RESUMEN
Background Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. The kinin B2 receptor agonist, bradykinin, has been implicated in AAA pathogenesis through promoting inflammation. Bradykinin is generated from high- and low-molecular-weight kininogen by the serine protease kallikrein-1. The aims of this study were first to examine the effect of neutralizing kallikrein-1 on AAA development in a mouse model and second to test how blocking kallikrein-1 affected cyclooxygenase-2 and prostaglandin E2 in human AAA explants. Methods and Results Neutralization of kallikrein-1 in apolipoprotein E-deficient (ApoE-/-) mice via administration of a blocking antibody inhibited suprarenal aorta expansion in response to angiotensin (Ang) II infusion. Kallikrein-1 neutralization decreased suprarenal aorta concentrations of bradykinin and prostaglandin E2 and reduced cyclooxygenase-2 activity. Kallikrein-1 neutralization also decreased protein kinase B and extracellular signal-regulated kinase 1/2 phosphorylation and reduced levels of active matrix metalloproteinase 2 and matrix metalloproteinase 9. Kallikrein-1 blocking antibody reduced levels of cyclooxygenase-2 and secretion of prostaglandin E2 and active matrix metalloproteinase 2 and matrix metalloproteinase 9 from human AAA explants and vascular smooth muscle cells exposed to activated neutrophils. Conclusions These findings suggest that kallikrein-1 neutralization could be a treatment target for AAA.
Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/terapia , Dinoprostona/metabolismo , Músculo Liso Vascular/patología , Calicreínas de Tejido/antagonistas & inhibidores , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Biopsia , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismoRESUMEN
Abdominal aortic aneurysm (AAA) rupture is an important cause of death in older adults. In clinical practice, the most established predictor of AAA rupture is maximum AAA diameter. Aortic diameter is commonly used to assess AAA severity in mouse models studies. AAA rupture occurs when the stress (force per unit area) on the aneurysm wall exceeds wall strength. Previous research suggests that aortic wall structure and strength, biomechanical forces on the aorta and cellular and proteolytic composition of the AAA wall influence the risk of AAA rupture. Mouse models offer an opportunity to study the association of these factors with AAA rupture in a way not currently possible in patients. Such studies could provide data to support the use of novel surrogate markers of AAA rupture in patients. In this review, the currently available mouse models of AAA and their relevance to the study of AAA rupture are discussed. The review highlights the limitations of mouse models and suggests novel approaches that could be incorporated in future experimental AAA studies to generate clinically relevant results.
Asunto(s)
Aorta Abdominal/fisiopatología , Aneurisma de la Aorta Abdominal/fisiopatología , Rotura de la Aorta/fisiopatología , Modelos Cardiovasculares , Animales , Aneurisma de la Aorta Abdominal/prevención & control , Fenómenos Biomecánicos , Simulación por Computador , Modelos Animales de Enfermedad , Humanos , Ratones , Factores de Riesgo , Estrés MecánicoRESUMEN
Vitamin D deficiency has been associated with human abdominal aortic aneurysm (AAA); however, its role in AAA pathogenesis is unclear. The aim of the present study was to investigate the effect of vitamin D deficiency on AAA development and examine if administering cholecalciferol (CCF) could limit growth of established AAA within the angiotensin-II (AngII) infused apolipoprotein E-deficient mouse model. Mice were rendered vitamin D deficiency through dietary restriction and during AngII infusion developed larger AAAs as assessed by ultrasound and ex vivo morphometry that ruptured more commonly (48% vs. 19%; P=0.028) than controls. Vitamin D deficiency was associated with increased aortic expression of osteopontin and matrix metalloproteinase-2 and -9 than controls. CCF administration to mice with established aortic aneurysms limited AAA growth as assessed by ultrasound (P<0.001) and ex vivo morphometry (P=0.036) and reduced rupture rate (8% vs. 46%; P=0.031). This effect was associated with up-regulation of circulating and aortic sclerostin. Incubation of human aortic smooth muscle cells with 1,25-dihyroxyvitamin D3 (the active metabolite of vitamin D) for 48 h induced up-regulation of sclerostin (P<0.001) and changed the expression of a range of other genes important in extracellular matrix remodeling. The present study suggests that vitamin D deficiency promotes development of large rupture-prone aortic aneurysms in an experimental model. CCF administration limited both growth and rupture of established aneurysms. These effects of vitamin D appeared to be mediated via changes in genes involved in extracellular matrix remodeling, particularly sclerostin.
Asunto(s)
Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Aneurisma de la Aorta Abdominal/etiología , Rotura de la Aorta/tratamiento farmacológico , Rotura de la Aorta/etiología , Colecalciferol/uso terapéutico , Suplementos Dietéticos , Progresión de la Enfermedad , Deficiencia de Vitamina D/complicaciones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiotensina II , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Abdominal/fisiopatología , Aneurisma de la Aorta Abdominal/fisiopatología , Rotura de la Aorta/fisiopatología , Apolipoproteínas E/deficiencia , Presión Sanguínea/efectos de los fármacos , Restricción Calórica , Colecalciferol/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
OBJECTIVE: Experimental studies suggest that fenofibrate prevents abdominal aortic aneurysm (AAA) development by lowering aortic osteopontin (OPN) concentration and reducing the number of macrophages infiltrating the aortic wall. The current study examined the effects of a short course of fenofibrate on AAA pathology in people with large AAAs awaiting aortic repair. METHODS: This randomised double blind parallel trial included male and female participants aged ≥ 60 years who had an asymptomatic AAA measuring ≥ 50 mm and were scheduled to undergo open AAA repair. Participants were allocated to fenofibrate (145 mg/day) or matching placebo for at least two weeks before elective AAA repair. Blood samples were collected at recruitment and immediately prior to surgery. AAA biopsies were obtained during aortic surgery. The primary outcomes were (1) AAA OPN concentration; (2) serum OPN concentration; and (3) number of AAA macrophages. Exploratory outcomes included circulating and aortic concentrations of other proteins previously associated with AAA. Outcomes assessed at a single time point were compared using logistic regression. Longitudinal outcomes were compared using linear mixed effects models. RESULTS: Forty-three participants were randomised. After three withdrawals, 40 were followed until the time of surgery (21 allocated fenofibrate and 19 allocated placebo). As expected, serum triglycerides reduced significantly from recruitment to the time of surgery in participants allocated fenofibrate. No differences in any of the primary and exploratory outcomes were observed between groups. CONCLUSION: A short course of 145 mg of fenofibrate/day did not lower concentrations of OPN or aortic macrophage density in people with large AAAs.
Asunto(s)
Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/cirugía , Aneurisma de la Aorta Abdominal/terapia , Fenofibrato/administración & dosificación , Procedimientos Quirúrgicos Vasculares , Anciano , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/patología , Biomarcadores/sangre , Progresión de la Enfermedad , Método Doble Ciego , Esquema de Medicación , Femenino , Fenofibrato/efectos adversos , Humanos , Macrófagos/patología , Masculino , Persona de Mediana Edad , Osteopontina/sangre , Queensland , Factores de Tiempo , Resultado del Tratamiento , Triglicéridos/sangre , Remodelación Vascular/efectos de los fármacos , Procedimientos Quirúrgicos Vasculares/efectos adversosRESUMEN
Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Chronic inflammation and excessive matrix remodelling are considered important in AAA pathogenesis. Kinins are bioactive peptides important in regulating inflammation. Stimulation of the kinin B2 receptor has been previously reported to promote AAA development and rupture in a mouse model. The endogenous B2 receptor agonist, bradykinin, is generated from the kallikrein-kinin system following activation of plasma kallikrein by Factor XII (FXII). In the current study whole-body FXII deletion, or neutralisation of activated FXII (FXIIa), inhibited expansion of the suprarenal aorta (SRA) of apolipoprotein E-deficient mice in response to angiotensin II (AngII) infusion. FXII deficiency or FXIIa neutralisation led to decreased aortic tumor necrosis factor-α-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-α-converting enzyme) (ADAM-17)) activity, plasma kallikrein concentration, and epithelial growth factor receptor (EGFR) phosphorylation compared with controls. FXII deficiency or neutralisation also reduced Akt1 and Erk1/2 phosphorylation and decreased expression and levels of active matrix metalloproteinase (Mmp)-2 and Mmp-9. The findings suggest that FXII, kallikrein, ADAM-17, and EGFR are important molecular mediators by which AngII induces aneurysm in apolipoprotein E-deficient mice. This could be a novel pathway to target in the design of drugs to limit AAA progression.
Asunto(s)
Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Factor XII/antagonistas & inhibidores , Proteína ADAM17/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta Abdominal/metabolismo , Modelos Animales de Enfermedad , Factor XII/metabolismo , RatonesRESUMEN
Peripheral arterial disease (PAD) develops due to the narrowing or blockage of arteries supplying blood to the lower limbs. Surgical and endovascular interventions are the main treatments for advanced PAD but alternative and adjunctive medical therapies are needed. Currently the main preclinical experimental model employed in PAD research is based on induction of acute hind limb ischemia (HLI) by a 1-stage procedure. Since there are concerns regarding the ability to translate findings from this animal model to patients, we aimed to develop a novel clinically relevant animal model of PAD. HLI was induced in male Apolipoprotein E (ApoE-/-) deficient mice by a 2-stage procedure of initial gradual femoral artery occlusion by ameroid constrictors for 14 days and subsequent excision of the femoral artery. This 2-stage HLI model was compared to the classical 1-stage HLI model and sham controls. Ischemia severity was assessed using Laser Doppler Perfusion Imaging (LDPI). Ambulatory ability was assessed using an open field test, a treadmill test and using established scoring scales. Molecular markers of angiogenesis and shear stress were assessed within gastrocnemius muscle tissue samples using quantitative polymerase chain reaction. HLI was more severe in mice receiving the 2-stage compared to the 1-stage ischemia induction procedure as assessed by LDPI (p = 0.014), and reflected in a higher ischemic score (p = 0.004) and lower average distance travelled on a treadmill test (p = 0.045). Mice undergoing the 2-stage HLI also had lower expression of angiogenesis markers (vascular endothelial growth factor, p = 0.004; vascular endothelial growth factor- receptor 2, p = 0.008) and shear stress response mechano-transducer transient receptor potential vanilloid 4 (p = 0.041) within gastrocnemius muscle samples, compared to animals having the 1-stage HLI procedure. Mice subjected to the 2-stage HLI receiving an exercise program showed significantly greater improvement in their ambulatory ability on a treadmill test than a sedentary control group. This study describes a novel model of HLI which leads to more severe and sustained ischemia than the conventionally used model. Exercise therapy, which has established efficacy in PAD patients, was also effective in this new model. This new model maybe useful in the evaluation of potential novel PAD therapies.
Asunto(s)
Miembro Posterior/fisiopatología , Isquemia/patología , Enfermedad Arterial Periférica/patología , Animales , Modelos Animales de Enfermedad , Arteria Femoral/cirugía , Fibrosis , Humanos , Isquemia/diagnóstico por imagen , Isquemia/metabolismo , Masculino , Ratones , Ratones Noqueados para ApoE , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Imagen de Perfusión , Enfermedad Arterial Periférica/metabolismo , Condicionamiento Físico Animal , Índice de Severidad de la Enfermedad , Resistencia al Corte , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Background There is no drug therapy for abdominal aortic aneurysm ( AAA ). FAME-2 (Fenofibrate in the Management of Abdominal Aortic Aneurysm 2) was a placebo-controlled randomized trial designed to assess whether administration of 145 mg of fenofibrate/d for 24 weeks favorably modified circulating markers of AAA. Methods and Results Patients with AAA s measuring 35 to 49 mm and no contraindication were randomized to fenofibrate or identical placebo. The primary outcome measures were the differences in serum osteopontin and kallistatin concentrations between groups. Secondary analyses compared changes in the circulating concentration of AAA -associated proteins, and AAA growth, between groups using multivariable linear mixed-effects modeling. A total of 140 patients were randomized to receive fenofibrate (n=70) or placebo (n=70). By the end of the study 3 (2.1%) patients were lost to follow-up and 18 (12.9%) patients had ceased trial medication. A total of 85% of randomized patients took ≥80% of allocated tablets and were deemed to have complied with the medication regimen. Patients' allocated fenofibrate had expected reductions in serum triglycerides and estimated glomerular filtration rate, and increases in serum homocysteine. No differences in serum osteopontin, kallistatin, or AAA growth were observed between groups. Conclusions Administering 145 mg/d of fenofibrate for 24 weeks did not significantly reduce serum concentrations of osteopontin and kallistatin concentrations, or rates of AAA growth in this trial. The findings do not support the likely benefit of fenofibrate as a treatment for patients with small AAA s. Clinical Trial Registration URL : www.anzctr.org.au . Unique identifier: ACTRN 12613001039774.
Asunto(s)
Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Fenofibrato/administración & dosificación , Osteopontina/sangre , Serpinas/sangre , Anciano , Anciano de 80 o más Años , Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/diagnóstico , Biomarcadores/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Hipolipemiantes/administración & dosificación , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del Tratamiento , UltrasonografíaRESUMEN
BACKGROUND AND AIMS: Diets enriched with tree nuts have been demonstrated to reduce the risk of atherosclerosis-related cardiovascular events. Abdominal aortic aneurysm (AAA) shares common risk factors with atherosclerosis and AAA patients commonly have atherosclerosis related cardiovascular events. AAA has some distinct pathological and clinical characteristics to those of atherosclerosis. No previous study has examined the effect of a diet enriched with tree nuts on experimental or clinical AAA. This study investigated the effect of a diet enriched with tree nuts on the development and severity of AAA within an experimental rodent model. METHODS: Male apolipoprotein E deficient mice were allocated to a diet enriched with tree nuts or control diet for 56 days (nâ¯=â¯17 per group). After 28 days, all mice were infused with angiotensin II whilst being maintained on their respective diets. The primary outcome was AAA severity assessed by the supra-renal aortic diameter, measured by ultrasound and ex vivo morphometric analysis. The severity of atherosclerosis was assessed by computer-aided analysis of Sudan IV stained aortic arches and sections of brachiocephalic arteries prepared with Van Gieson's stain. RESULTS: The diet enriched with tree nuts did not influence aortic diameter or aortic rupture incidence. Mice receiving the diet enriched with tree nuts had significantly less atherosclerosis within the brachiocephalic artery (pâ¯=â¯0.033) but not in the aortic arch. CONCLUSIONS: This experimental study suggests that a diet enriched with tree nuts does not reduce the severity of AAA, but does reduce the severity of atherosclerosis within the brachiocephalic artery. The study was not powered to identify a moderate effect of the diet on the primary outcome and therefore this cannot be excluded.
Asunto(s)
Angiotensina II , Alimentación Animal , Aneurisma de la Aorta Abdominal/prevención & control , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Ácidos Grasos Omega-3/administración & dosificación , Nueces , Polifenoles/administración & dosificación , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patología , Dilatación Patológica , Modelos Animales de Enfermedad , Masculino , Ratones Noqueados para ApoE , Valor Nutritivo , Placa Aterosclerótica , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
BACKGROUND: An abdominal aortic aneurysm (AAA) is a focal dilation of the abdominal aorta and is associated with a risk of fatal rupture. Experimental studies suggest that myo-inositol may exert beneficial effects on AAAs through favourable changes to biological pathways implicated in AAA pathology. The aim of the Inositol in the MAnaGemENt of abdominal aortic aneurysm (IMAGEN) trial is to assess if myo-inositol will reduce AAA growth. METHODS/DESIGN: IMAGEN is a multi-centre, prospective, parallel-group, randomised, double-blind, placebo-controlled trial. A total of 164 participants with an AAA measuring ≥ 30 mm will be randomised to either 2 g of myo-inositol or identical placebo twice daily for 12 months. The primary outcome measure will be AAA growth estimated by increase in total infrarenal aortic volume measured on computed tomographic scans. Secondary outcome measures will include AAA diameter assessed by computed tomography and ultrasound, AAA peak wall stress and peak wall rupture index, serum lipids, circulating AAA biomarkers, circulating RNAs and health-related quality of life. All analysis will be based on the intention-to-treat principle at the time of randomisation. All patients who meet the eligibility criteria, provide written informed consent and are enrolled in the study will be included in the primary analysis, regardless of adherence to dietary allocation. DISCUSSION: Currently, there is no known medical therapy to limit AAA progression. The IMAGEN trial will be the first randomised trial, to our knowledge, to assess the value of myo-inositol in limiting AAA growth. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12615001209583 . Registered on 6 November 2015.
Asunto(s)
Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Protocolos Clínicos , Inositol/uso terapéutico , Método Doble Ciego , Humanos , Evaluación de Resultado en la Atención de Salud , Estudios ProspectivosRESUMEN
OBJECTIVE: Recent evidence suggests an important role for angiotensin-converting enzyme 2 (ACE2) in limiting abdominal aortic aneurysm (AAA). This study examined the effect of ACE2 deficiency on AAA development and the efficacy of resveratrol to upregulate ACE2 in experimental AAA. APPROACH AND RESULTS: Ace2 deletion in apolipoprotein-deficient mice (ApoE-/-Ace2-/y ) resulted in increased aortic diameter and spontaneous aneurysm of the suprarenal aorta associated with increased expression of inflammation and proteolytic enzyme markers. In humans, serum ACE2 activity was negatively associated with AAA diagnosis. ACE2 expression was lower in infrarenal biopsies of patients with AAA than organ donors. AAA was more severe in ApoE-/-Ace2-/y mice compared with controls in 2 experimental models. Resveratrol (0.05/100-g chow) inhibited growth of pre-established AAAs in ApoE-/- mice fed high-fat chow and infused with angiotensin II continuously for 56 days. Reduced suprarenal aorta dilatation in mice receiving resveratrol was associated with elevated serum ACE2 and increased suprarenal aorta tissue levels of ACE2 and sirtuin 1 activity. In addition, the relative phosphorylation of Akt and ERK (extracellular signal-regulated kinase) 1/2 within suprarenal aorta tissue and gene expression for nuclear factor of kappa light polypeptide gene enhancer in B cells 1, angiotensin type-1 receptor, and metallopeptidase 2 and 9 were significantly reduced. Upregulation of ACE2 in human aortic smooth muscle cells by resveratrol in vitro was sirtuin 1-dependent. CONCLUSIONS: This study provides experimental evidence of an important role for ACE2 in limiting AAA development and growth. Resveratrol upregulated ACE2 and inhibited AAA growth in a mouse model.
Asunto(s)
Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Rotura de la Aorta/prevención & control , Peptidil-Dipeptidasa A/deficiencia , Estilbenos/farmacología , Angiotensina II , Enzima Convertidora de Angiotensina 2 , Animales , Aorta Abdominal/enzimología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Rotura de la Aorta/enzimología , Rotura de la Aorta/genética , Rotura de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Células Cultivadas , Dieta Alta en Grasa , Dilatación Patológica , Modelos Animales de Enfermedad , Inducción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Subunidad p50 de NF-kappa B/metabolismo , Peptidil-Dipeptidasa A/biosíntesis , Peptidil-Dipeptidasa A/genética , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Resveratrol , Sirtuina 1/metabolismo , Factores de TiempoRESUMEN
Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE-/- mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm.
Asunto(s)
Antitrombinas/farmacología , Aneurisma de la Aorta/prevención & control , Aterosclerosis/prevención & control , Receptor PAR-2/metabolismo , Transducción de Señal , Animales , Antitrombinas/administración & dosificación , Antitrombinas/uso terapéutico , Aneurisma de la Aorta/tratamiento farmacológico , Aneurisma de la Aorta/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Inhibidores del Factor Xa/administración & dosificación , Inhibidores del Factor Xa/farmacología , Inhibidores del Factor Xa/uso terapéutico , Regulación de la Expresión Génica , Infusiones Parenterales , Masculino , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Noqueados para ApoE , Receptor PAR-2/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Trombina/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. APPROACH AND RESULTS: SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E-deficient (ApoE-/-) mice (SOSTTg .ApoE-/-) and administration of recombinant mouse Sost inhibited angiotensin II-induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg .ApoE-/- mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg .ApoE-/- mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/ß-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg .ApoE-/- mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/ß-catenin pathway was activated in human AA samples. The cytosine-phosphate-guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. CONCLUSIONS: This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta/prevención & control , Aterosclerosis/prevención & control , Proteínas Morfogenéticas Óseas/metabolismo , Glicoproteínas/administración & dosificación , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Anciano , Anciano de 80 o más Años , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aneurisma de la Aorta/inducido químicamente , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/metabolismo , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Citocinas/metabolismo , Epigénesis Genética/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intercelular , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fenotipo , Remodelación Vascular/efectos de los fármacosRESUMEN
BACKGROUND: Arteriovenous fistula (AVF) failure is a significant cause of morbidity and expense in patients on maintenance haemodialysis (HD). Circulating biomarkers could be valuable in detecting patients at risk of AVF failure and may identify targets to improve AVF outcome. Currently there is little consensus on the relationship between circulating biomarkers and AVF failure. The aim of this systematic review was to identify circulating biomarkers associated with AVF failure. METHODS: Studies evaluating the association between circulating biomarkers and the presence or risk of AVF failure were systematically identified from the MEDLINE, EMBASE and Cochrane Library databases. No restrictions on the type of study were imposed. Concentrations of circulating biomarkers of routine HD patients with and without AVF failure were recorded and meta-analyses were performed on biomarkers that were assessed in three or more studies with a composite population of at least 100 participants. Biomarker concentrations were synthesized into inverse-variance random-effects models to calculate standardized mean differences (SMD) and 95% confidence intervals (CI). RESULTS: Thirteen studies comprising a combined population of 1512 participants were included after screening 2835 unique abstracts. These studies collectively investigated 48 biomarkers, predominantly circulating molecules which were assessed as part of routine clinical care. Meta-analysis was performed on twelve eligible biomarkers. No significant association between any of the assessed biomarkers and AVF failure was observed. CONCLUSION: This paper is the first systematic review of biomarkers associated with AVF failure. Our results suggest that blood markers currently assessed do not identify an at-risk AVF. Further, rigorously designed studies assessing biological plausible biomarkers are needed to clarify whether assessment of circulating markers can be of any clinical value. PROSPERO registration number CRD42016033845.
Asunto(s)
Derivación Arteriovenosa Quirúrgica/efectos adversos , Complicaciones Posoperatorias/sangre , Diálisis Renal/efectos adversos , Biomarcadores/sangre , Estudios de Casos y Controles , Humanos , Diálisis Renal/métodosRESUMEN
During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.
Asunto(s)
Conexinas/deficiencia , Endotelio Vascular/metabolismo , Hipertensión/etiología , Hipertensión/fisiopatología , Condicionamiento Físico Animal/efectos adversos , Animales , Presión Sanguínea/fisiología , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Uniones Comunicantes/fisiología , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Vasodilatación/fisiología , Proteína alfa-5 de Unión ComunicanteRESUMEN
Mutant forms of connexin40 (Cx40) exist in the human population and predispose carriers to atrial fibrillation. Since endothelial expression of Cx40 is important for electrical and chemical communication within the arterial wall, carriers of mutant Cx40 proteins may be predisposed to peripheral arterial dysfunction and dysregulation of blood pressure. We have therefore studied mice expressing either a chemically dysfunctional mutant, Cx40T202S, or wild-type Cx40, with native Cx40, specifically in the endothelium. Blood pressure was measured by telemetry under normal conditions and during cardiovascular stress induced by locomotor activity, phenylephrine or nitric oxide blockade (N(É·)-nitro-L-arginine methyl ester hydroxide, L-NAME). Blood pressure of Cx40T202STg mice was significantly elevated at night when compared with wild-type or Cx40Tg mice, without change in mean heart rate, pulse pressure or locomotor activity. Analysis over 24 h showed that blood pressure of Cx40T202STg mice was significantly elevated at rest and additionally during locomotor activity. In contrast, neither plasma renin concentration nor pressor responses to phenylephrine or L-NAME were altered, the latter indicating that nitric oxide bioavailability was normal. In isolated, pressurised mesenteric arteries, hyperpolarisation and vasodilation evoked by SKA-31, the selective modulator of SKCa and IKCa channels, was significantly reduced in Cx40T202STg mice, due to attenuation of the SKCa component. Acetylcholine-induced ascending vasodilation in vivo was also significantly attenuated in cremaster muscle arterioles of Cx40T202STg mice, compared to wild-type and Cx40Tg mice. We conclude that endothelial expression of the chemically dysfunctional Cx40T202S reduces peripheral vasodilator capacity mediated by SKCa-dependent hyperpolarisation and also increases blood pressure.
Asunto(s)
Conexinas/metabolismo , Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Canales de Potasio Calcio-Activados/metabolismo , Vasodilatación/fisiología , Animales , Presión Sanguínea , Conexinas/genética , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Proteína alfa-5 de Unión ComunicanteRESUMEN
Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.
Asunto(s)
Células Endoteliales/metabolismo , Expresión Génica , Represoras Lac/fisiología , Animales , Conexinas/biosíntesis , Conexinas/genética , Endotelio Vascular/citología , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Homocigoto , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas , Receptor TIE-2/genética , Activación Transcripcional , Transgenes , Proteína alfa-5 de Unión ComunicanteRESUMEN
The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by â¼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.