The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:200-203 (2008)
© 2008 The Gerontological Society of America
Effects of Ubiquitin-Proteasome System Deregulation on the Vascular Senescence and Atherosclerosis Process in Elderly Patients
Raffaele Marfella,
Clara Di Filippo,
Maria Teresa Laieta,
Rosanna Vestini,
Michelangela Barbieri,
Paolo Sangiulo,
Basilio Crescenzi,
Franca Ferraraccio,
Francesco Rossi,
Michele D'Amico and
Giuseppe Paolisso
Departments of 1 Geriatrics and Metabolic Diseases, 2 Experimental Medicine, and 3 Clinical, Public and Preventive Medicine, Second University of Naples, Italy.
4 Cardiovascular Surgery Unit, Monaldi Hospital, Naples, Italy.
Address correspondence to Raffaele Marfella, MD, PhD, Via Emilio Scaglione 141, 80145 Napoli, Italy. E-mail: raffaele.marfella{at}unina2.it
Abstract
Background. The role of the ubiquitin-proteasome system in the vascular senescence and atherosclerotic progression of elderly patients is unclear. We evaluated ubiquitin-proteasome activity in carotid plaques of asymptomatic elderly and adult patients.
Methods. Plaques were obtained from 28 elderly and 18 adult patients undergoing carotid endarterectomy. Plaques were analyzed for ubiquitin levels, proteasome 20S activity, p16 and p53, nitrotyrosine, matrix metalloproteinase-9 (MMP-9) and collagen content (immunohistochemistry and enzyme-linked immunosorbent assay). Serial sections were incubated with specific antibodies anti–human leukocyte antigen (HLA)-DR, anti CD68 and anti-CD3.
Results. Compared to plaques obtained from adult patients, plaques of elderly patients had more ubiquitin levels (257.4 ± 118.9 ng/mg vs 110 ± 14.4 ng/mg, p <.001), nitrotyrosine (3.8 ± 0.55 nmol/pg vs 1.1 ± 0.19 nmol/pg, p <.001), p53 and p16 staining (p <.01), and MMP-9 levels (14.6 ± 2.5 µg/mg vs 3.2 ± 0.1.8 µg/mg, p <.001), along with a lesser collagen content (21.9 ± 4.8% vs 7.1 ± 2.8%, p <.05) and less proteasome 20S activity (24.2 ± 6.9 pmol/mg vs 78.4 ± 10.3 pmol/mg, p <.001).
Conclusions. Our data suggest that reduction of proteasome activity promotes vascular cell senescence, thereby contributing to the pathogenesis of human atherosclerosis.
Key Words: Atherosclerosis • Ubiquitin-proteasome
EPIDEMIOLOGICAL studies have shown that age is a dominant risk factor for atherosclerotic cardiovascular diseases (1,2). The incidence and prevalence of atherothrombotic diseases, including coronary heart disease and stroke, both increase with advancing age (1,2). However, the molecular mechanisms underlying the increased risk of such diseases that is conferred by aging remain unclear. For example, arterial stiffness increases with age because of structural changes of the arterial walls as well as endothelial dysfunction, but convincing molecular explanations for these age-associated alterations of vascular structure and function have not yet been reported. Because aging includes various biological phenomena, it is difficult to attribute age-related changes of the vasculature or the organism to a certain molecule. Moreover, there are no accurate biomarkers for aging, which makes it problematic to study vascular aging. However, a number of studies have shown that many of the changes of senescent vascular cells are consistent with those seen in human atherosclerosis, suggesting a critical role of cellular senescence in vascular pathophysiology. These include components of the DNA-repair system, the tumor suppressor pathway, and the telomere maintenance system (3,4). Moreover, both nitric oxide (NO) production and endothelial NO synthase activity are reduced in senescent human vascular endothelial cells (5). The increment of NO production in response to shear stress is also smaller in senescent vascular endothelial cells. Production of reactive oxygen species (ROS) is increased in senescent cells (6,7), leading to a decrease in the bioavailability of NO and increased formation of peroxynitrite.
However, emerging evidence suggests that the ubiquitin-proteasome system, the major pathway for nonlysosomal intracellular protein degradation in eukaryotic cells, may play a role in all these changes in elderly persons (8). Indeed, ubiquitination of endogenous proteins is one of the key regulatory steps of protein degradation followed by regulation of proteasome activity. In recent years, evidence has increased that proteasome activity is decreased during the aging process in various model systems and that these changes might be causally related to aging and aged-related diseases (9). Depending on mono- and polyubiquitination and on how ubiquitin chains are linked together, ubiquitination is involved in negative regulators of the cell cycle such as p53 and p16 (10), in increased telomere shortening (11), and in accelerated entry into senescencelike growth arrest (12) as well as in increased formation of peroxynitrite (13). Moreover, the regulatory function of ubiquitination in DNA repair might be of high importance for aging, not only with regard to replicative senescence, but also with regard to segmental progeroid syndromes, the symptoms of which are largely caused by defects in the DNA repair system (14). Thus, it is conceivable that the ubiquitin-proteasome activity deregulation seen in elderly people might thereby be important in the pathogenesis of atherosclerosis progression in elderly patients. According to such evidences, we evaluated ubiquitin-proteasome activity as well as the staining of p16, p53, and nitrotyrosine (an index of the nitrosylation of proteins by peroxynitrite and/or ROS) in carotid plaques from elderly asymptomatic patients compared to plaques from adult asymptomatic patients.
Plaques were obtained from 46 asymptomatic patients undergoing carotid endarterectomy (Table 1). In our study population, 1097 adult patients recruited from the outpatient department of the teaching hospital at the Second University of Naples (from 1999 to 2006), about 6% have asymptomatic carotid plaque, and this frequency increases with age. Only a small number of these patients (about 25%), however, have asymptomatic carotid stenosis > 75%. After surgery, the specimens were cut perpendicular to the long axis into two halves. The first half was frozen in liquid nitrogen for the following enzyme-linked immunosorbent assay (ELISA) analysis. A portion of the other half specimen was immediately immersion-fixed in 10% buffered formalin. Sections were serially cut at 5 µm, mounted on lysine-coated slides, and stained with hematoxylin and eosin and with the trichrome method. Carotid artery specimens were analyzed by light microscopy. Plaques were analyzed for ubiquitin levels (R&D Systems, Santa Cruz, CA), proteasome 20S activity (sodium dodecyl sulfate [SDS]-activation kit; Boston Biochem, Cambridge, MA), p16 and p53 (Santa Cruz), nitrotyrosine (Imgenex, San Diego, CA), matrix metalloproteinase-9 (MMP-9; R&D Systems), and collagen content (ELISA, immunohistochemistry, and Sirius red staining). Serial sections were incubated with specific antibodies anti–human leukocyte antigen (HLA)-DR, anti CD68, and anti-CD3 (Dako, Glostrup, Denmark). Analysis of immunohistochemistry was performed with a personal computer–based quantitative 24-bit color image analysis system (IM500; Leica Microsystems AG, Milano, Italy). Written informed consent was obtained from all patients before each examination. The local ethics review committee approved the study.
Compared to plaques obtained from adult patients (age 47 ± 11 years), plaques of elderly patients (age 72 ± 6 years) had higher ubiquitin levels (257.4 ± 118.9 ng/mg vs 110 ± 14.4 ng/mg, p <.01), more nitrotyrosine (3.8 ± 0.55 nmol/pg vs 1.1 ± 0.19 nmol/pg, p <.01) and p53 and p16 staining (p <.01), higher MMP-9 levels (14.6 ± 2.5 µg/mg vs 3.2 ± 0.1.8 µg/mg, p <.01), lower collagen content (21.9 ± 4.8% vs 7.1 ± 2.8%, p <.05), and less proteasome 20S activity (24.2 ± 6.9 pmol/mg vs 78.4 ± 10.3 pmol/mg, p <.05) (all data are expressed as mean ± standard deviation) (Figure 1).
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Figure 1. Immunostaining (x200) of representative sections show immunochemistry for nitrotyrosine, ubiquitin, proteasome 20S, p16, and p53 in plaque from elderly patients and in plaque from adult patients. In carotid atheromatous plaques of elderly patients, p53 and p16 immunoreactivity was present in the nuclei of virtually all cell types: macrophages, smooth muscle cells, and endothelial cells
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Our data suggest that ubiquitin-proteasome deregulation may be involved in atherogenesis progression favoring vascular cell senescence through the induction of negative regulators of the cell cycle such as p53 and p16. In particular, the increase of ubiquitin levels, evoked by impaired proteasome 20S activity, may be responsible for the increased levels of both p53 and p16. Cellular senescence was originally described as the finite replicative life span of human somatic cells in culture. Senescent cells enter an irreversible growth arrest, exhibit a flattened and enlarged morphology, and express a different set of genes, including negative regulators of the cell cycle such as p53 and p16. Although the whole picture of cellular senescence regulated by cell-cycle modulators is far from complete and the question of how many subpathways are involved has not yet been fully addressed, our data suggest that senescence in vascular cells occurs via the p53 and p16 pathways. Consistent with this notion, it has been demonstrated that senescent vascular endothelial cells are predominately localized in the plaque of human atherosclerosis but not in normal arteries and that vascular cell senescence results in endothelial dysfunction (15). Given that various atherogenic stimuli, including growth factors and oxidative stress, are mediated by ubiquitin-proteasome activity deregulation (16), one would assume that reduction of proteasome activity promotes vascular cell senescence and thereby reduces NO availability and increases formation of nitrotyrosine in aged plaques, contributing to the progression of atherosclerosis toward plaque instability phenotype, as evidenced by higher levels of MMP-9 and inflammation along a lesser collagen content in elderly patients. In contrast, in human studies the ubiquitin-proteasome pathway is impaired in symptomatic plaques (17) as well as in asymptomatic diabetic plaques (18). However, in these study populations, matched for age, the ubiquitin-proteasome overactivity seems to be associated with the plaque instability phenotype. This evident difference between elderly and adult patients could be explained by the evidence that age-related underactivity of proteasome may be responsible for the accumulation of oxidized and ubiquitinated protein aggregates that may enhance the damage and foster plaque instability. It is noteworthy that aging has been associated with an impairment of the proteasome activity in human fibroblasts and induction of proteasome expression reduces cell senescence (19). It is an open question whether, in human atherosclerosis, an overactivation or an underactivation of the system is correlated with clinical manifestation of the atherosclerotic disease. Several age-related degenerative diseases, such as Alzheimer's disease and Parkinson's disease, cataracts, and some cancers, are frequently associated with reduced proteasome activity (20). Our study suggests that a similar process may play a role in human atherosclerotic plaques from elderly patients, which can be considered to be a degenerative as well as an inflammatory disease. The importance of the ubiquitin-proteasome system for molecular and cellular biology (as well as for medical sciences) is ever increasing, and there are only a few cellular pathways left that at one step or another are not regulated by ubiquitin. With regard to the aging of cells and tissues, the example presented suggests that deregulation of the ubiquitin-proteasome activity might have vast implications for the aging of organisms as well as for aging-associated diseases, although in some cases direct evidence is still missing. However, several atherogenic stimuli function as mitogens for vascular cells, promoting cell proliferation and mitogenic signaling pathways that may induce telomere-dependent and telomere-independent vascular cell senescence. Therefore, the increased levels of p53 and p16 in plaques of elderly patients may represent a more extensive replication in these lesions, compared to plaques of young individuals. Moreover, unsaturated aldehydes, produced by ROS-mediated lipid peroxidation (21), may be responsible for the proteasome function impairment demonstrated in plaques of elderly patients.
Study Limitations
Considering the relatively small population, it was not possible to evaluate the role of the vascular risk factors in the atherosclerosis progression of the adult patients in comparison with the elderly patients. Probably, vascular risk factors such as hypertension and smoking are important in early plaque formation, but when plaques become stenosing, local hemodynamic factors such as turbulence are the major factors in arterial remodeling (1). Moreover, the associative nature of the present work does not allow drawing definitive conclusions on the role of a dysregulation of the ubiquitin-proteasome system in the pathophysiology of atherosclerosis in the elderly population. In any case, further understanding of the influence of the ubiquitin-proteasome system and its related molecules on the aging process might help to identify targets for prevention of deleterious loss of cell and tissue function and pathogenesis of atherosclerosis.
Footnotes
Decision Editor: Luigi Ferrucci, MD, PhD
Received February 13, 2007
Accepted May 22, 2007
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