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1.
Blood ; 141(15): 1871-1883, 2023 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-36706361

RESUMO

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.


Assuntos
Anemia Falciforme , Fator XII , Animais , Camundongos , Anemia Falciforme/complicações , Anemia Falciforme/metabolismo , Fator XII/metabolismo , Inflamação , Acidente Vascular Cerebral , Trombose/metabolismo
2.
Curr Opin Hematol ; 30(5): 153-158, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37462409

RESUMO

PURPOSE OF REVIEW: This review provides an update on recent advances in mechanistic studies of thromboinflammatory mechanisms that contribute to the disease pathology in sickle cell disease (SCD). There is a focus on novel pathways, clinical relevance, and translational potential of these findings. We hope to encourage more advances in this area to reduce organ damage in young patients prior to gene therapy, and to serve the aging SCD patient population. RECENT FINDINGS: Novel insights into the roles of neutrophils, the ADAMTS-13/VWF axis, oxidative stress, and the intrinsic coagulation cascade, as well as relevant clinical trials, are discussed. SUMMARY: Several studies implicate dysregulation of the ADAMTS-13/VWF axis as playing a major role in vaso-occlusive events (VOE) in SCD. Another highlight is reducing iron overload, which has beneficial effects on erythrocyte and neutrophil function that reduce VOE and inflammation. Multiple studies suggest that targeting HO-1/ROS in erythrocytes, platelets, and endothelium can attenuate disease pathology. New insights into coagulation activation identify intrinsic coagulation factor XII as a central regulator of many thromboinflammatory pathologies in SCD. The complement cascade and modulators of neutrophil function and release of neutrophil extracellular traps are also discussed.


Assuntos
Anemia Falciforme , Ferro , Humanos , Proteína ADAMTS13 , Fator de von Willebrand , Anemia Falciforme/tratamento farmacológico , Inflamação
3.
Blood ; 137(11): 1538-1549, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33512489

RESUMO

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anemia Falciforme/metabolismo , Anexina A1/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Transdução de Sinais , Trombose/metabolismo , Adulto , Anemia Falciforme/complicações , Anemia Falciforme/patologia , Animais , Feminino , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Neutrófilos/patologia , Trombose/etiologia , Trombose/patologia , Adulto Jovem
4.
Blood ; 138(3): 259-272, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-33827130

RESUMO

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.


Assuntos
Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fibrinolisina/metabolismo , Fibrinólise/efeitos dos fármacos , Cininogênios/metabolismo , Proteólise/efeitos dos fármacos , Acetaminofen/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fator XII/genética , Fator XII/metabolismo , Feminino , Fibrinolisina/genética , Humanos , Cininogênios/genética , Masculino , Camundongos , Camundongos Knockout , Pré-Calicreína/genética , Pré-Calicreína/metabolismo
5.
Blood ; 135(20): 1783-1787, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31977004

RESUMO

Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.


Assuntos
Anemia Falciforme/metabolismo , Transtornos da Coagulação Sanguínea/etiologia , Receptor PAR-1/metabolismo , Trombina/metabolismo , Anemia Falciforme/complicações , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Constrição Patológica/genética , Constrição Patológica/metabolismo , Modelos Animais de Doenças , Feminino , Hemoglobina Falciforme/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microvasos/metabolismo , Microvasos/patologia , Receptor PAR-1/genética , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
6.
Blood ; 133(23): 2529-2541, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30952675

RESUMO

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa-independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.


Assuntos
Anemia Falciforme/complicações , Anemia Falciforme/patologia , Eritrócitos Anormais/patologia , Trombose/patologia , Trombose Venosa/patologia , Anemia Falciforme/sangue , Animais , Eritrócitos/patologia , Humanos , Camundongos , Trombose/sangue , Trombose Venosa/sangue , Trombose Venosa/etiologia
7.
Circulation ; 140(4): 319-335, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31154815

RESUMO

BACKGROUND: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. METHODS: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. RESULTS: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbß3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbß3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.


Assuntos
Anexina A1/genética , Plaquetas/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Inflamação/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais
8.
Blood ; 123(11): 1747-56, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24449213

RESUMO

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.


Assuntos
Anemia Falciforme/complicações , Modelos Animais de Doenças , Fator Xa/metabolismo , Inflamação/etiologia , Trombina/metabolismo , Doenças Vasculares/etiologia , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Benzimidazóis/farmacologia , Transplante de Medula Óssea , Dabigatrana , Inibidores do Fator Xa , Feminino , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Receptor PAR-1/fisiologia , Receptor PAR-2/fisiologia , Rivaroxabana , Tiofenos/farmacologia , Trombina/antagonistas & inibidores , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia
9.
Nat Chem Biol ; 10(4): 248-50, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24561662

RESUMO

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.


Assuntos
Anticoagulantes/antagonistas & inibidores , Anticoagulantes/síntese química , Heparina de Baixo Peso Molecular/antagonistas & inibidores , Heparina de Baixo Peso Molecular/síntese química , Animais , Anticoagulantes/farmacologia , Antitrombinas/metabolismo , Antitrombinas/farmacologia , Sequência de Carboidratos , Moléculas de Adesão Celular Neuronais/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores do Fator Xa , Hemorragia/tratamento farmacológico , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Indicadores e Reagentes , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Protaminas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Radioisótopos de Enxofre
10.
Haematologica ; 100(3): 308-14, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25596265

RESUMO

An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild-type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic or endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.


Assuntos
Anemia Hemolítica/genética , Anemia Falciforme/genética , Coagulação Sanguínea/efeitos dos fármacos , Heme/administração & dosagem , Tromboplastina/genética , Anemia Hemolítica/sangue , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/patologia , Anemia Falciforme/sangue , Anemia Falciforme/patologia , Animais , Anticorpos/farmacologia , Coagulação Sanguínea/genética , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Fator XI/genética , Fator XI/metabolismo , Fator XIIa/antagonistas & inibidores , Fator XIIa/genética , Fator XIIa/metabolismo , Feminino , Deleção de Genes , Expressão Gênica , Hemopexina/farmacologia , Humanos , Injeções Intravenosas , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Suínos , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo
11.
Res Pract Thromb Haemost ; 8(3): 102395, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38699410

RESUMO

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

13.
Front Med (Lausanne) ; 10: 1141020, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37497271

RESUMO

Sickle Cell Disease (SCD) is a group of inherited hemoglobinopathies. Sickle cell anemia (SCA) is caused by a homozygous mutation in the ß-globin generating sickle hemoglobin (HbS). Deoxygenation leads to pathologic polymerization of HbS and sickling of erythrocytes. The two predominant pathologies of SCD are hemolytic anemia and vaso-occlusive episodes (VOE), along with sequelae of complications including acute chest syndrome, hepatopathy, nephropathy, pulmonary hypertension, venous thromboembolism, and stroke. SCD is associated with endothelial activation due to the release of danger-associated molecular patterns (DAMPs) such as heme, recurrent ischemia-reperfusion injury, and chronic thrombin generation and inflammation. Endothelial cell activation is mediated, in part, by thrombin-dependent activation of protease-activated receptor 1 (PAR1), a G protein coupled receptor that plays a role in platelet activation, endothelial permeability, inflammation, and cytotoxicity. PAR1 can also be activated by activated protein C (APC), which promotes endothelial barrier protection and cytoprotective signaling. Notably, the APC system is dysregulated in SCD. This mini-review will discuss activation of PAR1 by APC and thrombin, the APC-EPCR-PAR1 axis, and their potential roles in SCD.

14.
Am J Physiol Gastrointest Liver Physiol ; 302(7): G748-57, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22223132

RESUMO

The liver is sensitive to pathological conditions associated with tissue hypoxia (Hx) and the presence of activated neutrophils that secrete the serine protease elastase (EL). We demonstrated previously that cotreatment of rat hepatocytes with nontoxic levels of Hx and EL caused synergistic cell death. Hx is sensed by hypoxia-inducible factor (HIF)-1α, a transcription factor that heterodimerizes with HIF-1ß/aryl hydrocarbon receptor nuclear translocator and directs expression of many genes, including the pro-cell death gene Bcl-2/adenovirus E1B-interacting protein 3 (BNIP3). Since cell death from EL or Hx also requires MAPK activation, we tested the hypothesis that the cytotoxic interaction of Hx and EL depends on MAPK and HIF-1α signaling. Treatment of Hepa1c1c7 cells with EL in the presence of Hx (2% O(2)) resulted in synergistic cell death. EL reduced phosphorylated ERK in O(2)-replete and Hx-exposed cells, and ERK inhibition enhanced the cytotoxicity of EL alone. Hx-EL cotreatment caused an additive increase in phosphorylated p38, and p38 inhibition attenuated cell death caused by this cotreatment. EL enhanced Hx-induced HIF-1α accumulation and transcription of the HIF-1α-mediated cell death gene BNIP3, and p38 inhibition attenuated BNIP3 expression and production. Cytotoxicity and BNIP3 expression from EL-Hx cotreatment were reduced in HIF-1ß-deficient HepaC4 cells compared with Hepa1c1c7 cells. These results suggest that p38 signaling contributes to Hx-EL cotreatment-induced cell death via modulation of HIF-1α-mediated gene transcription. Finally, lipid peroxidation was enhanced in Hx-EL-cotreated cells compared with cells treated with EL or Hx alone. Vitamin E treatment attenuated lipid peroxidation and protected cells from the cytotoxicity of Hx and EL, suggesting that lipid peroxidation plays a role.


Assuntos
Morte Celular/efeitos dos fármacos , Hepatócitos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Elastase de Leucócito/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oxigênio/farmacologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
15.
J Pharmacol Exp Ther ; 338(2): 492-502, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21576378

RESUMO

Hypoxia-inducible factor-1α (HIF-1α) is a critical transcription factor that controls oxygen homeostasis in response to hypoxia, inflammation, and oxidative stress. HIF has been implicated in the pathogenesis of liver injury in which these events play a role, including acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in the United States. APAP overdose has been reported to activate HIF-1α in mouse livers and isolated hepatocytes downstream of oxidative stress. HIF-1α signaling controls many factors that contribute to APAP hepatotoxicity, including mitochondrial cell death, inflammation, and hemostasis. Therefore, we tested the hypothesis that HIF-1α contributes to APAP hepatotoxicity. Conditional HIF-1α deletion was generated in mice using an inducible Cre-lox system. Control (HIF-1α-sufficient) mice developed severe liver injury 6 and 24 h after APAP overdose (400 mg/kg). HIF-1α-deficient mice were protected from APAP hepatotoxicity at 6 h, but developed severe liver injury by 24 h, suggesting that HIF-1α is involved in the early stage of APAP toxicity. In further studies, HIF-1α-deficient mice had attenuated thrombin generation and reduced plasminogen activator inhibitor-1 production compared with control mice, indicating that HIF-1α signaling contributes to hemostasis in APAP hepatotoxicity. Finally, HIF-1α-deficient animals had decreased hepatic neutrophil accumulation and plasma concentrations of interleukin-6, keratinocyte chemoattractant, and regulated upon activation normal T cell expressed and secreted compared with control mice, suggesting an altered inflammatory response. HIF-1α contributes to hemostasis, sterile inflammation, and early hepatocellular necrosis during the pathogenesis of APAP toxicity.


Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Hepatócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia
16.
ACS Cent Sci ; 6(7): 1199-1207, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32724854

RESUMO

Chondroitin sulfate E (CS-E) is a sulfated polysaccharide that contains repeating disaccharides of 4,6-disulfated N-acetylgalactosamine and glucuronic acid residues. Here, we report the enzymatic synthesis of three homogeneous CS-E oligosaccharides, including CS-E heptasaccharide (CS-E 7-mer), CS-E tridecasaccharide (CS-E13-mer), and CS-E nonadecasaccharide (CS-E 19-mer). The anti-inflammatory effect of CS-E 19-mer was investigated in this study. CS-E 19-mer neutralizes the cytotoxic effect of histones in a cell-based assay and in mice. We also demonstrate that CS-E 19-mer treatment improves survival and protects against organ damage in a mouse model of endotoxemia induced by bacterial lipopolysaccharide (LPS). CS-E19-mer directly interacts with circulating histones in the plasma from LPS-challenged mice. CS-E 19-mer does not display anticoagulant activity nor react with heparin-induced thrombocytopenia antibodies isolated from patients. The successful synthesis of CS-E oligosaccharides provides structurally defined carbohydrates for advancing CS-E research and offers a potential therapeutic agent to treat life-threatening systemic inflammation.

17.
J Thromb Haemost ; 18(9): 2329-2340, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32573897

RESUMO

BACKGROUND: Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein-kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation. OBJECTIVE: We hypothesize that HK contributes to the hypercoagulable and pro-inflammatory state that causes end-organ damage and early mortality in sickle mice. METHODS: We evaluated the role of HK in the Townes mouse model of SCD. RESULTS/CONCLUSIONS: We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK-deficient background. We found that short-term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long-term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.


Assuntos
Anemia Falciforme , Cininogênio de Alto Peso Molecular , Anemia Falciforme/complicações , Animais , Coagulação Sanguínea , Humanos , Rim , Camundongos , Trombina
18.
Sci Transl Med ; 12(535)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188725

RESUMO

Acetaminophen/paracetamol (APAP) overdose is the leading cause of drug-induced acute liver failure (ALF) in the United States and Europe. The progression of the disease is attributed to sterile inflammation induced by the release of high mobility group box 1 (HMGB1) and the interaction with receptor for advanced glycation end products (RAGE). A specific, effective, and safe approach to neutralize the proinflammatory activity of HMGB1 is highly desirable. Here, we found that a heparan sulfate (HS) octadecasaccharide (18-mer-HP or hepatoprotective 18-mer) displays potent hepatoprotection by targeting the HMGB1/RAGE axis. Endogenous HS proteoglycan, syndecan-1, is shed in response to APAP overdose in mice and humans. Furthermore, purified syndecan-1, but not syndecan-1 core protein, binds to HMGB1, suggesting that HMGB1 binds to HS polysaccharide side chains of syndecan-1. Last, we compared the protection effect between 18-mer-HP and N-acetyl cysteine, which is the standard of care to treat APAP overdose. We demonstrated that 18-mer-HP administered 3 hours after a lethal dose of APAP is fully protective; however, the treatment of N-acetyl cysteine loses protection. Therefore, 18-mer-HP may offer a potential therapeutic advantage over N-acetyl cysteine for late-presenting patients. Synthetic HS provides a potential approach for the treatment of APAP-induced ALF.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Falência Hepática Aguda , Acetaminofen/toxicidade , Animais , Anti-Inflamatórios , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Europa (Continente) , Heparitina Sulfato , Humanos , Fígado , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL
19.
J Pharmacol Exp Ther ; 331(1): 114-21, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19638570

RESUMO

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-alpha (TNF-alpha) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-alpha receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-alpha. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNF-alpha augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-alpha can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Sulindaco/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sinergismo Farmacológico , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/toxicidade , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Ratos , Ratos Sprague-Dawley , Sulindaco/toxicidade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue
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