke outcomes.
Assuntos
Antígenos CD , Apirase , Barreira Hematoencefálica , Infarto da Artéria Cerebral Média , AVC Isquêmico , Molécula 1 de Adesão de Célula Vascular , Animais , Humanos , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antígenos CD/metabolismo , Apirase/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Fibrinolíticos/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Allogenic hematopoietic stem cell transplant (allo-HSCT) can lead to sinusoidal obstruction syndrome (SOS) and graft-versus-host disease (GvHD) in some individuals. GvHD is characterised by an immune triggered response that arises due to donor T cells recognizing the recipient tissue as "foreign". SOS results in impaired liver function due to microvascular thrombosis and consequent obstruction of liver sinusoids. Endothelial damage occurs following chemotherapy and allo-HSCT and is strongly associated with GvHD onset as well as hepatic SOS. Animal models of GvHD are rarely clinically relevant, and endothelial dysfunction remains uncharacterised. Here we established and characterised a clinically relevant model of GvHD wherein Balb/C mice were subjected to myeloablative chemotherapy followed by transplantation of bone marrow (BM) cells± splenic T-cells from C57Bl6 mice, resulting in a mismatch of major histocompatibility complexes (MHC). Onset of disease indicated by weight loss and apoptosis in the liver and intestine was discovered at day 6 post-transplant in mice receiving BM+T-cells, with established GvHD detectable by histology of the liver within 3 weeks. Together with significant increases in pro-inflammatory cytokine gene expression in the liver and intestine, histopathological signs of GvHD and a significant increase in CD4+ and CD8+ effector and memory T-cells were seen. Endothelial activation including upregulation of vascular cell adhesion molecule (VCAM)- 1 and downregulation of endothelial nitric oxide synthase (eNOS) as well as thrombosis in the liver indicated concomitant hepatic SOS. Our findings confirm that endothelial activation is an early sign of acute GvHD and SOS in a clinically relevant mouse model of GvHD based on myeloablative chemotherapy. Preventing endothelial activation may be a viable therapeutic strategy to prevent GvHD.
Assuntos
Células Endoteliais/metabolismo , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T/transplante , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Agonistas Mieloablativos/toxicidade , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodosRESUMO
Global coagulation assays (GCAs) may provide a more comprehensive individual hemostatic profiling. We aim to evaluate GCAs (thromboelastography, thrombin generation) in healthy controls, and correlate results with age, gender, lipid status, tissue factor pathway inhibitor (TFPI) and P-selectin. Blood samples were collected from healthy controls (> 18 years of age) not taking anticoagulation or antiplatelet agents and without known cardiovascular disease. Thromboelastography (TEG) was performed on citrated whole blood while calibrated automated thrombogram (CAT), P-selectin (endothelial marker) and TFPI (principle inhibitor of tissue factor-initiated coagulation) were performed on platelet-poor plasma. 153 healthy controls (mean age 42 years, 98 females (64%)) were recruited. Female controls demonstrated more hypercoagulable TEG and CAT parameters while those over 50 years of age demonstrated more hypercoagulable TEG parameters despite comparable thrombin generation. Paradoxically, individuals with "flattened" thrombin curves (lower velocity index (rate of thrombin generation) despite preserved endogenous thrombin potential (amount of thrombin)) were more likely to be male (49% vs 20%, p = 0.003) with increased low-density lipoprotein cholesterol (3.3 vs 2.6 mmol/L, p = 0.003), P-selectin (54.2 vs 47.3 ng/mL, p = 0.038) and TFPI (18.7 vs 8.6 ng/ml, p = 0.001). In addition to reduced velocity index and thrombin peak, controls in the highest TFPI tertile also demonstrated a poorer lipid profile. GCAs can detect subtle changes of the hemostatic profile. Interestingly, reduced thrombin generation was paradoxically associated with increased cardiovascular risk factors, possibly attributable to increased TFPI. This finding may suggest compensation by the coagulation system in response to endothelial activation and represent a biomarker for early cardiovascular disease. A larger prospective study evaluating these assays in the cardiovascular disease population is ongoing.
Assuntos
Doenças Cardiovasculares , Hemostáticos , Testes de Coagulação Sanguínea , Feminino , Humanos , Masculino , Selectina-P , Estudos Prospectivos , TrombinaRESUMO
Stroke is caused by obstructed blood flow (ischaemia) or unrestricted bleeding in the brain (haemorrhage). Global brain ischaemia occurs after restricted cerebral blood flow e.g. during cardiac arrest. Following ischaemic injury, restoration of blood flow causes ischaemia-reperfusion (I/R) injury which worsens outcome. Secondary injury mechanisms after any stroke are similar, and encompass inflammation, endothelial dysfunction, blood-brain barrier (BBB) damage and apoptosis. We developed a new model of transient global forebrain I/R injury (dual carotid artery ligation; DCAL) and compared the manifestations of this injury with those in a conventional I/R injury model (middle-cerebral artery occlusion; MCAo) and with intracerebral haemorrhage (ICH; collagenase model). MRI revealed that DCAL produced smaller bilateral lesions predominantly localised to the striatum, whereas MCAo produced larger focal corticostriatal lesions. After global forebrain ischaemia mice had worse overall neurological scores, although quantitative locomotor assessment showed MCAo and ICH had significantly worsened mobility. BBB breakdown was highest in the DCAL model while apoptotic activity was highest after ICH. VCAM-1 upregulation was specific to ischaemic models only. Differential transcriptional upregulation of pro-inflammatory chemokines and cytokines and TLRs was seen in the three models. Our findings offer a unique insight into the similarities and differences in how biological processes are regulated after different types of stroke. They also establish a platform for analysis of therapies such as endothelial protective and anti-inflammatory agents that can be applied to all types of stroke.
Assuntos
Circulação Cerebrovascular/fisiologia , Acidente Vascular Cerebral Hemorrágico/patologia , AVC Isquêmico/patologia , Prosencéfalo/irrigação sanguínea , Traumatismo por Reperfusão/patologia , Animais , Anti-Inflamatórios/uso terapêutico , Apoptose/imunologia , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Artérias Carótidas/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Colagenases/administração & dosagem , Colagenases/efeitos adversos , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Acidente Vascular Cerebral Hemorrágico/tratamento farmacológico , Acidente Vascular Cerebral Hemorrágico/imunologia , Acidente Vascular Cerebral Hemorrágico/fisiopatologia , Humanos , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/imunologia , AVC Isquêmico/fisiopatologia , Ligadura , Locomoção/fisiologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Artéria Cerebral Média/fisiopatologia , Prosencéfalo/diagnóstico por imagem , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/patologia , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/fisiopatologia , Receptores Toll-Like/genética , Ativação Transcricional/imunologiaRESUMO
BACKGROUND: Arterial thrombosis models are important for preclinical evaluation of antithrombotics but how anesthetic protocol can influence experimental results is not studied. OBJECTIVES: We studied how three most commonly used rodent anesthetics affect the induction of thrombosis and thrombus resolution with antiplatelet agent integrilin (Eptifibatide). METHODS: The Folts, electrolytic, and FeCl3 models of carotid artery thrombosis were evaluated. The extent of blood flow reduction required to elicit cyclic flow reductions (CFR) was examined in the Folts model. The occlusion time and stability following electrolytic or FeCl3 injury was assessed. The efficacy of Eptifibatide was studied in each cohort and clot composition following FeCl3 application was assessed histologically. RESULTS: Isoflurane and ketamine-xylazine (ket-x) elicited higher basal blood flow velocities. For reliable CFR in the Folts model, a higher degree of blood flow reduction was required under ket-x and isoflurane. For the FeCl3 and electrolytic models, injury severity had to be increased in mice under ket-x anesthesia to achieve rapid occlusion. FeCl3-injured artery sections from ket-x and isoflurane-treated mice showed vessel dilatation and clots that were more fibrin/red-cell rich compared to pentobarbitone. Integrilin led to cycle abolishment for all three Folts-injury cohorts but for the electrolytic model a 2.5-fold higher dose was required to restore blood flow under pentobarbitone. Integrilin after FeCl3 arterial injury was partially ineffective in isoflurane-treated mice. CONCLUSIONS: Anesthesia impacts rodent carotid artery occlusion experiments and alters integrilin efficacy. It is important to consider anesthetic protocols in animal experiments involving pharmacological agents for treatment of atherothrombosis.
RESUMO
OBJECTIVE: Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS. METHODS: We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity. RESULTS: aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression. CONCLUSION: We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.
Assuntos
Aborto Espontâneo/imunologia , Anticorpos Antifosfolipídeos/metabolismo , Antígenos CD/metabolismo , Síndrome Antifosfolipídica/imunologia , Apirase/metabolismo , Complicações na Gravidez/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adulto , Animais , Antígenos CD/genética , Apirase/genética , Complemento C3d/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunização Passiva , Inflamação , Mediadores da Inflamação/metabolismo , Peroxidação de Lipídeos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Tromboplastina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Kidney ischemia-reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.
Assuntos
Antígenos CD/farmacologia , Apirase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fibrinolíticos/farmacologia , Rim/efeitos dos fármacos , Traumatismo por Reperfusão , Animais , Plaquetas/efeitos dos fármacos , Microambiente Celular/fisiologia , Humanos , Rim/irrigação sanguínea , Glicoproteínas de Membrana/farmacologia , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose â¼10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.
Assuntos
Antígenos CD/uso terapêutico , Apirase/uso terapêutico , Fibrinolíticos/uso terapêutico , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Trombose/tratamento farmacológico , Difosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Sistemas de Liberação de Medicamentos , Fibrinolíticos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genética , Trombose/metabolismo , Trombose/patologiaRESUMO
BACKGROUND: The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH. RESULTS: We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity. CONCLUSIONS: The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.
RESUMO
OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.
Assuntos
Difosfato de Adenosina/sangue , Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacologia , Hipotermia Induzida , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Análise de Variância , Animais , Antígenos CD/sangue , Antígenos CD/farmacologia , Apirase/sangue , Apirase/farmacologia , Plaquetas/metabolismo , Fibrinogênio/metabolismo , Humanos , Hidrólise , Hipotermia Induzida/efeitos adversos , Leucopenia/sangue , Leucopenia/etiologia , Glicoproteínas de Membrana/sangue , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/sangue , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y1/sangue , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Receptores Purinérgicos P2Y12/sangue , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Trombocitopenia/sangue , Trombocitopenia/etiologia , Trombose/sangue , Trombose/etiologia , Fator de von Willebrand/metabolismoRESUMO
Deletion of the long arm of one chromosome 20 (del(20q)) is a well-recognized abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and is presumed to cause loss of a tumor suppressor gene at 20q12. In a previously published series of MDS and AML cases, which had lost this region via unbalanced translocation, around 40% of cases were shown to have additional copies of the chromosome 20 abnormalities, with resulting gain or amplification of the retained parts of chromosome 20, most often 20q11.2. We have used FISH and array comparative genomic hybridization, to define further the retained and amplified regions. We now report targeted amplification of 20q11.21 in four of the 22 cases selected for further study and in one new case. The shortest amplified region of 250 kb in a series of five patients with three to ten copies of the 20q11.21 region contained the complete HCK, TM9SF4, PLAGL2, and POFUT1 genes. By RT-PCR we have shown that there is correlation between amplification and increased expression of these four genes in most cases. Localized and high level amplification of the common 250 kb region is evidence for activation of an oncogene in this region in these MDS and AML cases. Cases with 20q11.21 amplification tended to have a high proportion of erythroblasts in the marrow, with two cases diagnosed as erythroleukemia (AML-M6). Chromosome sub-band 20q11.21 amplification may therefore prove to be a marker of a specific subset of AML/MDS with a significant erythroid component.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 20 , Síndromes Mielodisplásicas/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Glycosylation of cell surface proteins is important in thymocyte maturation. In particular, the level of sialylation of key glycoproteins such as CD45 is believed to play a major role in regulating TCR signaling, adhesion and apoptosis of developing thymocytes. We show here that transgenic expression of human alpha1-2 fucosyltransferase (hFUT1) in mice resulted in a marked shift from sialylation to fucosylation of thymocyte glycoproteins. This was associated with a significant reduction in thymocyte number, an increased rate of apoptosis in double positive and single positive thymocytes, and a maturation arrest at TCR-dependent developmental transitions reminiscent of CD45 deficiency. Indeed, CD45RB dimerization was elevated in hFUT1 thymocytes, consistent with its hyposialylation, and there was a corresponding increase in phosphorylation of the TCR-associated protein Lck. However, contrary to the reduced TCR signaling in CD45 null mice, basal and stimulated TCR signaling was higher in hFUT1 thymocytes than in wild type thymocytes. Our results therefore demonstrate that aberrant expression of a single glycosyltransferase can profoundly affect thymopoiesis, although the relative involvement of CD45-dependent and -independent mechanisms is yet to be determined.
Assuntos
Apoptose , Diferenciação Celular , Fucosiltransferases/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/enzimologia , Animais , Anexina A5 , Dimerização , Glicosilação , Humanos , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ácido N-Acetilneuramínico , Fosforilação , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
An N-ethyl-N-nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein-N-acetylgalactosamine-3-beta-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1/plt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1-null background. gpIbalpha and podocalyxin were found to be major underglycosylated proteins in plt1/plt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1/plt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.
Assuntos
Galactosiltransferases/metabolismo , Nefropatias/metabolismo , Trombocitopenia/metabolismo , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Linhagem Celular , Proliferação de Células , Feminino , Galactosiltransferases/genética , Glicosilação , Humanos , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Mutação/genética , Taxa de Sobrevida , Trombocitopenia/genética , Trombocitopenia/patologiaRESUMO
Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.
Assuntos
Colágeno Tipo VI/química , Matriz Extracelular/metabolismo , Microfibrilas/química , Adolescente , Linhagem Celular Tumoral , Células Cultivadas , Complemento C5/química , Meios de Cultura/metabolismo , Fibroblastos/metabolismo , Humanos , Masculino , Microfibrilas/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in myotubularin result in the human disease X-linked myotubular myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human myotubularin were identified. Association between 3-PAP and myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with myotubularin led to attenuation of the plasma membrane phenotype, associated with myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an "adapter" for myotubularin, regulating myotubularin intracellular location and thereby altering the phenotype resulting from myotubularin overexpression.
Assuntos
Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Humanos , Técnicas In Vitro , Células K562 , Dados de Sequência Molecular , Mutação , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Fenótipo , Monoéster Fosfórico Hidrolases/genética , Subunidades Proteicas , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the dominantly inherited disorder, Bethlem myopathy. Glycine mutations that interrupt the Gly-X-Y repetitive amino acid sequence that forms the characteristic collagen triple helix have been defined in four families; however, the effects of these mutations on collagen VI biosynthesis, assembly, and structure have not been determined. In this study, we examined the consequences of Bethlem myopathy triple helical glycine mutations in the alpha1(VI) and alpha2(VI) chains, as well as engineered alpha3(VI) triple helical glycine mutations. Although the Bethlem myopathy and introduced mutations that are toward the N terminus of the triple helix did not measurably affect collagen VI intracellular monomer, dimer, or tetramer assembly, or secretion, the introduced mutation toward the C terminus of the helix severely impaired association of the mutant alpha3(VI) chain with alpha1(VI) and alpha2(VI). Association of the three chains was not completely prevented, however; and some non-disulfide bonded tetramers were secreted. Examination of the secreted Bethlem myopathy and engineered mutant collagen VI by negative staining electron microscopy revealed the striking finding that in all the cell lines a significant proportion of the tetramers contained a kink in the supercoiled triple helical region. Collagen VI tetramers from all of the mutant cell lines also showed a reduced ability to form microfibrils. These results provide the first evidence of the biosynthetic consequences of collagen VI triple helical glycine mutations and indicate that Bethlem myopathy results not only from the synthesis of reduced amounts of structurally normal protein but also from the presence of mutant collagen VI in the extracellular matrix.