RESUMO
Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Serina-Treonina Quinases/genética , Trombofilia/genética , Trombose Venosa/genética , Adulto , Células Endoteliais/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Lipoproteínas/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Fatores de Risco , Trombofilia/patologia , Tromboembolia Venosa/genética , Tromboembolia Venosa/patologia , Trombose Venosa/patologia , Sequenciamento do ExomaRESUMO
RESEARCH QUESTION: Are there genetic determinants shared by unrelated women with unexplained recurrent early miscarriage (REM)? DESIGN: Thirty REM cases and 30 controls were selected with extreme phenotype among women from Eastern Brittany (France), previously enrolled in an incident case-control study on thrombophilic mutations. Cases and controls were selected based on the number of early miscarriages or live births, respectively. Peripheral blood was collected for DNA extraction at initial visit. The burden of low-frequency variants in the coding part of the genes was compared using whole exome sequencing (WES). RESULTS: Cases had 3 to 17 early miscarriages (20 cases: ≥5 previous losses). Controls had 1 to 4 live births (20 controls: ≥3 previous live births) and no miscarriages. WES data were available for 29 cases and 30 controls. A total of 209,387 variants were found (mean variant per patient: 59,073.05) with no difference between groups (Pâ¯=â¯0.68). The top five most significantly associated genes were ABCA4, NFAM1, TCN2, AL078585.1 and EPS15. Previous studies suggest the involvement of vitamin B12 deficiency in REM. TCN2 encodes for vitamin B12 transporter into cells. Therefore, holotranscobalamin (active vitamin B12) was measured for both cases and controls (81.2 ± 32.1 versus 92.9 ± 34.3 pmol/l, respectively, P = 0.186). Five cases but no controls were below 50 pmol/l (P = 0.052). CONCLUSIONS: This study highlights four new genes of interest in REM, some of which belong to known networks of genes involved in embryonic development (clathrin-mediated endocytosis and ciliary pathway). The study also confirms the involvement of TCN2 (vitamin B12 pathway) in the early first trimester of pregnancy.
Assuntos
Aborto Habitual/genética , Sequenciamento do Exoma , Aborto Habitual/sangue , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Gravidez , Transcobalaminas/genética , Deficiência de Vitamina B 12/complicações , Adulto JovemRESUMO
IMPORTANCE: The prevalence of pulmonary embolism in patients with chronic obstructive pulmonary disease (COPD) and acutely worsening respiratory symptoms remains uncertain. OBJECTIVE: To determine the prevalence of pulmonary embolism in patients with COPD admitted to the hospital for acutely worsening respiratory symptoms. DESIGN, SETTING, AND PARTICIPANTS: Multicenter cross-sectional study with prospective follow-up conducted in 7 French hospitals. A predefined pulmonary embolism diagnostic algorithm based on Geneva score, D-dimer levels, and spiral computed tomographic pulmonary angiography plus leg compression ultrasound was applied within 48 hours of admission; all patients had 3-month follow-up. Patients were recruited from January 2014 to May 2017 and the final date of follow-up was August 22, 2017. EXPOSURES: Acutely worsening respiratory symptoms in patients with COPD. MAIN OUTCOMES AND MEASURES: The primary outcome was pulmonary embolism diagnosed within 48 hours of admission. Key secondary outcome was pulmonary embolism during a 3-month follow-up among patients deemed not to have venous thromboembolism at admission and who did not receive anticoagulant treatment. Other outcomes were venous thromboembolism (pulmonary embolism and/or deep vein thrombosis) at admission and during follow-up, and 3-month mortality, whether venous thromboembolism was clinically suspected or not. RESULTS: Among 740 included patients (mean age, 68.2 years [SD, 10.9 years]; 274 women [37.0%]), pulmonary embolism was confirmed within 48 hours of admission in 44 patients (5.9%; 95% CI, 4.5%-7.9%). Among the 670 patients deemed not to have venous thromboembolism at admission and who did not receive anticoagulation, pulmonary embolism occurred in 5 patients (0.7%; 95% CI, 0.3%-1.7%) during follow-up, including 3 deaths related to pulmonary embolism. The overall 3-month mortality rate was 6.8% (50 of 740; 95% CI, 5.2%-8.8%). The proportion of patients who died during follow-up was higher among those with venous thromboembolism at admission than the proportion of those without it at admission (14 [25.9%] of 54 patients vs 36 [5.2%] of 686; risk difference, 20.7%, 95% CI, 10.7%-33.8%; P < .001). The prevalence of venous thromboembolism was 11.7% (95% CI, 8.6%-15.9%) among patients in whom pulmonary embolism was suspected (n = 299) and was 4.3% (95% CI, 2.8%-6.6%) among those in whom pulmonary embolism was not suspected (n = 441). CONCLUSIONS AND RELEVANCE: Among patients with chronic obstructive pulmonary disease admitted to the hospital with an acute worsening of respiratory symptoms, pulmonary embolism was detected in 5.9% of patients using a predefined diagnostic algorithm. Further research is needed to understand the possible role of systematic screening for pulmonary embolism in this patient population.
Assuntos
Algoritmos , Doença Pulmonar Obstrutiva Crônica/complicações , Embolia Pulmonar/diagnóstico , Idoso , Estudos Transversais , Feminino , Seguimentos , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Gravidade do Paciente , Prevalência , Embolia Pulmonar/epidemiologia , Embolia Pulmonar/etiologia , Tromboembolia Venosa/epidemiologia , Tromboembolia Venosa/etiologia , Trombose Venosa/etiologiaRESUMO
Bone repair after trauma or surgical intervention involves a tightly regulated cascade of events that starts with hemostasis and an inflammatory response, which are critical for successful healing. Nonsteroidal anti-inflammatory drugs (NSAID) are routinely prescribed for pain relief despite their potential inhibitory effect on bone repair. The goal of this study was to determine the impact of administration of the non-selective NSAID diclofenac in the inflammatory phase of bone repair in mice with or without lipopolysaccharide-induced systemic inflammation. Repair of femoral window defects was characterized using micro computed tomography imaging and histological analyses at 2 weeks postoperative. The data indicate (a) impaired bone regeneration associated with reduced osteoblast, osteoclast, and macrophage activity; (b) changes in the number, activity, and distribution of mast cells in regenerating bone; and (c) impaired angiogenesis due to a direct toxic effect of diclofenac on vascular endothelial cells. The results of this study provide strong evidence to support the conjecture that administration of NSAIDs in the first 2 weeks after orthopaedic surgery disrupts the healing cascade and exacerbates the negative effects of systemic inflammation on the repair process.
Assuntos
Diclofenaco/farmacologia , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Ferimentos e Lesões/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células Endoteliais/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Procedimentos Ortopédicos/efeitos adversos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Dor/diagnóstico por imagem , Dor/patologia , Ferimentos e Lesões/complicações , Ferimentos e Lesões/diagnóstico por imagem , Ferimentos e Lesões/patologia , Microtomografia por Raio-XRESUMO
Venous thromboembolism is a common complication of cancer. Based on recent evidence that (1) growth arrest-specific 6 (Gas6) regulates the expression of tissue factor during venous thrombosis, and (2) cancer promotes a procoagulant milieu, we hypothesize that Gas6 may be involved in cancer-induced coagulopathy. Venous thrombi were induced in both wild-type (WT) and Gas6-deficient ((-/-)) mice with cancer. WT mice with cancer developed larger thrombi than their healthy counterparts; these larger thrombi induced by cancer were not seen in Gas6(-/-) mice. Whole genome microarray analysis of differential gene expression in WT and Gas6(-/-) endothelial cells exposed to M27 murine lung carcinoma cells reveal that Gas6 increases prostaglandin E synthase (Ptges) expression in endothelial cells. This was confirmed using real-time polymerase chain reaction and immunofluorescence staining. Culture of WT endothelial cells with M27 increases the secretion of prostaglandin E2 (PGE2), the enzymatic product of Ptges, in WT but not in Gas6(-/-) endothelial cells. In WT endothelial cells, Ptges expression was regulated through extracellular signal-regulated kinase 1/2 phosphorylation (ERK1/2). In vitro, PGE2 activates platelets after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced venous thrombosis.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Oxirredutases Intramoleculares/genética , Neoplasias/complicações , Trombose Venosa/etiologia , Animais , Células Cultivadas , Dinoprostona/efeitos adversos , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Oxirredutases Intramoleculares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Prostaglandina-E Sintases , Regulação para Cima , Trombose Venosa/genética , Trombose Venosa/patologiaRESUMO
OBJECTIVE: Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. APPROACH AND RESULTS: Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6-/- mice contain less inflammatory (CCR2hiCX3CR1lo) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2hiCX3CR1lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). CONCLUSIONS: This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2hiCX3CR1lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis.
Assuntos
Quimiotaxia de Leucócito , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Monócitos/metabolismo , Receptores CCR2/metabolismo , Receptores de Quimiocinas/metabolismo , Veia Cava Inferior/metabolismo , Trombose Venosa/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Predisposição Genética para Doença , Inflamação/genética , Inflamação/patologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Comunicação Parácrina , Fenótipo , Receptores CCR2/genética , Transdução de Sinais , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/patologia , Trombose Venosa/genética , Trombose Venosa/patologia , Trombose Venosa/prevenção & controleRESUMO
RATIONALE: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. OBJECTIVE: We investigated the role of FHL2 in the regulation of EOCs. METHODS AND RESULTS: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/ß3, αv/ß5, and ß2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs. CONCLUSIONS: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.
Assuntos
Movimento Celular , Células Endoteliais/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/metabolismo , Comunicação Parácrina , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fatores de Transcrição/metabolismo , Cicatrização , Adulto , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Artérias Carótidas/citologia , Sobrevivência Celular , Células Cultivadas , Cortactina/genética , Cortactina/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Integrinas/genética , Integrinas/metabolismo , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/genética , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Baço/citologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial cells, vascular smooth muscle cells, and leukocytes. It regulates cell survival, migration, and inflammatory response, but its role in atherogenesis is unknown. APPROACH AND RESULTS: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient mice were crossed with ApoE-deficient mice, to generate ApoE/FHL2-/- mice. After high-fat diet, ApoE/FHL2-/- mice had significantly smaller atherosclerotic plaques than ApoE-/- mice in the aortic sinus, the brachiocephalic artery, and the aorta. This was associated with enhanced collagen and smooth muscle cell contents and a 2-fold reduction in macrophage content within the plaques of ApoE/FHL-2-/- versus ApoE-/- mice. This could be explained, in part, by the reduction in aortic ICAM-1 (intracellular adhesion molecule) mRNA and VCAM-1 (vascular cell adhesion molecule) protein expression in the plaque. Aortic gene expression of the chemokines CX3CL1 and CCL5 was increased in ApoE/FHL2-/- versus ApoE-/- mice. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of proinflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- versus ApoE-/- mice. Furthermore, mRNA levels of CX3CR1 were 2-fold higher in monocytes from ApoE/FHL2-/- versus ApoE-/- mice. Finally, we investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. After being irradiated, ApoE-/- or ApoE/FHL2-/- mice were transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After high-fat diet, both chimeric groups developed smaller plaques than ApoE-/- transplanted with ApoE-/- bone marrow. CONCLUSIONS: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting proinflammatory chemokine production, adhesion molecule expression, and proinflammatory monocyte recruitment.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas com Domínio LIM/deficiência , Animais , Aterosclerose/fisiopatologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Distribuição AleatóriaRESUMO
Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5 µM ADP and 10 µM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6(-/-) mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6(-/-) endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.
Assuntos
Vasos Sanguíneos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Tromboplastina/genética , Trombose/genética , Animais , Plaquetas/metabolismo , Vasos Sanguíneos/patologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Tromboplastina/metabolismo , Trombose/metabolismoRESUMO
BACKGROUND: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. METHODS AND RESULTS: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation in vitro (P<0.01). CONCLUSIONS: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.
Assuntos
Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Receptor 3 Toll-Like , Trombose Venosa , Animais , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Trombose Venosa/metabolismo , Trombose Venosa/genética , Trombose Venosa/patologia , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transdução de Sinais , Células HEK293 , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia , Neutrófilos/metabolismo , RNA/genética , Masculino , Camundongos , Poli I-C/farmacologia , Coagulação SanguíneaRESUMO
BACKGROUND: The prevalence of pulmonary embolism (PE) is approximately 11-17 % in patients with an acute exacerbation of chronic obstructive pulmonary disease (AE-COPD). The optimal diagnostic strategy for PE in these patients remains undetermined. AIMS: To evaluate the safety and efficacy of standard (revised Geneva and Wells PE scores combined with fixed D-dimer cut-off) and computed tomography pulmonary angiogram (CTPA)-sparing diagnostic strategies (ADJUST-PE, YEARS, PEGeD, 4PEPS) in patients with AE-COPD. METHOD: Post-hoc analyses of data from the multicenter prospective PEP study were performed. The primary outcome was the diagnostic failure rate of venous thromboembolism (VTE) during the entire study period. Secondary outcomes included diagnostic failure rate of PE and deep venous thrombosis (DVT), respectively, during the entire study period and the number of CTPA needed per diagnostic strategy. RESULTS: 740 patients were included. The revised Geneva and Wells PE scores combined with fixed D-dimer cut-off had a diagnostic failure rate of VTE of 0.7 % (95%CI 0.3 %-1.7 %), but >70.0 % of the patients needed imaging. All CTPA-sparing diagnostic algorithms reduced the need for CTPAs (-10.1 % to -32.4 %, depending on the algorithm), at the cost of an increased VTE diagnosis failure rate of up to 2.1 % (95%CI 1.2 %-3.4 %). CONCLUSION: Revised Geneva and Wells PE scores combined with fixed D-dimer cut-off were safe, but a high number of CTPA remained needed. CTPA-sparing algorithms would reduce imaging, at the cost of an increased VTE diagnosis failure rate that exceeds the safety threshold. Further studies are needed to improve diagnostic management in this population.
Assuntos
Doença Pulmonar Obstrutiva Crônica , Embolia Pulmonar , Tromboembolia Venosa , Humanos , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/epidemiologia , Estudos Prospectivos , Embolia Pulmonar/complicações , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/epidemiologia , Algoritmos , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Produtos de Degradação da Fibrina e do FibrinogênioRESUMO
Reduced blood flow velocity in the vein triggers inflammation and is associated with the release into the extracellular space of alarmins or damage-associated molecular patterns (DAMPs). These molecules include extracellular nucleic acids, extracellular purinergic nucleotides (ATP, ADP), cytokines and extracellular HMGB1. They are recognized as a danger signal by immune cells, platelets and endothelial cells. Hence, endothelial cells are capable of sensing environmental cues through a wide variety of receptors expressed at the plasma membrane. The endothelium is then responding by expressing pro-coagulant proteins, including tissue factor, and inflammatory molecules such as cytokines and chemokines involved in the recruitment and activation of platelets and leukocytes. This ultimately leads to thrombosis, which is an active pro-inflammatory process, tightly regulated, that needs to be properly resolved to avoid further vascular damages. These mechanisms are often dysregulated, which promote fibrinolysis defects, activation of the immune system and irreversible vascular damages further contributing to thrombotic and inflammatory processes. The concept of thrombo-inflammation is now widely used to describe the complex interactions between the coagulation and inflammation in various cardiovascular diseases. In endothelial cells, activating signals converge to multiple intracellular pathways leading to phenotypical changes turning them into inflammatory-like cells. Accumulating evidence suggest that endothelial to mesenchymal transition (EndMT) may be a major mechanism of endothelial dysfunction induced during inflammation and thrombosis. EndMT is a biological process where endothelial cells lose their endothelial characteristics and acquire mesenchymal markers and functions. Endothelial dysfunction might play a central role in orchestrating and amplifying thrombo-inflammation thought induction of EndMT processes. Mechanisms regulating endothelial dysfunction have been only partially uncovered in the context of thrombotic diseases. In the present review, we focus on the importance of the endothelial phenotype and discuss how endothelial plasticity may regulate the interplay between thrombosis and inflammation. We discuss how the endothelial cells are sensing and responding to environmental cues and contribute to thrombo-inflammation with a particular focus on venous thromboembolism (VTE). A better understanding of the precise mechanisms involved and the specific role of endothelial cells is needed to characterize VTE incidence and address the risk of recurrent VTE and its sequelae.
RESUMO
BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening complication of a pulmonary embolism (PE) whose incidence and predictors are not precisely determined. OBJECTIVE: To determine the frequency and predictors for CTEPH after a first unprovoked PE. PATIENTS/METHODS: In a randomized trial comparing an additional 18-month warfarin versus placebo in patients after a first unprovoked PE initially treated with vitamin K antagonist for 6 months, we applied recommended CTEPH screening strategies through an 8-year follow-up to determine cumulative incidence of CTEPH. CTEPH predictors were estimated using Cox models. Pulmonary vascular obstruction (PVO) and systolic pulmonary arterial pressure (sPAP) at PE diagnosis and 6 months were studied by receiver operating curves analysis. All CTEPH cases and whether they were incident or prevalent were adjudicated. RESULTS: During a median follow-up of 8.7 years, nine CTEPH cases were diagnosed among 371 patients, with a cumulative incidence of 2.8% (95% confidence interval [CI] 0.95-4.64), and of 1.31% (95% CI 0.01-2.60) after exclusion of five cases adjudicated as prevalent. At PE diagnosis, PVO > 45% and sPAP > 56 mmHg were associated with CTEPH with a hazard ratio (HR) of 33.00 (95% CI 1.64-667.00, p = .02) and 12.50 (95% CI 2.10-74.80, p < .01), respectively. Age > 65 years, lupus anticoagulant antibodies and non-O blood groups were also predictive of CTEPH. PVO > 14% and sPAP > 34 mmHg at 6 months were associated with CTEPH (HR 63.90 [95% CI 3.11-1310.00, p < .01]and HR 17.2 [95% CI 2.75-108, p < .01]). CONCLUSION: After a first unprovoked PE, CTEPH cumulative incidence was 2.8% during an 8-year follow-up. PVO and sPAP at PE diagnosis and at 6 months were the main predictors for CTEPH diagnosis.
Assuntos
Hipertensão Pulmonar , Embolia Pulmonar , Humanos , Idoso , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/complicações , Fatores de Risco , Doença Crônica , Embolia Pulmonar/complicações , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/epidemiologia , Anticoagulantes/uso terapêuticoRESUMO
Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.
Assuntos
Senescência Celular/genética , Células Endoteliais/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Sirtuína 1/metabolismo , Células-Tronco/enzimologia , Animais , Diferenciação Celular/genética , Regulação para Baixo , Feminino , Homocistinúria/genética , Hiper-Homocisteinemia/tratamento farmacológico , Hiper-Homocisteinemia/enzimologia , Hiper-Homocisteinemia/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Espasticidade Muscular/genética , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Transtornos Psicóticos/genética , Pterinas/farmacologia , Espécies Reativas de Oxigênio , Telômero/metabolismoRESUMO
RATIONALE: Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)-mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown. OBJECTIVES: We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) would decrease cellular effects induced by aldosterone. METHODS AND RESULTS: We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT(1a)R knockout mice. CONCLUSION: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.
Assuntos
Aldosterona/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais , Angiotensina II/metabolismo , Animais , Células Cultivadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Receptor Cross-Talk , Receptor Tipo 1 de Angiotensina/deficiência , Receptor Tipo 1 de Angiotensina/genética , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Epidemiologic studies indicate that early life arsenic exposures are linked to an increased risk of cardiovascular diseases. Different oxidation and methylation states of arsenic exist in the environment and are formed in vivo via the action of arsenic (+3 oxidation state) methyltransferase (As3MT). Methylated arsenicals are pro-atherogenic postnatally, but pre- and perinatal effects are unclear. This is particularly important because methylated arsenicals are known to cross the placenta. OBJECTIVES: We tested the effects of early life exposure to inorganic and methylated arsenicals on atherosclerotic plaque formation and its composition in apolipoprotein E knock-out (apoE-/-) mice and evaluated whether apoE-/- mice lacking As3MT expression were susceptible to this effect. METHODS: We exposed apoE-/- or apoE-/-/As3MT-/- mice to 200 ppb inorganic or methylated arsenic in the drinking water from conception to weaning and assessed atherosclerotic plaques in the offspring at 18 wk of age. Mixed regression models were used to estimate the mean difference in each outcome relative to controls, adjusting for sex and including a random effects term to account for within-litter clustering. RESULTS: Early life exposure to inorganic arsenic, and more profoundly methylated arsenicals, resulted in significantly larger plaques in the aortic arch and sinus in both sexes. Lipid levels in these plaques were higher without a substantial difference in macrophage numbers. Smooth muscle cell content was not altered, but collagen content was lower. Importantly, there were sex-specific differences in these observations, where males had higher lipids and lower collagen in the plaque, but females did not. In mice lacking As3MT, arsenic did not alter the plaque size, although the size was highly variable. In addition, control apoE-/-/As3MT-/- mice had significantly larger plaque size compared with control apoE-/-. CONCLUSION: This study shows that early life exposure to inorganic and methylated arsenicals is pro-atherogenic with sex-specific differences in plaque composition and a potential role for As3MT in mice. https://doi.org/10.1289/EHP8171.
Assuntos
Arsênio , Placa Aterosclerótica , Efeitos Tardios da Exposição Pré-Natal , Animais , Arsênio/toxicidade , Arsenicais , Feminino , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Placa Aterosclerótica/induzido quimicamente , Gravidez , Fatores SexuaisRESUMO
PURPOSE: The use of 68Ga-labelled carbon nanoparticles has been proposed for lung ventilation PET/CT imaging. However, no study has assessed the physical properties of 68Ga-labelled carbon nanoparticles. The aim of this study therefore was to evaluate the shape and size of 68Ga-labelled carbon nanoparticles, and to determine the composition of the aerosol, as opposed to 99mTc-labelled carbon nanoparticles aerosol. PROCEDURES: 99mTc- and 68Ga-labelled carbon nanoparticles, stable gallium carbon nanoparticles, 0.9 % NaCl and 0.1 N HCl-based carbon nanoparticles were produced using an unmodified Technegas® generator, following the usual technique used for clinical Technegas® production. The shape and size of particles were studied by transmission electron microscopy (TEM) after decay of the radioactive samples. The composition of the 68Zn- and 99Tc-labelled carbon nanoparticles aerosols was assessed using scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) analysis after decay of the 68Ga- and 99mTc-labelled carbon nanoparticles, respectively. RESULTS: On TEM, all samples showed similar shape with hexagonally structured primary particles, agglomerated in clusters. The mean diameters of primary stable gallium carbon nanoparticles, 99Tc- and 68Zn-labelled carbon nanoparticles were 22.4 ± 10 nm, 20.9 ± 7.2 nm and 19.8 ± 11.7 nm, respectively. CONCLUSION: Using Technegas® generator in the usual clinical way, 99mTc- and 68Ga-labelled carbon nanoparticles demonstrated similar shape and diameters in the same size range size. These results support the use of 68Ga-labelled carbon nanoparticles for the assessment of regional lung ventilation function with PET imaging.
Assuntos
Carbono/química , Radioisótopos de Gálio/química , Nanopartículas/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ventilação Pulmonar , Pertecnetato Tc 99m de Sódio/química , Coloração e Rotulagem , Aerossóis , Celulose/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Cloreto de Sódio/química , Espectrometria por Raios XRESUMO
BACKGROUND: We aimed to assess whether high pulmonary vascular obstruction index (PVOI) measured at the time of pulmonary embolism (PE) diagnosis is associated with an increased risk of recurrent venous thromboembolism (VTE). STUDY DESIGN AND METHODS: French prospective cohort of patients with a symptomatic episode of PE diagnosed with spiral computerized tomography pulmonary angiography (CTPA) or ventilation-perfusion (V/Q) lung scan and a follow-up of at least 6 months after anticoagulation discontinuation. PVOI was assessed based on the available diagnostic exam (V/Q lung scan or CTPA). All patients had standardized follow-up and independent clinicians adjudicated all deaths and recurrent VTE events. Main outcome was recurrent VTE after stopping anticoagulation. RESULTS: A total of 418 patients with PE were included. During a median follow-up period of 3.6 (1.2-6.0) years, 109 recurrences occurred. In multivariate analysis, PVOI ≥ 40% was an independent risk factor for recurrence (hazard ratio 1.77, 95% confidence interval 1.20-2.62, p < 0.01), whether PE was provoked by a major transient risk factor or not. A threshold at 41% was identified as the best value associated with the risk of recurrence 6 months after stopping anticoagulation (area under curve = 0.64). CONCLUSION: PVOI ≥ 40% at PE diagnosis was an independent risk factor for recurrence VTE. Further prospective validation studies are needed.
Assuntos
Anticoagulantes/farmacologia , Embolia Pulmonar/diagnóstico , Tromboembolia Venosa/diagnóstico , Adulto , Idoso , Angiografia , Estudos de Coortes , Feminino , Seguimentos , França , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , Estudos Prospectivos , Embolia Pulmonar/complicações , Recidiva , Fatores de Risco , Resultado do Tratamento , Tromboembolia Venosa/complicaçõesRESUMO
Vascular cells are very sensitive to their hemodynamic environment. Any change in blood pressure or blood flow can be sensed by endothelial and vascular smooth muscle cells and ultimately results in structural modifications within the vascular wall that accommodate the new conditions. In the case of hypertension, the increase in arterial stretch stimulates vessel thickening to normalize the tensile forces. This process requires modification of the extracellular matrix and of cell-matrix interactions, which mainly involves extracellular proteases. In hypertension, chronic exposure of the arterial wall to stretch leads to vascular remodeling, arterial stiffness and calcification, which finally affect target organ function. This review surveys how mechanical stretch regulates extracellular proteases, considering the signaling pathways involved and the consequences on the cardiovascular system.
Assuntos
Matriz Extracelular/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Matriz Extracelular/enzimologia , Humanos , Hipertensão/genética , NF-kappa B/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Transdução de Sinais/genéticaRESUMO
Immune cells have been implicated in the pathogenesis of hypertension. We hypothesized that under the influence of chromosome (chr)2, T lymphocytes contribute to vascular inflammation in genetic salt-sensitive hypertension. Normotensive (Brown Norway), hypertensive (Dahl salt-sensitive), and consomic rats (SSBN2; in which chr2 has been transferred from Brown Norway to Dahl rats) were studied. Systolic blood pressure, measured by tail cuff, and aortic preproendothelin mRNA, measured by quantitative RT-PCR, were elevated in Dahl rats compared with Brown Norway rats and were reduced in SSBN2 rats compared with Dahl rats (P < 0.01). Compared with Brown Norway rats, Dahl rats exhibited increased inflammatory markers and mediators such as nuclear translocation of the aortic p65 subunit of NF-kappaB as well as VCAM-1, ICAM-1, chemokine (C-C motif) receptor 5, and CD4 mRNA, all of which were reduced in SSBN2 rats. Aortic CD8 mRNA was equally increased in Dahl and SSBN2 rats relative to Brown Norway rats. CD4(+) T cell infiltration in the aorta of SSBN2 rats was reduced compared with Dahl rats, whereas the aortic protein expression of Foxp3b and immunosuppressors transforming growth factor (TGF)-beta(1) and IL-10, the three markers associated with the regulatory T cell lineage, were enhanced in SSBN2 rats. Activation in vitro of T cells demonstrated that CD4(+)CD25(+) and CD8(+)CD25(+) cells (Tregs) produce IL-10 in SSBN2 rats. Thus, increased vascular inflammatory responses and hypertension in a genetic salt-sensitive hypertensive rodent model are reduced by transfer of chr2 from a normotensive strain, and this is associated with enhanced levels of immunosuppressive mediators.