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1.
J Cardiovasc Dev Dis ; 7(1)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197497

RESUMO

BACKGROUND: The timing for initiation of effective antithrombotic therapy relative to the onset of arterial thrombosis may influence outcomes. This report investigates the hypothesis that early administration of heparin anticoagulation relative to the onset of thrombotic occlusion will effect a reduction in occlusion. METHODS: A standard rat model of experimental thrombosis induction was used, injuring the carotid artery exposure with FeCl3-saturated filter paper, followed by flow monitoring for onset of occlusion and subsequent embolization events. Intravenous heparin administration (200 units/mL) was timed relative to the initiation of injury or onset of near occlusion, compared with controls (no heparin administration). RESULTS: No occlusion was found for delivery of heparin 5 min prior to thrombus induction, whereas all vessels occluded without heparin. Unstable (embolic) thrombi were seen with heparin given at or shortly after initial occlusion. Only 9% (1/11) of the vessels had permanent occlusion when heparin was given at the time of thrombotic onset (p < 0.0001 vs. unheparinized), while 50% occluded when heparin was delayed by 5 min (p > 0.05). CONCLUSIONS: These findings provide evidence that antithrombotic therapy may need to be administered prior to the onset of anticipated loss of patency, with less effectiveness when given after occlusion has occurred.

2.
Haematologica ; 105(1): 218-225, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31048354

RESUMO

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

3.
Nat Commun ; 10(1): 4192, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519896

RESUMO

Lymph node (LN) metastases correspond with a worse prognosis in nearly all cancers, yet the occurrence of cancer spreading from LNs remains controversial. Additionally, the mechanisms explaining how cancers survive and exit LNs are largely unknown. Here, we show that breast cancer patients frequently have LN metastases that closely resemble distant metastases. In addition, using a microsurgical model, we show how LN metastasis development and dissemination is regulated by the expression of a chromatin modifier, histone deacetylase 11 (HDAC11). Genetic and pharmacologic blockade of HDAC11 decreases LN tumor growth, yet substantially increases migration and distant metastasis formation. Collectively, we reveal a mechanism explaining how HDAC11 plasticity promotes breast cancer growth as well as dissemination from LNs and suggest caution with the use of HDAC inhibitors.


Assuntos
Neoplasias da Mama/metabolismo , Histona Desacetilases/metabolismo , Linfonodos/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Imunoprecipitação da Cromatina , Metilação de DNA/genética , Metilação de DNA/fisiologia , Citometria de Fluxo , Células HEK293 , Histona Desacetilases/genética , Humanos , Linfonodos/patologia , Metástase Linfática/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
4.
Blood Adv ; 2(21): 2848-2861, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30381401

RESUMO

Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction-induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI-mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet-fed conditions comparable to that seen in chow diet-fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.


Assuntos
Plaquetas/metabolismo , Antígenos CD36/metabolismo , Caspases/metabolismo , Fibrina/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fosfatidilserinas/metabolismo , Animais , Plaquetas/citologia , Antígenos CD36/antagonistas & inibidores , Venenos de Crotalídeos/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/patologia , Lectinas Tipo C , Lipoproteínas LDL/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombose/etiologia , Trombose/patologia , Quinases da Família src/metabolismo
5.
Blood Coagul Fibrinolysis ; 29(7): 636-643, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30113321

RESUMO

: The murine FeCl3 model is a widely used model for studying arterial thrombosis, yet provides limited information from each mouse, often only a single time point for the onset of occlusion (defined as the time to occlusion; TTO). To optimize data from the murine ferric chloride model of thrombosis. FeCl3 injury was induced in the carotid arteries of wild-type and Factor IX (FIX) knockout mice, with infusion of recombinant FIX (rFIX) to normalize FIX deficiency at various times around FeCl3 injury. The TTO was recorded as a percentage of baseline flow as occlusion continued to zero flow, with identification of reflow events. The TTO among the treatment groups of FIX-deficient mice showed no statistical differences, except with physiological saline-treated FIX-deficient mice and those receiving delayed treatment. Incidences of occlusion were 100% for wild-type mice and FIX-deficient mice receiving slow infusions of rFIX at early times around the FeCl3 application. In contrast, only 68% of FIX-deficient mice achieved occlusion with preinfusion of rFIX and none occluded with delayed rFIX infusion. A majority of occluded vessels exhibited reflow events, with significantly lower incidence for slow infusion of rFIX starting 4 min after FeCl3 application in comparison with preinjury bolus, demonstrating characterization of a differential response to timing and infusion rates of treatment. Simple use of the time to occlusion may not maximize data available from the FeCl3 arterial thrombosis model. Inclusion of documenting reflow events can extend the useful data obtained with application of this model.


Assuntos
Fator IX/administração & dosagem , Trombose/tratamento farmacológico , Animais , Artérias Carótidas/patologia , Cloretos , Compostos Férricos , Hemofilia B/tratamento farmacológico , Camundongos , Camundongos Knockout , Trombose/induzido quimicamente , Resultado do Tratamento
6.
Arterioscler Thromb Vasc Biol ; 38(4): 816-828, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29419409

RESUMO

OBJECTIVE: PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS-FIXa interaction in hemostasis. APPROACH AND RESULTS: We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein-protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa. CONCLUSIONS: Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.


Assuntos
Plaquetas/metabolismo , Fator IXa/metabolismo , Hemofilia B/sangue , Hemostasia , Heparina/metabolismo , Proteína S/metabolismo , Trombose Venosa/prevenção & controle , Animais , Sítios de Ligação , Ligação Competitiva , Coagulantes/administração & dosagem , Modelos Animais de Doenças , Fator IX/genética , Fator IX/metabolismo , Fator IXa/administração & dosagem , Fator IXa/genética , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Hemostasia/efeitos dos fármacos , Humanos , Infusões Intravenosas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Trombose Venosa/sangue , Trombose Venosa/genética
7.
Blood Adv ; 2(1): 25-35, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29344582

RESUMO

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIIIplasma) as a heterotetramer of A2 and B2 subunits and platelets (FXIIIplt) as an A2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIIIplasma, but not FXIIIplt, produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIIIplasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.


Assuntos
Eritrócitos/patologia , Fator XIII/fisiologia , Trombose/patologia , Trombose Venosa/patologia , Animais , Plaquetas , Fibrina/metabolismo , Hemorragia/etiologia , Camundongos , Plasma/química , Trombina/biossíntese
8.
Blood Adv ; 1(18): 1398-1408, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-29104956

RESUMO

Actin reorganization regulates key processes in platelet activation. Here we examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet function. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage (Arpc2fl/flPF4-Cre). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Arpc2fl/flPF4-Cre platelets exhibited alterations in their actin cytoskeleton and their peripheral microtubule coil. Thrombocytopenia was alleviated following clodronate liposome-induced macrophage depletion in Arpc2fl/flPF4-Cre mice. Arpc2fl/flPF4-Cre platelets failed to spread and showed a mild defect in integrin activation and aggregation. However, no significant differences in hemostasis or thrombosis were observed between Arpc2fl/flPF4-Cre and control mice. Thus, Arp2/3 is critical for platelet homeostasis but plays only a minor role for vascular hemostasis.

9.
Blood ; 129(21): 2917-2927, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28336528

RESUMO

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.


Assuntos
Plaquetas/imunologia , Antígenos CD36/imunologia , Hiperlipidemias/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 7 Ativada por Mitógeno/imunologia , Ativação Plaquetária/imunologia , Trombose/imunologia , Aloenxertos , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Plaquetas/patologia , Transplante de Medula Óssea , Antígenos CD36/genética , Humanos , Hiperlipidemias/genética , Hiperlipidemias/patologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Mutantes , Proteína Quinase 7 Ativada por Mitógeno/genética , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Ativação Plaquetária/genética , Trombose/genética , Trombose/patologia
10.
Blood ; 129(18): 2537-2546, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28251913

RESUMO

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro, and in silico. Compared with controls, mice with elevated hematocrit (RBCHIGH) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBCHIGH mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans.


Assuntos
Antitrombina III/metabolismo , Artérias , Coagulação Sanguínea , Peptídeo Hidrolases/metabolismo , Trombose/metabolismo , Lesões do Sistema Vascular/metabolismo , Animais , Artérias/lesões , Artérias/metabolismo , Plaquetas , Feminino , Hematócrito , Humanos , Masculino , Camundongos , Resistência ao Cisalhamento
11.
Arterioscler Thromb Vasc Biol ; 36(9): 1838-46, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27417588

RESUMO

OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.


Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Hemostasia , Ativação Plaquetária , Trombose/prevenção & controle , Animais , Modelos Animais de Doenças , Genótipo , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos Transgênicos , Mutação , Fenótipo , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Trombose/sangue , Trombose/genética , Fatores de Tempo , Proteínas rap1 de Ligação ao GTP/sangue
12.
Thromb Res ; 145: 159-60, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27373598

RESUMO

Whereas the extrinsic pathway of coagulation seals off bleeding at the cut tissue edges, it is proposed that the intrinsic pathway exploits the dirt from the skin surface to generate an outer coagulum of the oozing blood. Activated Factor XII (FXIIa) in this outer cap generates Factor XIa, which triggers clotting, and kallikrein that feeds back to form more FXIIa to promote the process. This dirty-wound hypothesis of coagulation function by the intrinsic pathway is supported by the use of dirt-based compounds in activated partial thromboplastin time assays as well as the evolutionary record where marine life that do not have skin-adherent dirt lack Factor XII, including marine mammals that have returned to sea life.


Assuntos
Coagulação Sanguínea , Humanos
13.
Circ Res ; 118(9): 1363-79, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-27126647

RESUMO

Thrombosis is a leading cause of morbidity and mortality worldwide. Animal models are used to understand the pathological pathways involved in thrombosis and to test the efficacy and safety of new antithrombotic drugs. In this review, we will first describe the central role a variety of animal models of thrombosis and hemostasis has played in the development of new antiplatelet and anticoagulant drugs. These include the widely used P2Y12 antagonists and the recently developed orally available anticoagulants that directly target factor Xa or thrombin. Next, we will describe the new players, such as polyphosphate, neutrophil extracellular traps, and microparticles, which have been shown to contribute to thrombosis in mouse models, particularly venous thrombosis models. Other mouse studies have demonstrated roles for the factor XIIa and factor XIa in thrombosis. This has spurred the development of strategies to reduce their levels or activities as a new approach for preventing thrombosis. Finally, we will discuss the emergence of zebrafish as a model to study thrombosis and its potential use in the discovery of novel factors involved in thrombosis and hemostasis. Animal models of thrombosis from zebrafish to nonhuman primates are vital in identifying pathological pathways of thrombosis that can be safely targeted with a minimal effect on hemostasis. Future studies should focus on understanding the different triggers of thrombosis and the best drugs to prevent each type of thrombotic event.


Assuntos
Anticoagulantes/uso terapêutico , Fibrinolíticos/uso terapêutico , Inibidores da Agregação de Plaquetas/uso terapêutico , Trombose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Camundongos , Primatas , Trombose/genética , Trombose/metabolismo , Trombose/patologia , Peixe-Zebra
14.
Thromb Res ; 140: 149-152, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26860966

RESUMO

Deep vein thrombosis (DVT) and its sequela, pulmonary embolism, occur at a rate of 1 per 1000 person/year. Experimental models for evaluation of DVT have many short-comings, such as mechanical occlusion or stenosis to cause thrombosis, rather than the clinical scenario of thrombosis causing occlusion/stenosis. The goal of this study was to develop a model of flow-based large-vein thrombosis with resistance to resolution, to model clinical DVT behavior. Adult male C57Bl/6 mice underwent thrombus induction via an electrolytic injury to the femoral vein (3V positive current for 90s), with subsequent intra-vital fluorescence quantitation of platelet and fibrin accumulation through the first 60 min, and final histomorphometric volume evaluation at 1, 7, 14, and 28 days. Platelet accumulation at the injury site was comparable to a milder electrolytic injury, whereas fibrin was greatly augmented by 60 min in the more severe injury model. Thrombi showed persistent presence at 1 and 7 days, with remodeling to a stenotic fibrosis that encroached into the lumen at 14 and 28 days. The thrombotic/fibrotic volume within the femoral vein fell by 23% from 1 to 7 days, but had a residual presence at 28 days that was 31% the 1-day volume. This new model may provide an alternative approach to evaluating DVT persistence and therapeutic inhibition, to develop a better understanding of the clinical progression of DVT to thrombophlebitis.


Assuntos
Veia Femoral/patologia , Trombose Venosa/patologia , Animais , Plaquetas/patologia , Modelos Animais de Doenças , Fibrina/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
Int J Pept Res Ther ; 22(3): 317-324, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28989333

RESUMO

Acute myocardial infarction (AMI) results in systolic dysfunction, myocarditis and fibrotic remodeling, which causes irreversible pathological remodeling of the heart. Associated cell death and inflammation cause cytokine release, which activates the p38 MAPK signaling pathway to propagate damaging signals via MAPKAP kinase 2 (MK2). Previously we showed that intraperitoneal injection of a cell permeable peptide inhibitor of MK2, MMI-0100, protects against fibrosis, apoptosis and systolic dysfunction in a mouse model of AMI induced by left-anterior descending coronary artery (LAD) ligation. Here we tested a new route of administration of MMI-0100: inhalation of nebulized peptide. When given within 30 min of AMI and daily for 2 weeks thereafter, both inhaled and injected MMI-0100 improved cardiac function as measured by conscious echocardiography. Limited fibrosis was observed after 2 weeks by Massons trichrome staining, suggesting that MMI-0100 protects the heart prior to the formation of significant fibrosis. These results support a nebulized route of administration of MMI-0100 can protect the myocardium from ischemic damage.

16.
Blood ; 125(26): 4078-84, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-25954015

RESUMO

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.


Assuntos
Desenvolvimento Embrionário/fisiologia , Lipoproteínas/metabolismo , Camundongos/embriologia , Ativação Plaquetária/fisiologia , Trombina/metabolismo , Animais , Camundongos Knockout
17.
Thromb Res ; 135(5): 939-43, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25764909

RESUMO

BACKGROUND: The mechanism of thrombotic induction in experimental models can vary greatly, as can the applied evaluative measures, making comparisons among models difficult. OBJECTIVES: This study comparatively evaluated the arterial thrombodynamic response among injury mechanisms. METHODS: Thrombotic responses were induced in mouse carotid arteries, with subsequent intravital imaging using rhodamine-6G-labeled platelets to quantitate platelet accumulation over 30minutes. Nine induction methods were evaluated: brief pinch, temporary hard ligation, cautery/heat, needle puncture, intralumenal wire (scratch), intralumenal adventitia/collagen (2 different models), and brief exposures to either iron-based surface electrolytic injury or ferric chloride. RESULTS: The accumulation of platelets was variable among induction methods, with a greater response to more severe injury mechanisms, free radical injury, and exposed collagen. Temporal profiles were generated by normalizing data to peak platelet accumulation for each run; rapid platelet development and subsequent detachment were found for mechanical injuries that maintained vessel integrity (pinch and ligation injuries), with more sustained growth for more severe mechanical (wire) injury or breach of the vessel (needle puncture or intralumenal collagen). A delayed but extended temporal response was seen with free radical injury (both electrolytic and ferric chloride). CONCLUSIONS: These findings demonstrate a dependence of platelet thrombodynamics on the method of induction, with collagen exposure causing greater, more prolonged activity, while free-radical injury effected a delayed but sustained platelet thrombus formation with slower resolution. A better understanding of how these various injury models relate to clinical causes of arterial thrombosis is needed for optimal translational interpretation of murine models of thrombosis.


Assuntos
Plaquetas/patologia , Artérias Carótidas/patologia , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/patologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica
18.
Microsurgery ; 34(8): 653-6, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24848809

RESUMO

Microvascular training models for vein grafting most often use the rat epigastric vein interpositioned to the femoral artery. We describe the rat posterior facial vein as an alternative vein graft model; it has at least a 2:1 diametric ratio to the femoral artery and a tougher connective tissue, making it more similar to clinical vein grafting for reconstructive microsurgery. A series of 24 grafts interpositioned to the femoral artery were done using 11-12 sutures per end-to-end anastomosis and yielded early patency rates of 96% at 20 min and 92% at 2 and 4 weeks for subsets of 12 grafts. As a training model the diametric disparity provides unique challenges with clinical relevance, for which a number of different techniques for matching arterial to venous circumferences can be done.


Assuntos
Face/irrigação sanguínea , Artéria Femoral/cirurgia , Microcirurgia/educação , Enxerto Vascular/educação , Veias/transplante , Anastomose Cirúrgica/educação , Animais , Modelos Anatômicos , Modelos Animais , Ratos , Ratos Endogâmicos Lew
19.
Sci Transl Med ; 6(227): 227ra34, 2014 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-24622514

RESUMO

Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage-tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor-ß (TGF-ß) signaling by TGF-ß neutralizing antibody, short hairpin RNA-mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell-specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-ß-Smad2/3-Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Veias/crescimento & desenvolvimento , Veias/transplante , Animais , Anticorpos Neutralizantes/farmacologia , Linhagem da Célula/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Neointima/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Veias/efeitos dos fármacos
20.
J Hand Surg Am ; 38(9): 1784-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23891176

RESUMO

PURPOSE: To evaluate the hypothesis that platelets and fibrin differentially accrue at microvascular anastomoses in arteries versus veins and under different pharmacologic conditions. METHODS: We evaluated mouse arterial and venous anastomoses with intravital fluorescence imaging, using fluorophore-labeled platelets and anti-fibrin antibodies to measure the extent of thrombus component development in the intraluminal anastomotic site. We evaluated systemic heparin or eptifibatide (platelet aggregation inhibitor) to determine their relative influences on thrombus composition. RESULTS: Platelets accumulated rapidly in both arterial and venous repairs, and then fell in number after 10 to 30 minutes of reflow. Fibrin had a relatively steady development over 60 minutes in veins, with a more variable increase in arteries. Heparin reduced platelet accumulation in arteries and fibrin development in veins. Eptifibatide reduced platelets in both arteries and veins and had an apparent effect on lowering the amount of fibrin in veins. CONCLUSIONS: These findings show that platelets have a rapid, transient response, whereas fibrin has a slower, more sustained accrual in both arterial and venous anastomoses. Furthermore, inhibition of either coagulation or platelet aggregation can influence presumably non-targeted components of thrombosis in vascular repairs of both arteries and veins. CLINICAL RELEVANCE: Preventing replantation failure using antithrombotic therapies requires a better understanding of the effect of each pharmacologic compound on the various aspects of thrombogenesis.


Assuntos
Fibrina/efeitos dos fármacos , Fibrinolíticos/farmacologia , Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trombose/fisiopatologia , Anastomose Cirúrgica , Animais , Eptifibatida , Fibrinolíticos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirurgia , Peptídeos/uso terapêutico , Reimplante , Trombose/prevenção & controle , Grau de Desobstrução Vascular/efeitos dos fármacos
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