RESUMEN
Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated ß-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Senescencia Celular , Células Endoteliales/patología , Endotelio Vascular/fisiopatología , Inflamación/patología , Neutrófilos/metabolismo , Daño por Reperfusión/fisiopatología , Angiotensinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Coagulación Sanguínea/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Micropartículas Derivadas de Células/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Neutrófilos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas Wistar , Bazo/efectos de los fármacos , Bazo/patología , Porcinos , Acetato de Tetradecanoilforbol/farmacología , Tromboplastina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment-most likely via a cyclooxygenase-dependent mechanism.
Asunto(s)
Envejecimiento/patología , Ácidos Docosahexaenoicos/administración & dosificación , Ácido Eicosapentaenoico/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Arteria Femoral/efectos de los fármacos , Vena Femoral/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/química , Enfermedades Vasculares/tratamiento farmacológico , Administración Oral , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Arteria Femoral/metabolismo , Arteria Femoral/patología , Vena Femoral/metabolismo , Vena Femoral/patología , Masculino , Ratas , Ratas Wistar , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2 O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.
Asunto(s)
Angiotensina II/farmacología , Senescencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Glucosa/metabolismo , Oxidación-Reducción/efectos de los fármacos , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Envejecimiento/efectos de los fármacos , Envejecimiento/metabolismo , Animales , Compuestos de Bencidrilo/farmacología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Glucósidos/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , PorcinosRESUMEN
BACKGROUND In organ transplantation, particularly pancreas transplantation, donor age is a determinant factor for graft survival. Physiological aging is crucial in the progressive deterioration of organs in adulthood. We compared the senescence and function features of pancreas and vascular tissues in young rats and middle-aged rats. MATERIAL AND METHODS Islet morphology and the area of cells secreting insulin or glucagon was investigated using immunohistology in young rats (12 weeks) and middle-aged rats (52 weeks) (n=8). Senescence markers, oxidative stress (ROS), and tissue factor (TF) were measured in the rat pancreases. Circulating microparticles (MPs) were measured as surrogates of vascular cell injury. Vascular function was studied in mesenteric arterial rings. RESULTS Larger islets were twice as frequent in young rats versus middle-aged rats. In middle-aged rats there was a significant decrease of the ß-cells/islet area ratio. Western blot analysis showed an increased expression of p53, p21, and p16 senescence markers (2-, 7- and 3-fold respectively) with no modification in caspase-3 activation. A 30% decrease of endothelial nitric oxide synthase (eNOS) was observed together with a 4-fold increase in TF expression. ROS formation increased significantly (2-fold) in middle-aged rats and their main source, determined by pharmacological inhibition, was NADPH oxidase and uncoupled nitric-oxide (NO) synthase. No sign of vascular injury (microparticles) or dysfunction was evidenced. CONCLUSIONS Modification in islet morphology and function were detected in middle-aged rats before any measurement of macro-vascular dysfunction. The data indicate a pancreatic senescence in the process of aging associated with uncontrolled accumulation of oxidative species that suggests a determining role of donor age in transplantation.
Asunto(s)
Envejecimiento/fisiología , Endotelio Vascular/fisiología , Estrés Oxidativo/fisiología , Páncreas/metabolismo , Animales , Caspasa 3/metabolismo , Glucagón/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Endothelial senescence has been suggested to promote endothelial dysfunction in age-related vascular disorders. This study evaluated the prothrombotic properties of senescent endothelial cells (ECs) and the underlying mechanism. Serial passaging from passage (P)1 to P4 (replicative senescence) of porcine coronary artery ECs, or treatment of P1 ECs with the endothelial nitric oxide synthase (eNOS) inhibitor L-NAME (premature senescence) induced acquisition of markers of senescence including increased senescence-associated-ß-galactosidase (SA-ß-gal) activity and p53, p21, p16 expression. Approximately 55% of P3 cells were senescent with a high level oxidative stress, and decreased eNOS-derived nitric oxide (NO) formation associated with increased expression of NADPH oxidase subunits (gp91phox, p47phox), cyclooxygenase (COX)-2 but not COX-1, and a decreased eNOS expression leading to a reduced ability of ECs to inhibit platelet aggregation. P3 cells also presented increased expression and activity of tissue factor (TF), a key initiator of the coagulation cascade. Treatment of senesecent cells with a NADPH oxidase inhibitor (VAS-2870) or by a COX inhibitor (indomethacin) reduced oxidative stress, decreased TF activity and expression, and reduced the expression of gp91phox, p47phox and COX-2 and restored the ability of ECs to inhibit effectively platelet aggregation. Thus, replicative endothelial senescence promotes a prothrombotic response involving the down-regulation of the protective NO pathway and the upregulation of the NADPH oxidase- and COXs-dependent oxidative stress pathway promoting TF expression and activity.
Asunto(s)
Senescencia Celular/fisiología , Endotelio Vascular/citología , NADPH Oxidasas/fisiología , Estrés Oxidativo/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , División Celular/fisiología , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Humanos , Óxido Nítrico/biosíntesis , Agregación Plaquetaria/fisiología , Sus scrofa , Tromboplastina/metabolismo , Trombosis/enzimología , Trombosis/patología , Regulación hacia Arriba/fisiologíaRESUMEN
Endothelial senescence, characterized by an irreversible cell cycle arrest, oxidative stress, and downregulation of endothelial nitric oxide synthase (eNOS), has been shown to promote endothelial dysfunction leading to the development of age-related vascular disorders. This study has assessed the possibility that the local angiotensin system promotes endothelial senescence in coronary artery endothelial cells and also the protective effect of the Crataegus extract WS1442, a quantified hawthorn extract. Serial passaging from P1 to P4 (replicative senescence) and treatment of P1 endothelial cells with the eNOS inhibitor L-NAME (premature senescence) promoted acquisition of markers of senescence, enhanced ROS formation, decreased eNOS expression, and upregulation of angiotensin-converting enzyme (ACE) and AT1 receptors. Increased SA-ß-gal activity and the upregulation of ACE and AT1R in senescent cells were prevented by antioxidants, an ACE inhibitor, and by an AT1 receptor blocker. WS1442 prevented SA-ß-gal activity, the downregulation of eNOS, and oxidative stress in P3 cells. These findings indicate that the impairment of eNOS-derived nitric oxide formation favors a pro-oxidant response triggering the local angiotensin system, which, in turn, promotes endothelial senescence. Such a sequence of events can be effectively inhibited by a standardized polyphenol-rich extract mainly by targeting the oxidative stress.