RESUMO
Background: In February 2021, a few cases of unusual, severe thrombotic events associated with thrombocytopenia reported after vaccination with ChAdOx1 nCoV-19 (Vaxzevria) or with Johnson & Johnson's Janssen vaccine raise concern about safety. The vaccine-induced thrombotic thrombocytopenia (VITT) has been related to the presence of platelet-activating antibodies directed against platelet Factor 4. Objectives: We investigated VITT subject genetic background by a high-throughput whole exome sequencing (WES) approach in order to investigate VITT genetic predisposition. Methods: Six consecutive patients (females of Caucasian origin with a mean age of 64 years) were referred to the Atherothrombotic Diseases Center (Department of Experimental and Clinical Medicine, Azienda Ospedaliero-Universitaria Careggi, Florence) with a diagnosis of definite VITT underwent WES analysis. WES analysis was performed on the Illumina NextSeq500 platform. Results:WES analysis revealed a total of 140,563 genetic variants. Due to VITT's rare occurrence, we focused attention on rare variants. The global analysis of all high-quality rare variants did not reveal a significant enrichment of mutated genes in biological/functional pathways common to patients analyzed. Afterwards, we focused on rare variants in genes associated with blood coagulation and fibrinolysis, platelet activation and aggregation, integrin-mediated signaling pathway, and inflammation with particular attention to those involved in vascular damage, as well as autoimmune thrombocytopenia. According to ACMG criteria, 47/194 (24.2%) rare variants were classified as uncertain significance variants (VUS), whereas the remaining were likely benign/benign. Conclusion: WES analysis identifies rare variants possibly favoring the prothrombotic state triggered by the exposure to the vaccine. Functional studies and/or extensions to a larger number of patients might allow a more comprehensive definition of these molecular pathways.
Assuntos
Sequenciamento do Exoma , Humanos , Pessoa de Meia-Idade , Feminino , Idoso , Trombocitopenia/genética , Trombocitopenia/induzido quimicamente , ChAdOx1 nCoV-19/efeitos adversos , Trombose/genética , Predisposição Genética para Doença , Fator Plaquetário 4/genética , Masculino , Vacinação/efeitos adversosRESUMO
Myocardial infarction (MI) is an event of heart attack due to the formation of plaques in the interior walls of the arteries. This study is conducted to explore the role of ubiquitin-specific peptidase 47 (USP47) in cardiac function and inflammatory immunity. MI mouse models were established, followed by an appraisal of cardiac functions, infarct size, pathological changes, and USP47 and NLRP3 levels. MI cell models were established in HL-1 cells using anoxia. Levels of cardiac function-associated proteins, USP7, interferon regulatory factor 1 (IRF1), platelet factor-4 (CXCL4), pyroptotic factors, and neutrophil extracellular traps (NETs) were determined. The bindings of IRF1 to USP47 and the CXCL4 promoter and the ubiquitination of IRF1 were analyzed. USP47 was upregulated in myocardial tissues of MI mice. USP47 inhibition alleviated cardiac functions, and decreased infarct size, pro-inflammatory cytokines, NETs, NLRP3, and pyroptosis. The ubiquitination and expression levels of IRF1 were increased by silencing USP47, and IRF1 bound to the CXCL4 promoter to promote CXCL4. Overexpression of IRF1 or CXCL4 in vitro and injection of Nigericin in vivo reversed the effect of silencing USP47 on alleviating pyroptosis and cardiac functions. Collectively, USP47 stabilized IRF1 and promoted CXCL4, further promoting pyroptosis, impairing cardiac functions, and aggravating immune inflammation through NLRP3 pathways.
Assuntos
Inflamassomos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Camundongos , Inflamassomos/metabolismo , Masculino , Piroptose , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/genética , Modelos Animais de Doenças , Linhagem Celular , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/genética , Ubiquitinação , HumanosRESUMO
BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.
Assuntos
Plaquetas , Ciclo-Oxigenase 1 , Modelos Animais de Doenças , Integrases , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária , Fator Plaquetário 4 , Receptores de LDL , Animais , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/deficiência , Agregação Plaquetária/efeitos dos fármacos , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Integrases/genética , Receptores de LDL/genética , Receptores de LDL/deficiência , Masculino , Camundongos , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/enzimologia , Aterosclerose/prevenção & controle , Aterosclerose/sangue , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/enzimologia , Fenótipo , Proteínas de Membrana , Complexo Glicoproteico GPIb-IX de PlaquetasRESUMO
Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.
Assuntos
Carcinoma Pulmonar de Lewis , Neoplasias do Colo , Exercício Físico , Fator Plaquetário 4 , Animais , Camundongos , Inibidores da Angiogênese , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/terapia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Fatores Imunológicos , Camundongos Endogâmicos BALB C , Fator Plaquetário 4/genética , RNA Mensageiro , Esplenomegalia/metabolismo , Exercício Físico/fisiologiaRESUMO
Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4-/- mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.
Assuntos
Ácidos Nucleicos Livres , Armadilhas Extracelulares , Sepse , Trombose , Humanos , Camundongos , Animais , Armadilhas Extracelulares/metabolismo , Fator Plaquetário 4/genética , Trombose/metabolismo , Inflamação/metabolismo , Trombina/metabolismo , Fatores Imunológicos , Ácidos Nucleicos Livres/metabolismoRESUMO
BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.
Assuntos
Trombocitopenia , Humanos , Camundongos , Animais , Trombocitopenia/induzido quimicamente , Heparina/efeitos adversos , Plaquetas , Anticoagulantes/efeitos adversos , Camundongos Transgênicos , Fenótipo , Fibrina/genética , Fator Plaquetário 4/genéticaRESUMO
Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.
Assuntos
Macrófagos , Miofibroblastos , Humanos , Fibrose , Ligantes , Macrófagos/metabolismo , Miofibroblastos/metabolismo , Osteopontina , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismoRESUMO
Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome characterized by high-titer anti-platelet factor 4 (PF4) antibodies, thrombocytopenia and arterial and venous thrombosis in unusual sites, as cerebral venous sinuses and splanchnic veins. VITT has been described to occur almost exclusively after administration of ChAdOx1 nCoV-19 and Ad26.COV2.S adenovirus vector- based COVID-19 vaccines. Clinical and laboratory features of VITT resemble those of heparin-induced thrombocytopenia (HIT). It has been hypothesized that negatively charged polyadenylated hexone proteins of the AdV vectors could act as heparin to induce the conformational changes of PF4 molecule that lead to the formation of anti-PF4/polyanion antibodies. The anti-PF4 immune response in VITT is fostered by the presence of a proinflammatory milieu, elicited by some impurities found in ChAdOx1 nCoV-19 vaccine, as well as by soluble spike protein resulting from alternative splice events. Anti-PF4 antibodies bind PF4, forming immune complexes which activate platelets, monocytes and granulocytes, resulting in the VITT's immunothrombosis. The reason why only a tiny minority of patents receiving AdV-based COVID-19 vaccines develop VITT is still unknown. It has been hypothesized that individual intrinsic factors, either acquired (i.e., pre-priming of B cells to produce anti-PF4 antibodies by previous contacts with bacteria or viruses) or inherited (i.e., differences in platelet T-cell ubiquitin ligand-2 [TULA-2] expression) can predispose a few subjects to develop VITT. A better knowledge of the mechanistic basis of VITT is essential to improve the safety and the effectiveness of future vaccines and gene therapies using adenovirus vectors.
Assuntos
COVID-19 , Púrpura Trombocitopênica Idiopática , Trombocitopenia , Trombose , Vacinas , Humanos , Complexo Antígeno-Anticorpo , Vacinas contra COVID-19/efeitos adversos , Ad26COVS1 , ChAdOx1 nCoV-19 , Ligantes , Glicoproteína da Espícula de Coronavírus , COVID-19/prevenção & controle , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Heparina/efeitos adversos , Trombocitopenia/induzido quimicamente , Vacinas/efeitos adversos , Púrpura Trombocitopênica Idiopática/induzido quimicamente , UbiquitinasRESUMO
Heparin, a widely used anticoagulant, carries the risk of an antibody-mediated adverse drug reaction, heparin-induced thrombocytopenia (HIT). A subset of heparin-treated patients produces detectable levels of antibodies against complexes of heparin bound to circulating platelet factor 4 (PF4). Using a genome-wide association study (GWAS) approach, we aimed to identify genetic variants associated with anti-PF4/heparin antibodies that account for the variable antibody response seen in HIT. We performed a GWAS on anti-PF4/heparin antibody levels determined via polyclonal enzyme-linked immunosorbent assays. Our discovery cohort (n = 4237) and replication cohort (n = 807) constituted patients with European ancestry and clinical suspicion of HIT, with cases confirmed via functional assay. Genome-wide significance was considered at α = 5 × 10-8. No variants were significantly associated with anti-PF4/heparin antibody levels in the discovery cohort at a genome-wide significant level. Secondary GWAS analyses included the identification of variants with suggestive associations in the discovery cohort (α = 1 × 10-4). The top variant in both cohorts was rs1555175145 (discovery ß = -0.112 [0.018], P = 2.50 × 10-5; replication ß = -0.104 [0.051], P = .041). In gene set enrichment analysis, 3 gene sets reached false discovery rate-adjusted significance (q < 0.05) in both discovery and replication cohorts: "Leukocyte Transendothelial Migration," "Innate Immune Response," and "Lyase Activity." Our results indicate that genomic variation is not significantly associated with anti-PF4/heparin antibody levels. Given our power to identify variants with moderate frequencies and effect sizes, this evidence suggests genetic variation is not a primary driver of variable antibody response in heparin-treated patients with European ancestry.
Assuntos
Fator Plaquetário 4 , Trombocitopenia , Anticorpos , Estudo de Associação Genômica Ampla , Heparina/efeitos adversos , Humanos , Fatores Imunológicos/efeitos adversos , Fator Plaquetário 4/genética , Trombocitopenia/induzido quimicamente , Trombocitopenia/genéticaRESUMO
Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. We performed a genome-wide association study (GWAS) with positive functional assay as the outcome in a large discovery cohort of patients divided into 3 groups: (1) functional assay-positive cases (n = 1269), (2) antibody-positive (functional assay-negative) controls (n = 1131), and (3) antibody-negative controls (n = 1766). Significant associations (α = 5 × 10-8) were investigated in a replication cohort (α = 0.05) of functional assay-confirmed HIT cases (n = 177), antibody-positive (function assay-negative) controls (n = 258), and antibody-negative controls (n = 351). We observed a strong association for positive functional assay with increasing PF4/heparin immunoglobulin-G (IgG) level (odds ratio [OR], 16.53; 95% confidence interval [CI], 13.83-19.74; P = 1.51 × 10-209) and female sex (OR, 1.15; 95% CI, 1.01-1.32; P = .034). The rs8176719 C insertion variant in ABO was significantly associated with positive functional assay status in the discovery cohort (frequency = 0.41; OR, 0.751; 95% CI, 0.682-0.828; P = 7.80 × 10-9) and in the replication cohort (OR, 0.467; 95% CI, 0.228-0.954; P = .0367). The rs8176719 C insertion, which encodes all non-O blood group alleles, had a protective effect, indicating that the rs8176719 C deletion and the O blood group were risk factors for HIT (O blood group OR, 1.42; 95% CI, 1.26-1.61; P = 3.09 × 10-8). Meta-analyses indicated that the ABO association was independent of PF4/heparin IgG levels and was stronger when functional assay-positive cases were compared with antibody-positive (functional assay-negative) controls than with antibody-negative controls. Sequencing and fine-mapping of ABO demonstrated that rs8176719 was the causal single nucleotide polymorphism (SNP). Our results clarify the biology underlying HIT pathogenesis with ramifications for prediction and may have important implications for related conditions, such as vaccine-induced thrombotic thrombocytopenia.
Assuntos
Estudo de Associação Genômica Ampla , Trombocitopenia , Sistema ABO de Grupos Sanguíneos/genética , Anticoagulantes/efeitos adversos , Feminino , Heparina/efeitos adversos , Humanos , Imunoglobulina G , Masculino , Fator Plaquetário 4/genética , Fatores de Risco , Trombocitopenia/induzido quimicamente , Trombocitopenia/genéticaRESUMO
CXCL chemokines (CXCLs) are small cytokines or signal proteins secreted by cells that have been proven to be linked to the occurrence and development of many kinds of cancer. However, the expression and diagnostic and prognostic value of CXCLs in diffuse large B-cell lymphoma (DLBCL) remain to be further studied. We obtained CXCL transcription and survival data of patients with DLBCL from Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), The Cancer Genome Atlas (TCGA), TIMER and cBioPortal databases. R software, STRING and EXCEL were used to process the data. This study discovered that the expression levels of CXCL9-14 in DLBCL were higher than those in normal tissues, while CXCL4, CXCL7 and CXCL8 were lower in tumor than in normal tissues. The expression levels of CXCL2, CXCL10 and CXCL11 were related to tumor stage. CXCL9-14 could be used as an auxiliary molecular marker for the diagnosis of DLBCL. CXCL17 might be a potential prognostic marker of DLBCL.
Assuntos
Biomarcadores Tumorais/genética , Quimiocinas CXC/genética , Linfoma Difuso de Grandes Células B/genética , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Quimiocina CXCL2/genética , Quimiocina CXCL9/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-8/genética , Linfoma Difuso de Grandes Células B/patologia , Masculino , Fator Plaquetário 4/genética , Prognóstico , Microambiente Tumoral/genética , beta-Tromboglobulina/genéticaRESUMO
Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.
Assuntos
Matriz Extracelular/patologia , Miofibroblastos/metabolismo , Fator Plaquetário 4/metabolismo , Fibrose Pulmonar/patologia , Escleroderma Sistêmico/patologia , Animais , Bleomicina/toxicidade , Linhagem Celular , Colágeno/biossíntese , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/citologia , Pericitos/metabolismo , Fator Plaquetário 4/genética , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
[Figure: see text].
Assuntos
Plaquetas/enzimologia , Peróxido de Hidrogênio/metabolismo , Integrina beta3/metabolismo , Mecanotransdução Celular , NADPH Oxidase 2/metabolismo , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Animais , Ativação Enzimática , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Integrina beta3/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Mecânico , Quinase Syk/metabolismoAssuntos
Proliferação de Células , Regulação para Baixo , Células-Tronco Hematopoéticas/citologia , Fator Plaquetário 4/genética , Policitemia Vera/genética , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Policitemia Vera/tratamento farmacológico , Transcriptoma/efeitos dos fármacosRESUMO
BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease that commonly causes kidney damage. Therefore, we measured plasma levels of cytokines that may be related to renal dysfunction in SLE patients. METHODS: To explore the differences between SLE patients with renal dysfunction and healthy volunteers, the levels of cytokines in plasma were screened using a human cytokine antibody array. Then, we chose fourteen of the elevated cytokines for verification with an expanded sample size by a human magnetic Luminex assay. Plasma samples were isolated from SLE patients (n = 72) and healthy volunteers (n = 8). RESULTS: Cytokine antibody array data showed elevated plasma cytokines in SLE patients with renal dysfunction compared with healthy volunteers. By using the human magnetic Luminex assay, we found that plasma levels of CHI3L1, GDF-15, IGFBP-2, MIF, ST2, TFF3, and uPAR were significantly higher in SLE patients than in healthy volunteers. Plasma levels of CXCL4 were significantly lower in the active group than in the inactive group, and plasma levels of CHI3L1, IGFBP-2, MIF, and MPO were significantly higher in the active group than in the inactive group. We also analyzed the correlation between plasma cytokine levels and the SLEDAI-2K, and our results showed that the plasma levels of the fourteen selected cytokines were weakly correlated or not correlated with the SLEDAI-2K. We further analyzed the correlation between cytokines and renal dysfunction. Plasma levels of GDF-15 and TFF3 were highly positively correlated with serum creatinine levels and 24-hour urine protein levels. CONCLUSION: Our data suggest that plasma levels of GDF-15 and TFF3 are potential renal dysfunction markers in SLE patients, but plasma levels of these cytokines are not correlated with the SLEDAI-2K. Further study is warranted to determine how these cytokines regulate inflammatory responses and renal dysfunction in SLE.
Assuntos
Biomarcadores/sangue , Citocinas/sangue , Fator 15 de Diferenciação de Crescimento/sangue , Nefropatias/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fator Plaquetário 4/sangue , Fator Trefoil-3/sangue , Adulto , China , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Fator Plaquetário 4/genética , Índice de Gravidade de DoençaRESUMO
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.
Assuntos
Medula Óssea/patologia , Fibrose/patologia , Inflamação/patologia , Transtornos Mieloproliferativos/complicações , Fator Plaquetário 4/metabolismo , Mielofibrose Primária/patologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Proliferação de Células , Progressão da Doença , Fibrose/etiologia , Fibrose/imunologia , Fibrose/metabolismo , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos , Camundongos , Camundongos Knockout , Mutação , Fator Plaquetário 4/genética , Mielofibrose Primária/etiologia , Mielofibrose Primária/imunologia , Mielofibrose Primária/metabolismoRESUMO
OBJECTIVE: Thrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients. CONCLUSIONS: Collectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.
Assuntos
Plaquetas/metabolismo , Endocitose , Infecções por HIV/sangue , HIV-1/patogenicidade , Ativação Plaquetária , Trombocitopenia/sangue , Receptores Toll-Like/sangue , Vírion , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/sangue , Fatores de Ribosilação do ADP/genética , Animais , Antirretrovirais/uso terapêutico , Plaquetas/virologia , Agregação Celular , Células Cultivadas , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Quinase I-kappa B/sangue , Quinase I-kappa B/genética , Leucócitos/metabolismo , Leucócitos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/sangue , Fator 88 de Diferenciação Mieloide/genética , Fator Plaquetário 4/sangue , Fator Plaquetário 4/genética , Trombocitopenia/diagnóstico , Trombocitopenia/virologia , Receptores Toll-Like/deficiência , Receptores Toll-Like/genética , Proteína 3 Associada à Membrana da Vesícula/sangue , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
Deep vein thrombosis (DVT) is a common complication after trauma. The development of markers to predict DVT in trauma patients is needed, and circulating microparticles (MPs) and their contents are possible candidates. In this study, we aimed to identify platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) mRNAs in circulating MPs as potential markers for DVT diagnosis in trauma patients. Fifteen trauma patients diagnosed with DVT and fifteen matched patients without DVT were included in this study. Fifteen healthy volunteers also were included as controls. Circulating MPs were obtained from the plasma of all study subjects. Annexin V+ MPs and platelet-derived MPs (PMPs) were quantified using flow cytometry. PF4 and ß-TG mRNAs in MPs were determined by qPCR, and the common logarithm of relative quantitation (RQ) was calculated using the comparative Ct method. Receiver-operating characteristic (ROC) curves were performed to analyze the diagnostic value of PF4 and ß-TG mRNAs. No significant differences were found in Annexin V+ MPs and PMPs levels between trauma patients with and without DVT. However, both PF4 and ß-TG mRNAs in MPs from the DVT group were significantly higher than the non-DVT group and healthy controls (P = 0.014 for PF4, P = 0.010 for ß-TG). The ROC curve analysis showed that both the PF4 mRNA (area-under curve (AUC) 0.756, P = 0.017) and the ß-TG mRNA (AUC 0.751, P = 0.019) had a positive predictive value for DVT. This finding indicates that the PF4 and ß-TG mRNAs in MPs may be used as potential biomarkers for DVT diagnosis in trauma patients.
Assuntos
Micropartículas Derivadas de Células/genética , Fator Plaquetário 4/genética , RNA Mensageiro/genética , Trombose Venosa/diagnóstico , beta-Tromboglobulina/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombose Venosa/sangue , Trombose Venosa/etiologia , Trombose Venosa/genética , Ferimentos e Lesões/complicaçõesRESUMO
Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.
Assuntos
Plaquetas/metabolismo , Proteína S/metabolismo , Trombose/sangue , Animais , Tempo de Sangramento , Coagulação Sanguínea/genética , Coagulação Sanguínea/fisiologia , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ativação Plaquetária/genética , Ativação Plaquetária/fisiologia , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Proteína S/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombose/etiologia , Trombose/genética , Trombose Venosa/sangue , Trombose Venosa/etiologia , Trombose Venosa/genéticaRESUMO
Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.