SummaryVascular endothelial dysfunction occurs during the human aging process, and it is considered as a crucial event in the development of many vasculopathies. We investigated the underlying mechanisms of this process, particularly those related with oxidative stress and inflammation, in the vasculature of subjects aged 18-91 years without cardiovascular disease or risk factors. In isolated mesenteric microvessels from these subjects, an age-dependent impairment of the endotheliumdependent relaxations to bradykinin was observed. Similar results were observed by plethysmography in the forearm blood flow in response to acetylcholine. In microvessels from subjects aged less than 60 years, most of the bradykinin-induced relaxation was due to nitric oxide release while the rest was sensitive to cyclooxygenase (COX) blockade. In microvessels from subjects older than 60 years, this COX-derived vasodilatation was lost but a COX-derived vasoconstriction occurred. Evidence for age-related vascular oxidant and inflammatory environment was observed, which could be related to the development of endothelial dysfunction. Indeed, aged microvessels showed superoxide anions (O 2 ) ) and peroxynitrite (ONOO ) ) formation, enhancement of NADPH oxidase and inducible NO synthase expression. Pharmacological interference of COX, thromboxane A 2 ⁄ prostaglandin H 2 receptor, O 2, inducible NO synthase, and NADPH oxidase improved the age-related endothelial dysfunction. In situ vascular nuclear factor-jB activation was enhanced with age, which correlated with endothelial dysfunction. We conclude that the age-dependent endothelial dysfunction in human vessels is due to the combined effect of oxidative stress and vascular wall inflammation.

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Vascular aging is a key process determining health status of aged population. Aging is an independent cardiovascular risk factor associated to an impairment of endothelial function, which is a very early and important event leading to cardiovascular disease. Vascular aging, formerly being considered an immutable and inexorable risk factor, is now viewed as a target process for intervention in order to achieve a healthier old age. A further knowledge of the mechanisms underlying the age-related vascular dysfunction is required to design an adequate therapeutic strategy to prevent or restore this impairment of vascular functionality. Among the proposed mechanisms that contribute to age-dependent endothelial dysfunction, this review is focused on the following aspects occurring into the vascular wall: (1) the reduction of nitric oxide (NO) bioavailability, caused by diminished NO synthesis and/or by augmented NO scavenging due to oxidative stress, leading to peroxynitrite formation (ONOO−); (2) the possible sources involved in the enhancement of oxidative stress; (3) the increased activity of vasoconstrictor factors; and (4) the development of a low-grade pro-inflammatory environment. Synergisms and interactions between all these pathways are also analyzed. Finally, a brief summary of some cellular mechanisms related to endothelial cell senescence (including telomere and telomerase, stress-induced senescence, as well as sirtuins) are implemented, as they are likely involved in the age-dependent endothelial dysfunction, as well as in the lower vascular repairing capacity observed in the elderly. Prevention or reversion of those mechanisms leading to endothelial dysfunction through life style modifications or pharmacological interventions could markedly improve cardiovascular health in older people.

BackgroundEndothelial dysfunction is a crucial early phenomenon in vascular diseases linked to diabetes mellitus and associated to enhanced oxidative stress. There is increasing evidence about the role for pro-inflammatory cytokines, like interleukin-1β (IL-1β), in developing diabetic vasculopathy. We aimed to determine the possible involvement of this cytokine in the development of diabetic endothelial dysfunction, analysing whether anakinra, an antagonist of IL-1 receptors, could reduce this endothelial alteration by interfering with pro-oxidant and pro-inflammatory pathways into the vascular wall.ResultsIn control and two weeks evolution streptozotocin-induced diabetic rats, either untreated or receiving anakinra, vascular reactivity and NADPH oxidase activity were measured, respectively, in isolated rings and homogenates from mesenteric microvessels, while nuclear factor (NF)-κB activation was determined in aortas. Plasma levels of IL-1β and tumor necrosis factor (TNF)-α were measured by ELISA. In isolated mesenteric microvessels from control rats, two hours incubation with IL-1β (1 to 10 ng/mL) produced a concentration-dependent impairment of endothelium-dependent relaxations, which were mediated by enhanced NADPH oxidase activity via IL-1 receptors. In diabetic rats treated with anakinra (100 or 160 mg/Kg/day for 3 or 7 days before sacrifice) a partial improvement of diabetic endothelial dysfunction occurred, together with a reduction of vascular NADPH oxidase and NF-κB activation. Endothelial dysfunction in diabetic animals was also associated to higher activities of the pro-inflammatory enzymes cyclooxygenase (COX) and the inducible isoform of nitric oxide synthase (iNOS), which were markedly reduced after anakinra treatment. Circulating IL-1β and TNF-α levels did not change in diabetic rats, but they were lowered by anakinra treatment.ConclusionsIn this short-term model of type 1 diabetes, endothelial dysfunction is associated to an IL-1 receptor-mediated activation of vascular NADPH oxidase and NF-κB, as well as to vascular inflammation. Moreover, endothelial dysfunction, vascular oxidative stress and inflammation were reduced after anakinra treatment. Whether this mechanism can be extrapolated to a chronic situation or whether it may apply to diabetic patients remain to be established. However, it may provide new insights to further investigate the therapeutic use of IL-1 receptor antagonists to obtain vascular benefits in patients with diabetes mellitus and/or atherosclerosis.

1 Alterations of the vessel structure, which is mainly determined by smooth muscle cells through cell growth and/or cell death mechanisms, are characteristic of diabetes complications. We analysed the in¯uence of high glucose (22 mM) on cultured human aortic smooth muscle cell growth and death, as hyperglycaemia is considered one of the main factors involved in diabetic vasculopathy. 2 Growth curves were performed over 96 h in medium containing 0.5% foetal calf serum. Cell number increased by 2 ± 4 fold over the culture period in the presence of 5.5 mM (low) glucose, while a 20% reduction in ®nal cell number was observed with high glucose. Under serum-free conditions, cell number remained constant in low glucose cultures, but a 40% decrease was observed in high glucose cultures, suggesting that high glucose may induce increased cell death rather than reduced proliferation. Reduced ®nal cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum. 3 The possible participation of oxidative stress was investigated by co-incubating high glucose with di erent reactive oxygen species scavengers. Only catalase reversed the e ect of high glucose. Intracellular H 2 O 2 content, visualized with 2',7'-dichloro¯uorescein and quanti®ed by¯ow cytometry, was increased after high glucose treatment. 4 To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quanti®ed. No di erences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures. 5 In conclusion, high glucose promotes necrotic cell death through H 2 O 2 formation, which may participate in the development of diabetic vasculopathy. British Journal of Pharmacology (2001) 133, 967 ± 974

Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase (SA‐β‐gal+) positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications.

Our data highlight a pivotal role for the receptor Mas in preserving normal vascular relaxation. Consequently, Mas agonists arise as a promising tool in the treatment of cardiovascular diseases characterized by endothelial dysfunction.

Visfatin, also known as extracellular pre–B-cell colony–enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 µmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 µmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 µmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 µmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 µmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 µmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.