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Short telomeres protect from diet-induced atherosclerosis in apolipoprotein E-null mice¹

ENRIC POCH^*,2,4, PAZ CARBONELL^*,4, SONIA FRANCO^,3, ANTONIO DÍEZ-JUAN^*, MARÍA A. BLASCO^,3 and VICENTE ANDRÉS^*,5

^* Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia-CSIC, Valencia 46010, Spain; and
Department of Oncology and Immunology, Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, Madrid 28049, Spain

⁵Correspondence: E-mail: vandres{at}ibv.csic.es

SPECIFIC AIMS

By imposing a replicative defect in most somatic cells, gradual telomere attrition during aging progressively impairs cellular function and viability and may contribute to age-related disease. Immune cells play important roles in all phases of atherosclerosis, a multifactorial disease that prevails within the elderly. Because shorter telomeres have been found in circulating blood leukocytes of human patients with advanced coronary atherosclerosis, it has been suggested that a tendency to telomere shortening may be a primary abnormality that predisposes the organism to atheroma development. In the present study, we investigate the impact of telomere attrition on atherogenesis induced by dietary cholesterol in apolipoprotein E (apoE)-deficient mice, a well-established model of experimental atherosclerosis that recapitulates important aspects of the human disease.

PRINCIPAL FINDINGS

1. Short telomeres protect from diet-induced atherosclerosis in apoE null mice
Previous studies have shown that the breeding of successive generations of mice deficient for the RNA component of telomerase (Terc) is necessary to reach critically short telomeres and associated phenotypes. Moreover, the mouse as a species is highly resistant to atherosclerosis. Thus, we generated for our studies fourth generation animals doubly deficient in Terc and apoE (G4Terc^-/-apoE^-/-) and counterparts singly deficient in apoE (G4Terc^+/+apoE^-/-). As revealed by quantitative fluorescence in situ hybridization with a fluorescent telomeric probe, the average telomere length in medial smooth muscle cells (SMCs) from the aorta of 2-month-old mice fed control chow was significantly reduced in G4Terc^-/-apoE^-/- mice compared with G4Terc^+/+apoE^-/- counterparts with intact telomerase (arbitrary units of fluorescence: 191.4±5.1 vs. 400±72.3, P<0.001).

We next challenged mice with a high-fat, cholesterol-rich diet for 6 weeks. Consistent with numerous studies in apoE null mice, atheromas in both groups of mice predominated within the aortic arch, as determined by Oil Red O staining (Fig. 1 A). Notably, G4Terc^-/-apoE^-/- mice disclosed a significant reduction in the area of atherosclerotic lesions compared with G4Terc^+/+apoE^-/- mice (Fig. 1A , P<0.0002). We also quantified in cross-sections of the aortic arch the area of atheroma (intimal lesion) relative to the tunica media (Fig. 1B, C ). Both the intimal area and the intima-to-media ratio were significantly reduced in fat-fed G4Terc^-/-apoE^-/- compared with G4Terc^+/+apoE^-/- mice. These studies demonstrate reduced atherosclerosis as a consequence of telomere exhaustion.

View larger version (38K): [in this window] [in a new window]   Figure 1. Short telomeres protect from diet-induced atherosclerosis. Two-month-old mice were challenged with a cholesterol-rich diet for 1.5 months. Atherosclerosis within the aortic arch region was quantified by computer-assisted planimetry to determine the area of atherosclerotic lesions after Oil Red O staining in whole-mounted arteries A), lipid-laden lesions in red, and to quantify medial and intimal areas in cross-sections stained with May-Grünwald Giemsa (B, C). The edge of the atherosclerotic plaque (intimal lesion) is drawn with a discontinuous line in (C). The number of mice analyzed (n) and gender distribution (males/females) are shown. The red line in the graph in A indicates the average value in each group. Statistical analysis was performed using 2-tailed, unpaired Student’s t test.

2. Atherosclerotic lesions are less advanced in apoE null mice with short telomeres
Numerous studies in both experimental animals and humans have shown that leukocytes, and in particular lipid-laden macrophages, are the main cellular components of incipient atheromas. Chemokines and cytokines secreted by neointimal leukocytes activate the underlying SMCs, which are induced to proliferate and migrate toward the subendothelial space to form fibrous cap lesions, a characteristic of more advanced lesions. Immunohistochemistry of the aortic arch in fat-fed G4Terc^+/+apoE^-/- and G4Terc^-/-apoE^-/- mice shows abundance of Mac-3-expressing macrophages within atherosclerotic lesions (Fig. 2 A, B, G), and SM-actin-immunoreactive SMCs in the media (Fig. 2C -F ). SM-actin-immunoreactivity in fibrous caps was also frequent in the atherosclerotic lesions of G4 Terc^+/+apoE^-/- mice (Fig. 2C, E, H ). However, expression of this SMC marker was virtually absent in the atheromas of G4Terc^-/-apoE^-/- mice (Fig. 2D, F, H ). Thus, atherosclerotic lesions in apoE-mice with short telomeres appear less advanced than those of control animals with intact telomerase.

View larger version (65K): [in this window] [in a new window]   Figure 2. Cellular composition of atherosclerotic lesions. Immunohistochemistry of aortic arch cross-sections of fat-fed mice showing in red Mac-3-immunoreactive macrophages (A, B) and SM-actin-immunoreactive SMCs (C–F). Specimens were counterstained with hematoxylin. The top and middle photomicrographs of each genotype show adjacent sections. Arrowheads in C) and E) point at SM-actin-positive fibrous caps. Atherosclerotic lesions were analyzed by computer-assisted planimetry to calculate the area occupied by Mac-3- and SM-actin-immunoreactive cells (G, H, respectively). The number of mice analyzed (n) and gender distribution (males/females) are shown.

3. G4Terc^-/-apoE^-/- leukocytes display reduced replicative potential
Plasma cholesterol level after fat feeding was slightly increased in G4Terc^-/-apoE^-/- compared with G4Terc^+/+apoE^-/- mice (3683±278 mg/dl, n=9, vs. 2848±189 mg/dl, n=25, respectively, P<0.03), indicating that diminished atherosclerosis in fat-fed G4Terc^-/-apoE^-/- mice is not secondary because of reduced plasma cholesterol content. We next investigated whether telomere exhaustion can reduce the proliferative capacity of macrophages and lymphocytes, an important step in atherosclerosis. Cultured splenocytes and bone marrow-derived macrophages isolated from G4Terc^-/-apoE^-/- mice disclosed a marked reduction in mitogen-induced proliferation compared with G4Terc^+/+apoE^-/- cells, as revealed by ³H-thymidine incorporation assays.

CONCLUSIONS AND SIGNIFICANCE

By comparing the development of atherosclerosis in fat-fed apoE null mice with an intact Terc gene and with targeted disruption of Terc, the present study demonstrates for the first time that telomere attrition protects from atherogenesis in spite of sustained hypercholesterolemia. We found that short telomeres impair the proliferation of both lymphocytes and macrophages, an important step in atherosclerosis development. Thus, we propose that telomere attrition resulting in replicative immunosenescence may serve as a mechanism for restricting atheroma progression in hypercholesterolemic mice (Fig. 3 ). A detailed analysis of atherosclerotic tissue by double immunohistochemistry and immunofluorescence using cell type-specific markers in combination with proliferation (i. e., proliferating cell nuclear antigen, Ki67) and apoptosis markers (i. e., TUNEL, bcl-2, caspases-1, -3, and –8, TRAIL and FAS proteins) will be necessary to shed further insight on the mechanisms underlying the atheroprotective effect of telomere exhaustion.

View larger version (74K): [in this window] [in a new window]   Figure 3. Diagram illustrating early events in atherosclerosis. Atherogenic stimuli lead to endothelial dysfunction, thus promoting the recruitment of blood circulating leukocytes and their proliferation within the growing atherosclerotic lesion. These processes are perpetuated by the release of chemokines and cytokines by neointimal leukocytes. These inflammatory mediators also activate the proliferation of SMCs and their migration toward the atherosclerotic lesion, thus further contributing to atheroma growth. In part by imposing replicative immunosenescence, telomere exhaustion can restrict atheroma progression.

Because telomere shortening is more obvious in highly proliferative somatic cells, it has been debated whether decreased telomere length in regions of human arteries highly susceptible to atherosclerosis and in leukocytes of patients with severe coronary atherosclerosis is merely a consequence of increased cell turnover induced by the chronic inflammatory response underlying the atherogenic process, or a primary abnormality that renders the organism more susceptible to atherosclerotic risk factors. Although it is important to be cautious when considering how findings that are made in mice relate to humans, the atheroprotective effect of telomere attrition reported herein appears to support the first possibility. However, a conclusive answer to this fundamental question must await the results of prospective epidemiological studies to ascertain if newborns with significantly shorter telomeres in blood circulating leukocytes can be identified, and if so whether they are at higher risk of developing coronary atherosclerosis in adulthood independently of known cardiovascular risk factors.

Notably, mice with critically short telomeres are also less predisposed to tumor development. Thus, telomere-based replicative senescence appears to play a critical role in the setting of atherosclerosis and cancer in mice. In this regard, it is remarkable that although human aging is associated with telomere erosion in somatic cells, both atherosclerosis and cancer are more prevalent in the elderly. These seemingly conflicting findings might be reconciled if one accepts the hypothesis that increasing cellular damage imposed by prolonged exposure to predisposing risk factors and accumulation of multiple mutations over time may ultimately exceed the protective effect of telomere shortening -and of other protecting mechanisms. Of interest in this regard, we have recently shown that 4 to 5-year-old rabbits display significantly smaller atherosclerotic lesions that 4 to 5-month-old counterparts, in spite of comparable hypercholesterolemia induced by the same dietary regimen.

In summary, we have shown that telomere exhaustion restricts atheroma progression in hypercholesterolemic mice, perhaps in part by imposing replicative immunosenescence. Future studies are warranted to investigate whether additional events involved in atheroma initiation and progression are also impaired by telomere exhaustion (i. e., interaction between blood circulating leukocyte and endothelial cells, transendothelial migration of leukocytes, synthesis of cytokines, chemokines, and extracellular matrix proteoglycans, SMC proliferation and migration, apoptosis) (Fig. 3) , and to assess whether genetic and environmental cardiovascular risk factors affect telomerase activity and/or telomere length.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0710fje;

² Present address: Department of Chemistry, Biochemistry and Molecular Biology, Universidad Cardenal Herrera-CEU, Moncada 46113, Spain

³ Present address: Telomeres and Telomerase Group, Centro Nacional de Investigaciones Oncológicas (CNIO) (Spanish National Cancer Center), Madrid 28029, Spain

⁴ These authors contributed equally to the work.

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