RESUMEN
AIMS/HYPOTHESIS: People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium-glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. METHODS: Male, 8-week-old LDL-receptor-deficient (Ldlr-/-) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. RESULTS: Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet-leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr-/- mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10-9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10-9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. CONCLUSIONS/INTERPRETATION: We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin-platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin's beneficial cardiovascular risk profile.
Asunto(s)
Compuestos de Bencidrilo/uso terapéutico , Enfermedad de la Arteria Coronaria/prevención & control , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucósidos/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Trombina/metabolismo , Adulto , Animales , Glucemia/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , HDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Citometría de Flujo , Voluntarios Sanos , Humanos , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Selectina-P/metabolismo , Recuento de Plaquetas , Reacción en Cadena en Tiempo Real de la Polimerasa , Conducta de Reducción del RiesgoRESUMEN
RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.
Asunto(s)
Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Receptor beta de Linfotoxina/deficiencia , Animales , Antígenos Ly/metabolismo , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/diagnóstico , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Trasplante de Médula Ósea , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Receptor beta de Linfotoxina/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Placa Aterosclerótica , Factores de Tiempo , Transcripción Genética , Quimera por TrasplanteRESUMEN
OBJECTIVE: Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non-vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. APPROACH AND RESULTS: Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E-deficient (ApoE(-/-)) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE(-/-) mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient (ApoE(-/-)/Bgn(-/0)) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE(-/-)/Bgn(-/0) mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE(-/-)/Bgn(-/0) mice with the thrombin inhibitor argatroban. Ultimately, ApoE(-/-)/Bgn(-/0) mice developed aggravated atherosclerosis. CONCLUSIONS: The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Biglicano/deficiencia , Inflamación/metabolismo , Trombina/metabolismo , Síndrome Coronario Agudo/sangre , Animales , Antitrombinas/farmacología , Aorta/efectos de los fármacos , Aorta/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/genética , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/prevención & control , Biglicano/sangre , Biglicano/genética , Citocinas/sangre , Modelos Animales de Enfermedad , Genotipo , Cofactor II de Heparina/metabolismo , Humanos , Inflamación/sangre , Inflamación/genética , Inflamación/prevención & control , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Activación Plaquetaria , Factores de TiempoRESUMEN
Interleukin-6 (IL-6) is a multifunctional cytokine that orchestrates the immune response to a wide variety of pathophysiologic challenges but also contributes to tissue homeostasis. Furthermore, IL-6 is elevated in patients with acute myocardial infarction. Hyaluronan (HA) is an extracellular carbohydrate that has been implicated in wound healing and accumulates after acute myocardial infarction (AMI). Aim of this study was to investigate the involvement of IL-6 in the regulation of the HA-matrix in the early phase of infarct healing. In the present study, we show by the use of a blocking anti-IL-6 antibody, that endogenous IL-6 rapidly but transiently increased HA-synthase (HAS) 1 and 2 expression resulting in the formation of a HA-rich matrix acutely after AMI in mice. In vitro, IL-6 induced HAS1 and 2 via STAT3 phosphorylation in cardiac fibroblasts (CF) and supported a myofibroblastic phenotype in a HA-dependent manner. Furthermore, CCL5 and MCP1 expression were dependent on IL-6, HA-synthesis and the HA-receptor CD44 as shown in cultured CF derived from CD44 knockout mice. In vivo after AMI, blocking IL-6 decreased HA-matrix formation in the peri-infarct region and alpha-smooth muscle actin-positive myofibroblasts. Blocking IL-6 also reduced neutrophil infiltration in infarcted left ventricles. Moreover, treatment with the blocking IL-6 antibody reduced cardiac ejection fraction and increased infarct size 3 weeks after AMI. These findings support a functionally important role for IL-6 in CF by transiently inducing a HA-rich matrix that in turn promotes a myofibroblastic phenotype and inflammatory responses, and ultimately establishes a cardioprotective program after AMI.
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Fibroblastos/fisiología , Ácido Hialurónico/fisiología , Interleucina-6/fisiología , Infarto del Miocardio , Animales , Matriz Extracelular/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Miofibroblastos/fisiología , FenotipoRESUMEN
BACKGROUND AND AIMS: The non-vitamin K oral anticoagulant dabigatran etexilate (dabigatran) is increasingly prescribed to patients with non-valvular atrial fibrillation and venous thromboembolism. Adipose tissue (AT) inflammation during obesity plays a crucial role in the development of insulin resistance, type II diabetes and atherogenesis. The aim of the present study was to investigate the effects of thrombin inhibition by dabigatran in a combined model of diet-induced obesity and atherosclerosis. METHODS: Female Low density lipoprotein receptor knockout (Lldr-/-) mice were fed a high-fat diet containing 5â¯mg/g dabigatran or matching control for 20 weeks. RESULTS: Dabigatran-treated animals showed increased adipocyte hypertrophy, but reduced numbers of pro-inflammatory M1-polarized macrophages in the adipose tissue. Abundance of pro-inflammatory M1 macrophages was also decreased in the aortic wall of dabigatran-fed mice. Multiple circulating cytokines were reduced, indicating an effect in systemically relevant secretory compartments such as the AT. CONCLUSIONS: Dabigatran treatment reduces pro-inflammatory M1 macrophages in atherosclerotic lesions, thereby contributing to plaque stabilizing and atheroprotective effects of the thrombin inhibitor. This finding is not restricted to the vascular wall but is also present in AT where dabigatran treatment reduced the release of pro-inflammatory cytokines and accumulation of M1 macrophages.
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Tejido Adiposo/patología , Aorta Torácica/patología , Aterosclerosis/tratamiento farmacológico , Dabigatrán/farmacología , Inflamación/tratamiento farmacológico , Activación de Macrófagos/fisiología , Macrófagos/patología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Antitrombinas/farmacología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.
Asunto(s)
Tejido Adiposo Pardo/efectos de los fármacos , Himecromona/farmacología , Termogénesis/efectos de los fármacos , Animales , Metabolismo Energético , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied. APPROACH AND RESULTS: It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 ß (IL-1ß) in an NFkB dependent manner and blocking antibodies to IL-1ß abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation. CONCLUSIONS: Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1ß, our results suggest that therapeutic anti-IL-1ß antibodies, recently tested in human clinical trials (CANTOS), may exert their beneficial effects on inflammation in post-myocardial infarction patients in part via effects on HAS3. TOC categorybasic study TOC subcategoryarteriosclerosis.
Asunto(s)
Apolipoproteínas E/deficiencia , Hialuronano Sintasas/metabolismo , Interleucina-1beta/metabolismo , Músculo Liso Vascular/citología , Placa Aterosclerótica/metabolismo , Animales , Polaridad Celular , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ácido Hialurónico/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , FN-kappa B/farmacocinética , Placa Aterosclerótica/genética , Placa Aterosclerótica/inmunología , Células TH1/citología , Células TH1/metabolismoRESUMEN
1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) inhibits the expression of an immediate-early gene, IEX-1, which is involved in the regulation of cellular growth and apoptosis in a variety of cells. 1alpha,25(OH)2D3 alters the subcellular localization of IEX-1 by causing an efflux of IEX-1 from the nucleus, and the sterol decreases the expression of IEX-1 messenger RNA in cells via a novel DR3 repeat-type DNA response element.