RESUMO
PURPOSE: Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. EXPERIMENTAL DESIGN: MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS: MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. CONCLUSIONS: We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.
Assuntos
Aminopiridinas , Benzimidazóis , Inibidores de Janus Quinases , Transtornos Mieloproliferativos , Animais , Camundongos , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismoRESUMO
BACKGROUND: Sickle cell disease (SCD) is a genetic hemoglobinopathy associated with high morbidity and mortality. The primary cause of hospitalization in SCD is vaso-occlusive crisis (VOC), mediated by alteration of red blood cells, platelets, immune cells and a pro-adhesive endothelium. OBJECTIVES: We investigated the potential therapeutic use of the plant-derived omega-3 alpha-linolenic acid (ALA) in SCD. METHODS: Berkeley mice were fed a low- or high-ALA diet for 4 weeks, followed by analysis of liver fibrosis, endothelial activation, platelet activation and formation of platelet-neutrophils aggregates. Aggregation of platelets over collagen under flow after high-ALA was compared to a blocking P-selectin Fab. RESULTS: Dietary high-ALA was able to reduce the number of sickle cells in blood smear, liver fibrosis, and the expression of adhesion molecules on the endothelium of aorta, lungs, liver and kidneys (VCAM-1, ICAM-1 and vWF). Specific parameters of platelet activation were blunted after high-ALA feeding, notably P-selectin exposure and the formation of neutrophil-platelet aggregates, along with a correspondingly reduced expression of PSGL-1 on neutrophils. By comparison, in vivo treatment of SCD mice with the anti-P-selectin Fab was able to similarly reduce the formation of neutrophil-platelet aggregates, but did not reduce GpIbα shedding nor the activation of the αIIb ß3 integrin in response to thrombin. Both ALA feeding and P-selectin blocking significantly reduced collagen-mediated cell adhesion under flow. CONCLUSIONS: Dietary ALA is able to reduce the pro-inflammatory and pro-thrombotic state occurring in the SCD mouse model and may represent a novel, inexpensive and readily available therapeutic strategy for SCD.
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Anemia Falciforme , Ácido alfa-Linolênico , Anemia Falciforme/tratamento farmacológico , Animais , Adesão Celular , Colágeno , Dieta , Camundongos , Ativação Plaquetária , Ácido alfa-Linolênico/farmacologia , Ácido alfa-Linolênico/uso terapêuticoRESUMO
Somatic mutations in JAK2, calreticulin, and MPL genes drive myeloproliferative neoplasms (MPN), and recent technological advances have revealed a heterogeneous genomic landscape with additional mutations in MPN. These mainly affect genes involved in epigenetic regulation and splicing and are of diagnostic and prognostic value, predicting the risk of progression and informing decisions on therapeutic management. Thus, genetic testing has become an integral part of the current state-of-the-art laboratory work-up for MPN patients and has been implemented in current guidelines for disease classification, tools for prognostic risk assessment, and recommendations for therapy. The finding that JAK2, CALR, and MPL driver mutations activate JAK2 signaling has provided a rational basis for the development of targeted JAK2 inhibitor therapies and has fueled their translation into clinical practice. However, the disease-modifying potential of JAK2 inhibitors remains limited and is further impeded by loss of therapeutic responses in a substantial proportion of patients over time. Therefore, the investigation of additional molecular vulnerabilities involved in MPN pathogenesis is imperative to advance the development of new therapeutic options. Combination of novel compounds with JAK2 inhibitors are of specific interest to enhance therapeutic efficacy of molecularly targeted treatment approaches. Here, we summarize the current insights into the genetic basis of MPN, its use as a diagnostic and prognostic tool in clinical settings, and the most recent advances in targeted therapies for MPN.
RESUMO
Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients' CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.
Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Transtornos Mieloproliferativos/tratamento farmacológico , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Proliferação de Células , Feminino , Humanos , Janus Quinase 2/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologiaRESUMO
BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.
Assuntos
Quimiocina CXCL12 , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Fator A de Crescimento do Endotélio Vascular , Animais , Modelos Animais de Doenças , Células Endoteliais , Membro Posterior , Músculo Esquelético , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The association of glycated hemoglobin (HbA1c) with venous thromboembolism (VTE) and death in the elderly is unknown. In the SWEETCO 65+ study we analyzed prospectively a Swiss Cohort of Elderly Patients with Venous Thromboembolism (SWITCO 65+). 888 patients were enrolled for the SWEETCO 65+ analysis. HbA1c was determined at baseline and divided into three categories (HbA1c < 5.7%, normal range; 5.7-6.49%, pre-diabetic range; and >6.5%, diabetic range). Median follow-up was 2.5 years. The primary endpoint was recurrent VTE. Secondary endpoints included all-cause mortality and major bleeds. The total prevalence of diabetes was 22.1%. The risk of recurrent VTE was similar in patients with HbA1c with pre-diabetes (adjusted subhazard ratio (aSHR) 1.07 [0.70 to 1.63]) and diabetes (aSHR 0.73 [0.39 to 1.37]) as compared to those with a HbA1c in the normal range. However, a HbA1c ≥ 6.5% (median IQ range 7.0 [6.70;7.60]) was significantly associated with a higher risk of all-cause mortality (adjusted hazard ratio [aHR] 1.83 [1.21 to 2.75]). In summary we found no association between HbA1c and major bleeding. Elevated HbA1c levels are not associated with recurrent VTE but with increased all-cause mortality in an elderly population with acute VTE.
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Hemoglobinas Glicadas/metabolismo , Mortalidade/tendências , Tromboembolia Venosa/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Tromboembolia Venosa/epidemiologiaRESUMO
AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
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Aterosclerose/metabolismo , Aterosclerose/patologia , Medula Óssea/patologia , Deleção de Genes , Receptores Depuradores Classe A/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Transplante de Medula Óssea , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hematopoese , Homozigoto , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Modelos Biológicos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células RAW 264.7RESUMO
Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2 Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-ß cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas , Ácido alfa-Linolênico , Animais , Plaquetas , Análise por Conglomerados , Tomografia com Microscopia Eletrônica , Humanos , Ácido alfa-Linolênico/farmacologia , Fator de von WillebrandRESUMO
Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.
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Neoplasias Hematológicas/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Substituição de Aminoácidos , Animais , Becaplermina/genética , Becaplermina/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismoRESUMO
BACKGROUND: Omega-3 fatty acids (nâ-â3 FA) may have blood pressure (BP)-lowering effects in untreated hypertensive and elderly patients. The effect of nâ-â3 FA on BP in young, healthy adults remains unknown. The Omega-3 Index reliably reflects an individuals' omega-3 status. We hypothesized that the Omega-3 Index is inversely associated with BP levels in young healthy adults. METHODS: The current study (nâ=â2036) is a cross-sectional study investigating the baseline characteristics of a cohort, which includes healthy adults, age 25-41 years. Individuals with cardiovascular disease, known diabetes or a BMI higher than 35âkg/m were excluded. The Omega-3 Index was determined in whole blood using gas chromatography. Association with office and 24-h BP was assessed using multivariable linear regression models adjusted for potential confounders. RESULTS: Median Omega-3 Index was 4.58% (interquartile range 4.08; 5.25). Compared with individuals in the lowest Omega-3 Index quartile, individuals in the highest had a SBP and DBP that was 4 and 2âmmHg lower, respectively (Pâ<â0.01). A significant linear inverse relationship of the Omega-3 Index with 24-h and office BP was observed. Per 1-U increase in log-transformed Omega-3 Index the lowering in BP (given as multivariable adjusted ß coefficients; 95% confidence interval) was -2.67âmmHg (-4.83; -0.51; Pâ=â0.02) and -2.30âmmHg (-3.92; -0.68; Pâ=â0.005) for 24-h SBP and DBP, respectively. CONCLUSION: A higher Omega-3 Index is associated with statistically significant, clinically relevant lower SBP and DBP levels in normotensive young and healthy individuals. Diets rich in nâ-â3 FA may be a strategy for primary prevention of hypertension.
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Pressão Sanguínea , Ácidos Graxos Ômega-3/sangue , Adulto , Estudos de Coortes , Estudos Transversais , Feminino , Voluntários Saudáveis , Humanos , MasculinoRESUMO
BACKGROUND: Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. METHODS AND RESULTS: After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. CONCLUSIONS: Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Endotélio Vascular/fisiopatologia , Derivação Gástrica/efeitos adversos , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Obesidade/cirurgia , Peptídeos/administração & dosagem , Vasodilatação/fisiologia , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Injeções Subcutâneas , Masculino , Fármacos Neuroprotetores , Obesidade/complicações , Obesidade/enzimologia , Estresse Oxidativo , Fosforilação , Ratos , Ratos WistarRESUMO
Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Furocumarinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/efeitos da radiação , Raios Ultravioleta , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Colágeno/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos da radiação , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos da radiação , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator de von Willebrand/metabolismoRESUMO
AIMS: The lectin-like oxLDL receptor-1 (LOX-1) promotes endothelial uptake of oxidized low-density lipoprotein (oxLDL) and plays an important role in atherosclerosis and acute coronary syndromes (ACS). However, its role in arterial thrombus formation remains unknown. We investigated whether LOX-1 plays a role in arterial thrombus formation in vivo at different levels of oxLDL using endothelial-specific LOX-1 transgenic mice (LOX-1TG) and a photochemical injury thrombosis model of the carotid artery. METHODS AND RESULTS: In mice fed a normal chow diet, time to arterial occlusion was unexpectedly prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity in carotid arteries of LOX-1TG mice were reduced by half. This effect was mediated by activation of octamer transcription factor 1 (Oct-1) leading to upregulation of the mammalian deacetylase silent information regulator-two 1 (SIRT1) via binding to its promoter and subsequent inhibition of NF-κB signaling. In contrast, intravenous injection of oxLDL as well as high cholesterol diet for 6 weeks led to a switch from the Oct-1/SIRT1 signal transduction pathway to the ERK1/2 pathway and in turn to an enhanced thrombotic response with shortened occlusion time. CONCLUSIONS: Thus, LOX-1 differentially regulates thrombus formation in vivo depending on the degree of activation by oxLDL. At low oxLDL levels LOX-1 activates the protective Oct-1/SIRT1 pathway, while at higher levels of the lipoprotein switches to the thrombogenic ERK1/2 pathway. These findings may be important for arterial thrombus formation in ACS and suggest that SIRT1 may represent a novel therapeutic target in this context.
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Coagulação Sanguínea , Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Lipoproteínas LDL/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Receptores Depuradores Classe E/metabolismo , Sirtuína 1/metabolismo , Trombose/enzimologia , Animais , Sítios de Ligação , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Colesterol na Dieta , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Receptores Depuradores Classe E/genética , Transdução de Sinais , Sirtuína 1/genética , Tromboplastina/metabolismo , Trombose/sangue , Trombose/genética , Trombose/prevenção & controle , Fatores de TempoRESUMO
BACKGROUND AND AIMS: High-density lipoprotein cholesterol (HDL-C) is inversely related to cardiovascular risk. HDL-C raising ester transfer protein (CETP) inhibitors, are novel therapeutics. We studied the effects of CETP inhibitors anacetrapib and evacetrapib on triglycerides, cholesterol and lipoproteins, cholesterol efflux, paraoxonase activity (PON-1), reactive oxygen species (ROS), and endothelial function in E3L and E3L.CETP mice. METHODS: Triglycerides and cholesterol were measured at weeks 5, 14 and 21 in E3L.CETP mice on high cholesterol diet and treated with anacetrapib (3 mg/kg/day), evacetrapib (3 mg/kg/day) or placebo. Cholesterol efflux was assessed ex-vivo in mice treated with CETP inhibitors for 3 weeks on a normal chow diet. Endothelial function was analyzed at week 21 in isolated aortic rings, and serum lipoproteins assessed by fast-performance liquid chromatography. RESULTS: Anacetrapib and evacetrapib increased HDL-C levels (5- and 3.4-fold, resp.) and reduced triglycerides (-39% vs. placebo, p = 0.0174). Total cholesterol levels were reduced only in anacetrapib-treated mice (-32%, p = 0.0386). Cholesterol efflux and PON-1 activity (+45% and +35% vs. control, p < 0.005, resp.) were increased, while aortic ROS production was reduced with evacetrapib (-49% vs. control, p = 0.020). Anacetrapib, but not evacetrapib, impaired endothelium dependent vasorelaxation (p < 0.05). In contrast, no such effects were observed in E3L mice for all parameters tested. CONCLUSIONS: Notwithstanding a marked rise in HDL-C, evacetrapib did not improve endothelial function, while anacetrapib impaired it, suggesting that CETP inhibition does not provide vascular protection. Anacetrapib exerts unfavorable endothelial effects beyond CETP inhibition, which may explain the neutral results of large clinical trials in spite of increased HDL-C.
Assuntos
Anticolesterolemiantes/farmacologia , Benzodiazepinas/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , HDL-Colesterol/sangue , Dislipidemias/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Oxazolidinonas/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Anticolesterolemiantes/toxicidade , Apolipoproteína E3/genética , Arildialquilfosfatase/sangue , Benzodiazepinas/toxicidade , Biomarcadores/sangue , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Dislipidemias/sangue , Dislipidemias/genética , Dislipidemias/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Predisposição Genética para Doença , Humanos , Camundongos Transgênicos , Oxazolidinonas/toxicidade , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Triglicerídeos/sangue , Regulação para CimaRESUMO
AIMS: The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated. METHODS AND RESULTS: Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-α); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-α-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries. CONCLUSION: Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor.
Assuntos
Adenosina/análogos & derivados , Coagulação Sanguínea/efeitos dos fármacos , Lesões das Artérias Carótidas/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Fibrinolíticos/farmacologia , Tromboplastina/metabolismo , Trombose/prevenção & controle , Ticlopidina/análogos & derivados , Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Células Cultivadas , Clopidogrel , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Inibidores da Agregação Plaquetária/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Receptores Purinérgicos P2Y12/metabolismo , Tromboplastina/genética , Trombose/sangue , Trombose/genética , Ticagrelor , Ticlopidina/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Roux-en-Y gastric bypass (RYGB) reduces body weight and cardiovascular mortality in morbidly obese patients. Glucagon-like peptide-1 (GLP-1) seems to mediate the metabolic benefits of RYGB partly in a weight loss-independent manner. The present study investigated in rats and patients whether obesity-induced endothelial and high-density lipoprotein (HDL) dysfunction is rapidly improved after RYGB via a GLP-1-dependent mechanism. METHODS AND RESULTS: Eight days after RYGB in diet-induced obese rats, higher plasma levels of bile acids and GLP-1 were associated with improved endothelium-dependent relaxation compared with sham-operated controls fed ad libitum and sham-operated rats that were weight matched to those undergoing RYGB. Compared with the sham-operated rats, RYGB improved nitric oxide (NO) bioavailability resulting from higher endothelial Akt/NO synthase activation, reduced c-Jun amino terminal kinase phosphorylation, and decreased oxidative stress. The protective effects of RYGB were prevented by the GLP-1 receptor antagonist exendin9-39 (10 µg·kg(-1)·h(-1)). Furthermore, in patients and rats, RYGB rapidly reversed HDL dysfunction and restored the endothelium-protective properties of the lipoprotein, including endothelial NO synthase activation, NO production, and anti-inflammatory, antiapoptotic, and antioxidant effects. Finally, RYGB restored HDL-mediated cholesterol efflux capacity. To demonstrate the role of increased GLP-1 signaling, sham-operated control rats were treated for 8 days with the GLP-1 analog liraglutide (0.2 mg/kg twice daily), which restored NO bioavailability and improved endothelium-dependent relaxations and HDL endothelium-protective properties, mimicking the effects of RYGB. CONCLUSIONS: RYGB rapidly reverses obesity-induced endothelial dysfunction and restores the endothelium-protective properties of HDL via a GLP-1-mediated mechanism. The present translational findings in rats and patients unmask novel, weight-independent mechanisms of cardiovascular protection in morbid obesity.
Assuntos
Peso Corporal/fisiologia , Endotélio Vascular/fisiologia , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Lipoproteínas HDL/fisiologia , Obesidade/cirurgia , Redução de Peso/fisiologia , Adulto , Animais , Antioxidantes/fisiologia , Estudos de Casos e Controles , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Endotélio Vascular/patologia , Feminino , Derivação Gástrica , Humanos , Masculino , Óxido Nítrico/fisiologia , Obesidade/fisiopatologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais , Resultado do TratamentoRESUMO
Venous thromboembolism (VTE) is a leading cause of cardiovascular death. Omega-3 fatty acids (n-3 FA) exhibit protective effects against cardiovascular disease. Others and our group have reported that the plant-derived n-3 FA alpha-linolenic acid (ALA) displays antiinflammatory, anticoagulant and antiplatelet effects, thereby reducing atherosclerosis and arterial thrombosis in mice fed a high ALA diet. Since procoagulant factors such as tissue factor and fibrin as well as platelets and leukocytes are crucially involved in the development of VTE, we investigated possible protective effects of dietary ALA on venous thrombus formation in a mouse model of stenosis- and furthermore, in a mouse model of endothelial injury-induced venous thrombosis. Four week old C57BL/6 mice underwent four weeks of high (7.3g%) or low ALA (0.03g%) treatment before being exposed to inferior vena cava (IVC) stenosis for 48 hours or laser injury of the endothelium of the internal jugular vein (IJV). Thrombus generation frequency, thrombus size and composition (IVC stenosis group) and time to thrombus formation (endothelial injury group) were assessed. In addition, plasma glycocalicin, a marker of platelet activation, platelet P-selectin and activated integrin expression as well as plasma thrombin generation was determined, but did not reveal any significant differences between the groups. Despite its protective properties against arterial thrombus formation, dietary ALA did not protect against venous thrombosis neither in the IVC stenosis nor the endothelial injury model, further indicating that the biological processes involved in arterial and venous thrombosis are different.
Assuntos
Suplementos Nutricionais , Trombose Venosa/prevenção & controle , Ácido alfa-Linolênico/administração & dosagem , Animais , Biomarcadores/sangue , Contagem de Células Sanguíneas , Coagulação Sanguínea , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Selectina-P/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/metabolismo , Fatores de Tempo , Trombose Venosa/sangue , Trombose Venosa/etiologiaRESUMO
Effects of Omega-3 fatty acids (n-3 FA) in particular on the development of cardiovascular disease (CVD) are of major interest. Many experimental studies reported their anti-inflammatory, anti-thrombotic and anti-atherosclerotic properties and suggested favourable effects on the prevention of CVD. While the majority of former studies showed a benefit of n-3 FA acid intake, recent clinical trials using n-3 supplements on top of established medication and prudent nutrition did not confirm these findings. The conflicting data may be due to several factors such as the selection of study population with different sizes or characteristics as well as choosing different doses or types of n-3 FA. The most recent meta-analyses observed clear benefits of fish consumption, but not of n-3 capsules intake. Furthermore, a nutrition rich in plant-derived n-3 FA alpha-linolenic acid has been found to have beneficial effects on the development of cardio- and cerebrovascular diseases.
Le rôle des acides gras oméga-3 dans la prévention de maladies cardiovasculaires a fait l'objet de recherches intenses. Ainsi plusieurs études expérimentales ont démontré leurs propriétés anti-inflammatoires et anti-thrombotiques. Cependant les récentes études épidémiologiques à grande échelle ont délivré des résultats ambivalents en ce qui concerne l'efficacité des acides gras oméga-3. En particulier, le choix de différentes sources d'acides gras, les différents dosages ainsi que les différences entre les populations étudiées semblent être à l'origine des divergences dans les résultats obtenus.
Assuntos
Anti-Inflamatórios/administração & dosagem , Ácidos Graxos Ômega-3/administração & dosagem , Anti-Inflamatórios/efeitos adversos , Doenças Cardiovasculares/prevenção & controle , Ensaios Clínicos como Assunto , Ácidos Graxos Ômega-3/efeitos adversos , Humanos , Neoplasias/prevenção & controle , Guias de Prática Clínica como Assunto , Insuficiência Renal/prevenção & controle , Acidente Vascular Cerebral/prevenção & controleRESUMO
Previously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbß3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α-converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.