RESUMO
The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.
Assuntos
Antimaláricos , Peptídeos , Plasmodium falciparum , Fator Plaquetário 4 , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/farmacologia , Antimaláricos/química , Humanos , Fator Plaquetário 4/química , Fator Plaquetário 4/farmacologia , Peptídeos/farmacologia , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
PF4 is a pro-atherosclerotic molecule. Endothelial CD40, upon binding to its ligand CD40L, induces endothelial cell (EC) activation, which is a vital pathophysiological process in the initiation and progression of atherosclerosis. However, the relationship between PF4 and endothelial CD40 remains elusive. This study aims to investigate whether and how PF4 affects endothelial CD40 expression using primary HAECs. PF4 treatment down-regulated sirtuin 1 (SIRT1) expression but upregulated the expression of acetylated NF-κB p65 (Ac-p65) and CD40 in HAECs in a concentration- and time-dependent manner. Pretreatment with SIRT1 agonist (SRT1720 or RSV) or SIRT1-overexpressing lentivirus attenuated PF4-induced Ac-p65 and CD40 expression in HAECs, whereas preincubation with SIRT1 antagonist (NAM or EX527) or SIRT1 shRNA had the opposite effect. To investigate whether NF-κB/p65 signaling pathway modulates CD40 expression in PF4-treated HAECs, PDTC, a NF-κB inhibitor, and p65-shRNA were introduced. PDTC or p65-shRNA treatment down-regulated Ac-p65 expression in HAECs. PDTC or p65-shRNA preincubation suppressed CD40 expression in HAECs after PF4 treatment. To better determine whether SIRT1 regulates CD40 expression in PF4-treated HAECs via the NF-κB/p65 signaling pathway, p65-knockdown HAECs were preincubated with SIRT1 agonists before PF4 treatment. SIRT1 agonist preincubation further decreased CD40 expression in p65-knockdown HAECs treated with PF4. Moreover, PF4 treatment promoted p65 nuclear translocation in HAECs. The results of dual luciferase assay demonstrated that four NF-κB binding sites in the promoter of human CD40 gene were activated in PF4-treated HAECs. In conclusion, our findings suggest that PF4 treatment facilitates CD40 expression in HAECs through the SIRT1/NF-κB/p65 pathway.
Assuntos
NF-kappa B , Sirtuína 1 , Humanos , Animais , NF-kappa B/metabolismo , Sirtuína 1/genética , Células Endoteliais , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/farmacologia , Transdução de Sinais , RNA Interferente Pequeno/metabolismoRESUMO
Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.
Assuntos
Envelhecimento , Cognição , Disfunção Cognitiva , Doenças Neuroinflamatórias , Nootrópicos , Fator Plaquetário 4 , Animais , Masculino , Camundongos , Envelhecimento/sangue , Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Cognição/efeitos dos fármacos , Cognição/fisiologia , Doenças Neuroinflamatórias/sangue , Doenças Neuroinflamatórias/complicações , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/prevenção & controle , Fator Plaquetário 4/sangue , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/farmacologia , Fator Plaquetário 4/uso terapêutico , Nootrópicos/sangue , Nootrópicos/metabolismo , Nootrópicos/farmacologia , Nootrópicos/uso terapêutico , Plasma/química , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Disfunção Cognitiva/sangue , Disfunção Cognitiva/complicações , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/prevenção & controle , Transcrição Gênica/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacosRESUMO
BACKGROUND: The effect of platelet factor 4 (PF4) on bone marrow mesenchymal stem cells (BMMSCs) and osteoporosis is poorly understood. Therefore, this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism. METHODS: First, in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry, respectively. Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S. Next, an osteoporotic mouse model was established by performing bilateral ovariectomy (OVX). Furthermore, the PF4 concentrations were obtained using enzyme-linked immunosorbent assay. The bone microarchitecture of the femur was evaluated using microCT and histological analyses. Finally, the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting. RESULTS: Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs. Furthermore, the levels of PF4 in the serum and bone marrow were generally increased, whereas bone microarchitecture deteriorated due to OVX. Moreover, in vivo mouse PF4 supplementation triggered bone deterioration of the femur. In addition, several key regulators of osteogenesis were downregulated, and the integrin α5-focal adhesion kinase-extracellular signal-regulated kinase (ITGA5-FAK-ERK) pathway was inhibited due to PF4 supplementation. CONCLUSIONS: PF4 may be attributed to OVX-induced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.
Assuntos
Integrina alfa5 , Osteogênese , Animais , Feminino , Humanos , Camundongos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/farmacologia , Sistema de Sinalização das MAP Quinases , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Arnebia euchroma (Royle) I.M.Johnst. (AE) is a Chinese medicinal herb that is traditionally used to treat various circulatory diseases. It exhibits certain effects, such as the promotion of blood circulation and cooling, rash clearance, and detoxification. AIM OF THE STUDY: This study was designed to explore the hepatoprotective and hemostatic effects of the ethyl acetate extract of AE in rats with carbon tetrachloride (CCl4)-induced liver injury. MATERIALS AND METHODS: Wistar rats were treated via oral gavage with different doses of the ethyl acetate extract of AE (3.5, 7, or 14 g kg-1·day-1) for 14 consecutive days, following which hemostatic and liver function tests were conducted. For the hemostatic tests, the platelet count, blood platelet aggregation, blood platelet adhesion to fibrinogen, platelet factor 4 (PF-4) secretion from blood platelets, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen levels were measured at the end of the treatment period. For the liver function tests, 0.25 mL/200 g (1.25 mL kg-1·day-1) of olive oil was injected into the abdominal cavity of the control rats, whereas 15% CCl4 plus olive oil (prescription: 7.5 mL CCl4 + 42.5 olive oil) was injected into that of the treated rats at 1 h after extract administration on day 6, 13, and 20. Additionally, food and water were withheld from all the animals. On the following day, the rats were anesthetized and their albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels were measured. Glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx) levels among the groups were determined using a one-way analysis of variance. RESULTS: The platelet count and blood platelet aggregation, blood platelet adhesion to fibrinogen and PF-4 secretion levels were significantly increased in the (3.5 g kg-1 day-1) AE group as compared to those in the control group (all p < 0.001; for the 7 and 14 g kg-1 day-1 AE groups, all p > 0.05, respectively). Although the PT and aPTT were not affected by the AE extract (all p > 0.05), the TT was reduced and the FIB levels were significantly increased in all AE groups (p < 0.05). Liver function tests showed that CCl4 caused significant liver damage, thereby decreasing the albumin, SOD, CAT, GSH, GST, GR, and GPx levels, while increasing the AST, ALT, ALP, SGOT, SGPT, GGT, LDH, ROS, and MDA levels (all p < 0.001). By contrast, treatment with the different doses of AE extract reversed the CCl4 effects on all these parameters. Compared with the levels in the CCl4 group, the GSH and GR levels in the three AE groups (3.5, 7, and 14 g kg-1·day-1) were significantly higher (p < 0.05, p < 0.01, and p < 0.001, respectively), whereas the differences in the other parameters for these three groups were all at the significance levels of p < 0.05, p < 0.05, and p < 0.01, respectively. CONCLUSIONS: AE extracts administered orally exhibited hepatoprotective activity by affecting platelet production and blood coagulation and ameliorating liver function-damaging modifications. Specifically, a dosage of 3.5 g kg-1·day-1 resulted in the most optimal effects.
Assuntos
Boraginaceae , Doença Hepática Induzida por Substâncias e Drogas , Hemostáticos , Plantas Medicinais , Acetatos , Alanina Transaminase , Albuminas/farmacologia , Aldeídos , Fosfatase Alcalina , Animais , Antioxidantes/farmacologia , Aspartato Aminotransferases , Tetracloreto de Carbono/farmacologia , Catalase , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fibrinogênio , Glutationa/farmacologia , Glutationa Peroxidase , Glutationa Redutase , Glutationa Transferase , Hemostáticos/farmacologia , Lactato Desidrogenases , Fígado , Metano/farmacologia , Azeite de Oliva , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Fator Plaquetário 4/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio , Superóxido Dismutase , gama-GlutamiltransferaseRESUMO
Intracerebral haemorrhage (ICH) is a lethal cerebrovascular disorder with a high mortality and morbidity. Although it is a major public health problem, there is no effective treatment for ICH. After ICH, the primary and secondary mechanisms are mentioned when discussing brain injury. The transcription factor, nuclear factor-kappa B (NF-kB), is an important regulator of inflammatory responses. The role of platelet factor 4 (PF4) in ICH is unclear. To study the effect of PF4 on inflammatory response of rats in ICH, a rat model of striatum ICH was established by injecting autologous blood from the autogenous femoral artery into the right striatum of rats. Forty-eight hours after ICH, the expression of PF4, NF-kB (P-P65) and inflammatory changes in rats were determined with WB and ELISA. Heme was used to induce PC12 cell damage, simulate the ICH model in vitro, and detect PF4, P-P65 and striatal inflammatory changes. Short hairpin RNA (shRNA-PF4) was used to knock-down the expression of PF4 in PC12 cells to detect changes in inflammatory factors. The results showed that 48 hours after surgery, the behavioural score of cerebral haemorrhage was the lowest. The expression of PF4 and P-P65 in the striatum of the ICH group was significantly higher compared with the sham surgery group. The expression of interleukin (IL)-6 and IL-1b in the ICH group was also greatly improved. After inhibiting NF-kB expression, PF4 expression was decreased. In short, ICH enhances the expression of PF4, which induces an inflammatory response in rats with cerebral haemorrhage through the NF-kB signalling pathway. Reducing the expression of PF4 can attenuate the inflammatory response.
Assuntos
Lesões Encefálicas , NF-kappa B , Ratos , Animais , NF-kappa B/metabolismo , Fator Plaquetário 4/farmacologia , Hemorragia Cerebral/metabolismo , Transdução de Sinais , Lesões Encefálicas/metabolismoRESUMO
The use of high-dose intravenous immune globulin (IVIG) plus anticoagulation is recommended for the treatment of vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare side effect of adenoviral vector vaccines against coronavirus disease 2019 (Covid-19). We describe the response to IVIG therapy in three of the first patients in whom VITT was identified in Canada after the receipt of the ChAdOx1 nCoV-19 vaccine. The patients were between the ages of 63 and 72 years; one was female. At the time of this report, Canada had restricted the use of the ChAdOx1 nCoV-19 vaccine to persons who were 55 years of age or older on the basis of reports that VITT had occurred primarily in younger persons. Two of the patients in our study presented with limb-artery thrombosis; the third had cerebral venous and arterial thrombosis. Variable patterns of serum-induced platelet activation were observed in response to heparin and platelet factor 4 (PF4), indicating the heterogeneity of the manifestations of VITT in serum. After the initiation of IVIG, reduced antibody-induced platelet activation in serum was seen in all three patients. (Funded by the Canadian Institutes of Health Research.).
Assuntos
Vacinas contra COVID-19/efeitos adversos , Imunoglobulinas Intravenosas , Trombocitopenia/terapia , Trombose/terapia , Idoso , ChAdOx1 nCoV-19 , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinogênio/análise , Heparina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Fator Plaquetário 4/farmacologia , Serotonina/sangue , Trombocitopenia/sangue , Trombocitopenia/etiologia , Trombose/etiologia , Trombose/imunologiaRESUMO
BACKGROUND: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. METHODS: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. RESULTS: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. CONCLUSIONS: CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.
Assuntos
Neoplasias da Mama/imunologia , Antígenos CD4/genética , Antígenos CD8/genética , Linfócitos T CD8-Positivos/transplante , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Fator Plaquetário 4/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Células Supressoras Mieloides/imunologia , Células Neoplásicas Circulantes/imunologia , Fator Plaquetário 4/administração & dosagem , Fator Plaquetário 4/farmacologia , Análise de Sobrevida , Transplante Isogênico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbß3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbß3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.
Assuntos
Plaquetas/efeitos dos fármacos , Galectina 1/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Plaquetas/metabolismo , Humanos , Ácido N-Acetilneuramínico/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000â¯ng/mL, 0â¯=â¯control) to NCSU-23 with 0.4â¯mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100-1000â¯ng/mL, 0â¯=â¯Control-PVA) to a BSA-free medium (NCSU-23 with 0.3â¯mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4â¯mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0⯱â¯10.9 to 70.0⯱â¯5.8%); of day 7-blastocyst rates (range: 46.6⯱â¯10.0 to 56.4⯱â¯8.2%) and of total day 7-blastocyst efficiency (range: 32.3⯱â¯8.3 to 37.2⯱â¯7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1⯱â¯23.2 to 50.5⯱â¯26.4). In Experiment 2, PF4 accelerated embryo development, increasing (Pâ¯<â¯0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500â¯ng/mL (range: 29.9⯱â¯7.8 to 31.8⯱â¯5.5%; Pâ¯<â¯0.05) compared with BSA-control (17.2⯱â¯8.2%) and PF4 1000â¯ng/mL (15.5⯱â¯7.9%); showing similar blastocyst rates (range: 42.0⯱â¯11.5 to 49.3⯱â¯10.0%), total efficiency (28.0⯱â¯8.2 to 32.3⯱â¯7.1%) total cell numbers (range: 42.6⯱â¯19.3 to 45.7⯱â¯23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions.
Assuntos
Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Fator Plaquetário 4/química , Albumina Sérica/química , Suínos/embriologia , Animais , Relação Dose-Resposta a Droga , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator Plaquetário 4/administração & dosagem , Fator Plaquetário 4/farmacologia , Albumina Sérica/administração & dosagem , Albumina Sérica/farmacologiaRESUMO
INTRODUCTION: This study focused on PF4 effects on caspase-3,-6, -7, -8 and -9 which regulate the apopotosis process in breast cancer. MATERIALS AND METHODS: Breast tumours were induced in forty 21-day-old female Sprague Dawley rats (SDRs) using MNU until tumour size reached 14.5 mm (SD: 0.5 mm). The rats were then divided into two groups: Group 1 (control injected with 0.9% saline; n = 20), and Group 2 (platelet factor 4 (PF4); n = 20). PF4 was administered through focal intralesional injection at 20 µg/lesion dose. Following 5-day treatment, the SDRs were sacrificed. Subsequently, representative sections from the tumour were obtained for haematoxylin and eosin (H&E) staining. The expressions of caspase-3, -6, -7, -8 and -9 were evaluated using immunohistochemistry (IHC) staining. RESULTS: The majority of breast tumour specimens were of aggressive types [ncontrol = 13 (65%); nPF4 = 12 (60%)]. Invasive ductal carcinoma not otherwise specified (IDC-NOS) was the most commonly observed breast tumour histology for control and PF4 groups (n = 8 (40%) in respective groups). PF4-treated group exhibited significant differences in the caspase-3, -6 and -8 expression levels compared to the control group (all p < 0.001). There were no significant differences in caspase-7 (p = 0.347) and caspase-9 (p = 0.373) expression levels between both groups. CONCLUSION: This study found that PF4 acts via the caspase-mediated extrinsic apoptosis pathway without the involvement of the intrinsic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Fator Plaquetário 4/farmacologia , Animais , Apoptose/fisiologia , Feminino , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.
Assuntos
Linfócitos B/imunologia , Via Clássica do Complemento/imunologia , Heparina/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária , Fator Plaquetário 4/imunologia , Complemento C3c/imunologia , Via Clássica do Complemento/efeitos dos fármacos , Heparina/farmacologia , Humanos , Fator Plaquetário 4/farmacologia , ProteômicaRESUMO
Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Fator Plaquetário 4/química , Fator Plaquetário 4/farmacologia , Adulto , Desenho de Fármacos , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Modelos MolecularesRESUMO
Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4's effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.
Assuntos
Heparina/análogos & derivados , Trombocitopenia/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Megacariócitos/citologia , Camundongos , Contagem de Plaquetas , Fator Plaquetário 4/sangue , Fator Plaquetário 4/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/etiologia , TrombopoeseRESUMO
CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.
Assuntos
Plaquetas/metabolismo , Células Endoteliais/fisiologia , Fator Plaquetário 4/metabolismo , Adulto , Idoso , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fator Plaquetário 4/genética , Fator Plaquetário 4/farmacologia , Isoformas de Proteínas , Proteínas RecombinantesRESUMO
Chemokines have been shown to play immune-modulatory functions unrelated to steering cell migration. CXCL4 is a chemokine abundantly produced by activated platelets and immune cells. Increased levels of circulating CXCL4 are associated with immune-mediated conditions, including systemic sclerosis. Considering the central role of dendritic cells (DCs) in immune activation, in this article we addressed the effect of CXCL4 on the phenotype and function of monocyte-derived DCs (moDCs). To this end, we compared innate and adaptive immune responses of moDCs with those that were differentiated in the presence of CXCL4. Already prior to TLR- or Ag-specific stimulation, CXCL4-moDCs displayed a more matured phenotype. We found that CXCL4 exposure can sensitize moDCs for TLR-ligand responsiveness, as illustrated by a dramatic upregulation of CD83, CD86, and MHC class I in response to TLR3 and TLR7/8-agonists. Also, we observed a markedly increased secretion of IL-12 and TNF-α by CXCL4-moDCs exclusively upon stimulation with polyinosinic-polycytidylic acid, R848, and CL075 ligands. Next, we analyzed the effect of CXCL4 in modulating DC-mediated T cell activation. CXCL4-moDCs strongly potentiated proliferation of autologous CD4+ T cells and CD8+ T cells and production of IFN-γ and IL-4, in an Ag-independent manner. Although the internalization of Ag was comparable to that of moDCs, Ag processing by CXCL4-moDCs was impaired. Yet, these cells were more potent at stimulating Ag-specific CD8+ T cell responses. Together our data support that increased levels of circulating CXCL4 may contribute to immune dysregulation through the modulation of DC differentiation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Células Dendríticas/imunologia , Ativação Linfocitária , Fator Plaquetário 4/imunologia , Fator de Necrose Tumoral alfa/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Escherichia coli/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Genes MHC Classe I , Humanos , Imidazóis/farmacologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Fenótipo , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/farmacologia , Poli I-C/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologia , Antígeno CD83RESUMO
BACKGROUND AND OBJECTIVE: Periodontitis is a highly prevalent chronic inflammatory disease that causes tooth loss, morbidity and confers an increased risk for systemic disease. Tissue destruction during periodontitis is due in large part to collagen-degrading matrix metalloproteinases (MMPs) released by resident cells of the periodontium in response to proinflammatory cytokines. Platelets are immune-competent blood cells with a newly recognized role in chronic inflammation; however, their role in the pathogenesis of periodontitis is undefined. Consequently, the objective of this study was to assess the effect of platelet factor 4 (PF4), a major platelet-derived cytokine, on MMP-1 (collagenase) expression in human gingival fibroblasts (HGFs). MATERIAL AND METHODS: HGFs were cultured in the presence or absence of recombinant PF4. Pro-MMP-1 secretion was quantified by enzyme-linked immunosorbent assay analysis of the cell culture supernatants. MMP-1 transcription was quantified by real-time polymerase chain reaction. Regulation of MMP-1 production by the p44/42 MAP kinase (MAPK) signaling pathway was examined in the presence or absence of PF4. RESULTS: Exposure to PF4 caused a ~ 2-3-fold increase in MMP-1 transcription and secretion from cultured HGFs. PF4 treatment also enhanced phosphorylation of p44/42 MAPK, which has been previously shown to induce MMP-1 expression in fibroblasts. Blockade of p44/42 MAPK signaling with the cell-permeant inhibitors PD98059 and PD184352 abrogated PF4-induced pro-MMP-1 transcription upregulation and release from cultured HGFs. CONCLUSION: We conclude that PF4 upregulates MMP-1 expression in HGFs in a p44/42 MAPK-dependent manner. These findings point to a previously unidentified role for platelets in the pathogenesis of periodontal diseases.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Gengiva/citologia , Metaloproteinase 1 da Matriz/metabolismo , Fator Plaquetário 4/farmacologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Regulação para CimaRESUMO
Upon inflammation, circulating monocytes leave the bloodstream and migrate into the tissues, where they differentiate after exposure to various growth factors, cytokines or infectious agents. The best defined macrophage polarization types are M1 and M2. However, the platelet-derived CXC chemokine CXCL4 induces the polarization of macrophages into a unique phenotype. In this study, we compared the effect of CXCL4 and its variant CXCL4L1 on the differentiation of monocytes into macrophages and into immature monocyte-derived dendritic cells (iMDDC). Differently to M-CSF and CXCL4, CXCL4L1 is not a survival factor for monocytes. Moreover, the expression of the chemokine receptors CCR2, CCR5 and CXCR3 was significantly higher on CXCL4L1-treated monocytes compared to M-CSF- and CXCL4-stimulated monocytes. IL-1 receptor antagonist (IL-1RN) expression was upregulated by CXCL4 and downregulated by CXCL4L1, respectively, whereas both chemokines reduced the expression of the mannose receptor (MRC). Furthermore, through activation of CXCR3, CXCL4L1-stimulated monocytes released significantly higher amounts of CCL2 and CXCL8 compared to CXCL4-treated monocytes, indicating more pronounced inflammatory traits for CXCL4L1. In contrast, in CXCL4L1-treated monocytes, the production of CCL22 was lower. Compared to iMDDC generated in the presence of CXCL4L1, CXCL4-treated iMDDC showed an enhanced phagocytic capacity and downregulation of expression of certain surface markers (e.g. CD1a) and specific enzymes (e.g. MMP-9 and MMP-12). CXCL4 and CXCL4L1 did not affect the chemokine receptor expression on iMDDC and cytokine production (CCL2, CCL18, CCL22, CXCL8, IL-10) by CXCL4- or CXCL4L1-differentiated iMDDC was similar. We can conclude that both CXCL4 and CXCL4L1 exert a direct effect on monocytes and iMDDC. However, the resulting phenotypes are different, which suggests a unique role for the two CXCL4 variants in physiology and/or pathology.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/classificação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteases/genética , Monócitos/citologia , Monócitos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Receptores de Quimiocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoAssuntos
Autoanticorpos/sangue , Plaquetas/metabolismo , Heparina/imunologia , Testes Imunológicos/métodos , Selectina-P/biossíntese , Ativação Plaquetária/imunologia , Fator Plaquetário 4/imunologia , Púrpura Trombocitopênica Idiopática/diagnóstico , Serotonina/metabolismo , Autoanticorpos/imunologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Selectina-P/sangue , Selectina-P/genética , Fator Plaquetário 4/farmacologia , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/imunologia , Estudos de Amostragem , Sensibilidade e Especificidade , Manejo de Espécimes , Temperatura , Fatores de TempoRESUMO
BACKGROUND: To elucidate the role of rapamycin and PF4 on apoptosis regulation via Bax (pro-apoptosis), Bcl-2 (anti-apoptosis) and survivin activation on the growth in the 1-methyl-1-nitrosourea -induced invasive breast carcinoma model. MATERIALS AND METHODS: Thirty five female Sprague Dawley rats at age 21-day old were divided into 4 groups; Group 1 (control, n=10), Group 2 (PF4, n=5), Group 3 (rapamycin, n=10) and Group 4 (rapamycin+PF4, n=10). MNU was administered intraperitionally, dosed at 70 mg/kg body weight. The rats were treated when the tumors reached the size of 14.5 ± 0.5 mm and subsequently sacrificed after 5 days. Rapamycin and PF4 were administered as focal intralesional injections at the dose of 20 µg/lesion. The tumor tissue was then subjected to histopathological examinations for morphological appraisal and immunohistochemical assessment of the pro-apoptotic marker, Bax and anti-apoptotic markers, Bcl-2 and survivin. RESULTS: The histopathological pattern of the untreated control cohort showed that the severity of the malignancy augments with mammary tumor growth. Tumors developing in untreated groups were more aggressive whilst those in treated groups demonstrated a transformation to a less aggressive subtype. Combined treatment resulted in a significant reduction of tumor size without phenotypic changes. Bax, the pro-apoptotic marker, was significantly expressed at higher levels in the rapamycin-treated and rapamycin+PF4-treated groups compared to controls (p<0.05). Consequently, survivin was also significantly downregulated in the rapamycin-treated and rapamycin+PF4-treated group and this was significantly different when compared to controls (p). CONCLUSIONS: In our rat model, it could be clearly shown that rapamycin specifically affects Bax and survivin signaling pathways in activation of apoptosis. We conclude that rapamycin plays a critical role in the induction of apoptosis in MNU-induced mammary carcinoma.