Role of telomere in endothelial dysfunction in atherosclerosis

PURPOSE OF REVIEW: Telomeres consist of repeats of G-rich sequence at the end of chromosomes. These DNA repeats are synthesized by enzymatic activity associated with an RNA protein complex called telomerase. In most somatic cells, telomerase activity is insufficient, and telomere length decreases with increasing cell division, resulting in an irreversible cell growth arrest, termed cellular senescence. Cellular senescence is associated with an array of phenotypic changes suggestive of aging. Until recently, cellular senescence has largely been studied as an in-vitro phenomenon; however, there is accumulating evidence that indicates a critical role of telomere function in the pathogenesis of human atherosclerosis. This review attempts to summarize recent work in vascular biology that supports the "telomere hypothesis". We discuss the possible relevance of telomere function to vascular aging and the therapeutic potential of telomere manipulation. RECENT FINDINGS: It has been reported that many of the changes in senescent vascular cell behavior are consistent with known changes seen in age-related vascular diseases. Introduction of telomere malfunction has been shown to lead to endothelial dysfunction that promotes atherogenesis, whereas telomere lengthening extends cell lifespan and protects against endothelial dysfunction associated with senescence. Indeed, recent studies have demonstrated that telomere attrition and cellular senescence occur in the blood vessels and are associated with human atherosclerosis. SUMMARY: Recent findings suggest that vascular cell senescence induced by telomere shortening may contribute to atherogenesis and may provide insights into a novel treatment of antisenescence to prevent atherosclerosis.     

Shared environmental factors associated with telomere length maintenance in elderly male twins

During aging, chromosome ends, or telomeres, gradually erode or shorten with each somatic cell division. Loss of telomere length homeostasis has been linked to age-related disease. Remarkably, specific environmental assaults, both physical and psychological, have been shown to correlate with shortened telomeres. However, the extent that genetic and/or environmental factors may influence telomere length during later stages of lifespan is not known. Telomere length was measured in 686 male US World War II and Korean War veteran monozygotic (MZ) and dizygotic (DZ) twins (including 181 MZ and 125 DZ complete pairs) with a mean age of 77.5 years (range 73-85 years). During the entire process of telomere length measurement, participant age and twin status were completely blinded. White blood cell mean telomere length shortened in this elderly population by 71 base pairs per year (P < 0.0001). We observed no evidence of heritable effects in this elderly population on telomere length maintenance, but rather find that telomere length was largely associated with shared environmental factors (P < 0.0001). Additionally, we found that individuals with hypertension and cardiovascular disease had significantly shorter telomeres (P = 0.0025 and 0.002, respectively). Our results emphasize that shared environmental factors can have a primary impact on telomere length maintenance in elderly humans.     

Telomere biology in mammalian germ cells and during development

The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on.     

The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence

The absence of telomerase in many eukaryotes leads to the gradual shortening of telomeres, causing replicative senescence. In humans, this proliferation barrier constitutes a tumor suppressor mechanism and may be involved in cellular aging. Yet the heterogeneity of the senescence phenotype has hindered the understanding of its onset. Here we investigated the regulation of telomere length and its control of senescence heterogeneity. Because the length of the shortest telomeres can potentially regulate cell fate, we focus on their dynamics in Saccharomyces cerevisiae. We developed a stochastic model of telomere dynamics built on the protein-counting model, where an increasing number of protein-bound telomeric repeats shift telomeres into a nonextendable state by telomerase. Using numerical simulations, we found that the length of the shortest telomere is well separated from the length of the others, suggesting a prominent role in triggering senescence. We evaluated this possibility using classical genetic analyses of tetrads, combined with a quantitative and sensitive assay for senescence. In contrast to mitosis of telomerase-negative cells, which produces two cells with identical senescence onset, meiosis is able to segregate a determinant of senescence onset among the telomerase-negative spores. The frequency of such segregation is in accordance with this determinant being the length of the shortest telomere. Taken together, our results substantiate the length of the shortest telomere as being the key genetic marker determining senescence onset in S. cerevisiae.     

Telomere Length Profiles in Humans: All Ends are Not Equal

Telomere length is an important parameter of telomere function since it determines number of aspects controlling chromosome stability and cell division. Since telomeres shorten with age in humans and premature aging syndromes are often associated with the presence of short telomeres, it has been proposed that telomere length is also an important parameter for organismal aging. How mean telomere lengths are determined in humans remains puzzling, but it is clear that genetic and epigenetic factors appear to be of great importance. Experimental evidence obtained from many different organisms has provided the basis for a widely accepted counting mechanism based on a negative feedback loop for telomerase activity at the level of individual telomeres. In addition, recent studies in both normal and pathological contexts point to the existence of chromosome-specific mechanisms of telomere length regulation determining a telomere length profile, which is inherited and maintained throughout life. In this review, we recapitulate the available data, propose a synthetic view of telomere length control mechanisms in humans and suggest new approaches to test current hypotheses.     

A critical role for telomeres in suppressing and facilitating carcinogenesis

Progressive telomere shortening occurs with the division of primary human cells and activates tumor suppressor pathways, triggering senescence and inhibiting tumorigenesis. Loss of p53 function, however, allows continued cell division despite increasing telomere dysfunction and entry into telomere crisis. Recent data suggest that the severe chromosomal instability of telomere crisis promotes secondary genetic changes that facilitate carcinogenesis. Reactivation of telomerase stabilizes telomere ends and allows continued tumor growth.     

Stochastic mechanism of cellular aging--abrupt telomere shortening as a model for stochastic nature of cellular aging

A strong stochastic component has been described for the appearance of senescent cells in cultures that have not completed their in vitro lifespan. The proliferative potential of individual clones show a bimodal distribution. Additionally, two cells arising from a single mitotic event can exhibit large differences in their doubling capacities. In this report we present a model and a computer simulation of the model that explains the observed stochastic phenomena. The model is based on both gradual and abrupt telomere shortening.Gradual telomere shortening (GTS) occurs during each cell division as a consequence of the inability of DNA polymerase to replicate the very ends of chromosomal DNA. It is responsible for the gradual decline in proliferative potential of a cell culture, but does not explain the stochastic aspects of cellular aging. Abrupt telomere shortening (ATS) occurs either through DNA recombination or nuclease digestion at the subtelomeric/telomeric border region of the chromosome. Recombination involves the invasion of a telomere single-strand three-prime protruding end at this border in the telomere of the same chromosome or in another subtelomeric/telomeric region. Shortening of one or more telomeres in the cell causes a sudden onset of cell senescence, referred to as sudden senescence syndrome (SSS). This is manifested as a stochastic and abrupt transition of cells from the larger to the smaller proliferative potential pool and can cause cell cycle arrest within one cell division. The computer simulation matches well with experimental data supporting the prediction that abrupt telomere shortening underlies the stochastic onset of cell senescence. Sudden senescence syndrome appears to be the most important mechanism in the control of the extent of proliferation of a cell culture because it prevents virtually every cell in the culture from reaching its maximum doubling capacity, that would otherwise be allowed by telomere shortening via the end-replication mechanism alone.     

In vitro aging of rat lung cells. Downregulation of telomerase activity and continuous decrease of telomere length are not incompatible with malignant transformation

Most normal mammalian somatic cells cultivated in vitro enter replicative senescence after a finite number of divisions, as a consequence of the progressive shortening of telomeres during proliferation that reflects one aspect of organism/cellular aging. The situation appears more complex in rodent cells due to physiological telomerase expression in most somatic normal tissues, great telomere length, and the difficulties of finding suitable in vitro culture conditions. To study in vitro aging of rat lung epithelial cells, we have developed primary culture conditions adapted to rat fresh lung explants and have studied for 1 year (50 passages) the changes in cellular proliferation and mortality, genetic instability, telomerase activity, telomere length, and tumorigenic potential. We have observed an absence of senescence and/or crisis, a transient genetic instability, the persistence of a differentiated Clara cell phenotype, a steady decrease in telomerase activity followed by a low residual activity together with a continuous decrease in telomere length, a constant rate of proliferation, and the acquisition of tumorigenic potential. The bypass of the growth arrest and the acquisition of long-term growth properties could be explained by the loss of p16(INK4a) expression, the ARF/p53 pathway not being altered. In conclusion, these results clearly indicate that, in rat lung epithelial cells, in vitro transformation and acquisition of tumorigenic properties can occur even if the telomere length is still decreasing and telomerase activity remains downregulated.     

Lack of telomere shortening with age in mouse resting zone chondrocytes

BACKGROUND AND AIM: Telomeres are hexameric repeat sequences that flank eukaryotic chromosomes. The telomere hypothesis of cellular aging proposes that replication of normal somatic cells leads to progressive telomere shortening which induces replicative senescence. Previous studies suggest that growth plate chondrocytes have a finite proliferative capacity in vivo. We therefore hypothesized that telomere shortening in resting zone chondrocytes leads to replicative senescence. METHOD: To test this hypothesis we compared the telomere restriction fragment (TRF) length of Mus casteneus at 1, 4, 8, and 56 weeks of age. RESULTS AND CONCLUSIONS: We found that TRF length did not diminish measurably with age, suggesting that telomere shortening in resting zone chondrocytes is not the mechanism that limits proliferation of growth plate chondrocytes in vivo.     

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice

Alzheimer′s disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d -galactose and aluminum chloride, and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ₄₂ oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p (miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ_1–42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.     

Childhood growth, IQ and education as predictors of white blood cell telomere length at age 49-51 years: the Newcastle Thousand Families Study

Background

Telomere length is emerging as a potential factor in the pathogenesis of cardiovascular disease. We investigated whether birth weight, infant growth, childhood cognition and adult height, as well as a range of lifestyle, socio-economic and educational factors, were associated with white blood cell telomere length at age 49–51 years.

Methods

The study included 318 members of the Newcastle Thousand Families Study, a prospectively followed birth cohort which includes all individuals born in Newcastle, England in May and June 1947, who attended for clinical examination at age 49–51 years, and had telomere length successfully measured using real-time PCR analyses of DNA extracted from peripheral blood mononuclear cells.

Results

No association was found between birth weight and later telomere length. However, associations were seen with other factors from early life. Education level was the only predictor in males, while telomere length in females was associated with gestational age at birth, childhood growth and childhood IQ.

Conclusions

While these findings may be due to chance, in particular where differing associations were seen between males and females, they do provide evidence of early life associations with telomere length much later in life. Our findings of sex differences in the education association may reflect the sex differences in achieved education levels in this generation where few women went to university regardless of their intelligence. Our findings do not support the concept of telomere length being on the pathway between very early growth and later disease risk.     

Cellular senescence and DNA repair

Aging is a complex process that results in functional decline and mortality of the organisms. On the cellular lever, cellular senescence has been used as a model for aging. Therefore, understanding cellular senescence has important health implications. Initial observations suggest that cellular senescence is the result of telomere shortening. Recent findings suggest that cellular senescence could be triggered by DNA damage. In fact, both telomere shortening and DNA-damage-induced cellular senescence share a common mechanism, the DNA damage response pathway. This review will discuss the link between DNA repair defects and cellular senescence.     

Historical claims and current interpretations of replicative aging

Replicative aging is the process by which most normal human cells "count" the number of times they have divided, eventually undergoing a growth arrest termed cellular senescence. This process is dependent on the shortening of telomeres, repeated sequences at the ends of the chromosomes. The loss of telomeric sequences with each cell division eventually induces a growth arrest that has a similar phenotype to that of cells stressed by inadequate culture or other conditions. Experiments over the past several years have identified species in which replicative aging does not occur and many examples in which a failure to proliferate has been misinterpreted as replicative senescence. Insights from these studies now permit a reevaluation of much of the seemingly contradictory data concerning replicative aging. There are good theoretical reasons for believing a limited proliferative capacity contributes to declining tissue homeostasis with increasing age. Although the presence of telomere shortening provides strong circumstantial evidence that replicative aging is occurring in vivo, thus far there is only very limited direct evidence for actual physiological effects of replicative aging.     

Computel: Computation of Mean Telomere Length from Whole-Genome Next-Generation Sequencing Data

Telomeres are the ends of eukaryotic chromosomes, consisting of consecutive short repeats that protect chromosome ends from degradation. Telomeres shorten with each cell division, leading to replicative cell senescence. Deregulation of telomere length homeostasis is associated with the development of various age-related diseases and cancers. A number of experimental techniques exist for telomere length measurement; however, until recently, the absence of tools for extracting telomere lengths from high-throughput sequencing data has significantly obscured the association of telomere length with molecular processes in normal and diseased conditions. We have developed Computel, a program in R for computing mean telomere length from whole-genome next-generation sequencing data. Computel is open source, and is freely available at https://github.com/lilit-nersisyan/computel. It utilizes a short-read alignment-based approach and integrates various popular tools for sequencing data analysis. We validated it with synthetic and experimental data, and compared its performance with the previously available software. The results have shown that Computel outperforms existing software in accuracy, independence of results from sequencing conditions, stability against inherent sequencing errors, and better ability to distinguish pure telomeric sequences from interstitial telomeric repeats. By providing a highly reliable methodology for determining telomere lengths from whole-genome sequencing data, Computel should help to elucidate the role of telomeres in cellular health and disease.     

Telomeres,cardiovascular aging,and potential intervention for cellular senescence

A consistent association has been observed between leukocyte telomere length (LTL) and atherosclerosis, but the mechanisms underlying these associations are still not well understood. Premature biology aging was evident in atherosclerotic plaques, characterized by reduced cell proliferation, irreversible growth arrest and apoptosis, and telomere attrition. As atherosclerosis is a state of chronic low-grade inflammation and increased oxidative stress, shortened LTL in patients with atherosclerosis might stem from the two sources, one is an accelerated rate in hematopoietic stem cells (HSCs) replication to replace leukocytes consumed in the inflammatory process, and another is the increase in the loss of telomere repeats per replication. Thus, diminished HSC reserves at birth and age-dependent telomere attrition afterward are mirrored in shortened LTL during the adulthood. In addition, the inter-individual variation of LTL in the general population can be partly explained by genetic factors regulating telomere maintenance and the rate of HSCs replication. Atherosclerosis is an aging-related disease, and practically all humans develop atherosclerosis if they live long enough. Here we overview the potential roles of LTL dynamics in the imbalance between injurious oxidative stress/inflammation and endothelial repair during the pathogenesis of age-related atherosclerosis, and discuss the possibility that preventing accelerated cellular senescence is a potential target in prevention of atherosclerosis.     

Cardiovascular mortality in twins and the fetal origins hypothesis.

The intrauterine growth patterns for twins are characterized by normal development during the first two trimesters and reduced growth during the third trimester. According to the fetal origins hypothesis this growth pattern is associated with risk factors for cardiovascular morbidity and mortality. We studied cause-specific mortality of 19,986 Danish twin individuals from the birth cohorts 1870-1930 followed from 1952 through 1993. Despite the large sample size and follow-up period we were not able to detect any difference between twins and the general population with regard to all-cause mortality or cardiovascular mortality. Hence, the intrauterine growth retardation experienced by twins does not result in any "fetal programming" of cardiovascular diseases. There is still an important role for twins (and other sibs) to play in the testing of the fetal origins hypothesis, namely in studies of intra-pair differences, which can assess the role of genetic confounding in the association between fetal growth and later health outcome.     

TZAP‐ing telomeres down to size

The phenomenon of gradual telomere shortening has become a paradigm for how we understand the biology of aging and cancer. Cell proliferation is accompanied by cumulative telomere loss, and the aged cell either senesces, dies or transforms toward cancer. This transformation requires the activation of telomere elongation mechanisms in order to restore telomere length such that cell death or senescence programs are not induced. Most of the time, this occurs through telomerase reactivation. In other rare cases, the Alternative lengthening of telomeres (ALT) pathway hijacks DNA recombination‐associated mechanisms to hyperextend telomeres, often to more than 50 kb. Why telomere length is restricted and what sets their maximal length has been a long‐standing puzzle in cell biology. Two recent studies published in this issue of EMBO Reports [1] and recently in Science [2] sought to address this important question. Both built on omics approaches that identified ZBTB48 as a potential telomere‐associated protein and reveal it to be a critical regulator of telomere length homeostasis by the telomere trimming mechanism. These discoveries provide fundamental insights for our understanding of telomere trimming and how it impacts telomere integrity in stem and cancer cells.     

A mechanistic understanding of ageing revealed by studying the young

A main focus within biomedical research is to understand how adverse environmental conditions experienced during early development affects lifelong health (Barker 1992). Within this context, extensive research in rodent models and humans has shown that intrauterine growth retardation (IUGR) caused by nutrient restriction during early development is often followed by post-natal 'catch-up' growth when access to food resources improves. However, this accelerated growth rate seems to come at a cost, as metabolic and endocrine processes that are programmed during this time cause later-life onset of diseases such as obesity, insulin resistance and cardiovascular disease (reviewed in Crespi & Denver 2005). In this issue Molecular Ecology, Geiger et al. (2012) asked what are the costs of catch-up growth in nutrient-restricted king penguin chicks (Fig. 1) by measuring lengths of telomeres, the protective DNA sequences at the end of chromosomes, before and after catch-up growth, as the amount and rate of telomere sequence loss over time has been associated with reduced lifespan in both model and nonmodel organisms (see reviews of Costantini et al. 2010; Haussmann & Marchetto 2010). Geiger et al. (2011) found that chicks entering the post-winter growth season at a smaller size exhibited increased growth rates (i.e. catch-up growth) at the cost of increased oxidative stress and reduced telomere lengths compared with the chicks entering the growth period at a larger size. Furthermore, chicks that did not survive had drastically shorter telomere lengths and reduced antioxidant capacities at the beginning of the growth period than all other chicks, thereby directly associating telomere length to mortality. These results suggest that while catch-up growth allows smaller chicks to head off into the world on equal footing with chicks that hatched at a larger size, it likely comes at the cost of a shortened lifespan. Thus, this study provides a mechanism that supports the antagonistic pleiotropy theory of senescence (Promislow 2004).     

Spontaneous senescence in the MDA-MB-231 cell line

Normal human somatic cells have a limited division potential when they grow in vitro. It is believed that shortening of telomeres, specialized structures at the ends of chromosomes, controls cell growth. When one telomere achieves a critical minimal length, the cell cycle control mechanism recognizes it as DNA damage and causes the cell's exit from the cycle in G1-phase. Because it is not possible to extend telomeres in normal cells, this non-dividing state is prolonged indefinitely, and is known as cellular senescence. The immortal cell line MDA-MB-231 has active telomerase, which prevents telomere shortening and allows cells' permanent divisions. However, there is a fraction of cells that do not divide over several days in culture as documented for some other tumour cell lines. Combination of methods has made it possible to isolate these non-growing cells and compare them with the fraction of fast-growing cells from the same culture. Although the non-growing fraction contains a significant percentage of typical senescent cells, both fractions have equal telomerase activity and telomere length. In this paper we discuss possible mechanisms that cause the appearance of this non-growing fraction of cells in cultures of MDA-MB-231, which indicate stress and genome instability rather than variation in telomerase activity or telomere shortening to affect individual cells.     

Telomere attrition and accumulation of senescent cells in cultured human endothelial cells

The human umbilical vein endothelial cell (HUVEC) is an important model of the human endothelium that is widely used in vascular research. HUVECs and the adult endothelium share many characteristics including progression into senescence as the cells age. Despite this, the shortening of telomeres and its relationship to the progression into senescence are poorly defined in HUVECs. In this study of several HUVEC lines we show notable consistency in their growth curves. There is a steady decline in the growth rate of HUVECs grown continually in culture and we estimate complete cessation of growth after approximately 70 population doublings. The HUVECs lose telomeric DNA at a consistent rate of 90 base pairs/population doubling and show a progressive accumulation of shortened telomeres (below 5 kilobases). This telomeric loss correlates with the accumulation of senescent HUVECs in culture as assessed by staining for beta-galactosidase activity at pH 6. Although the telomere length of a large population of cells is a relatively crude measure, we suggest that in HUVECs a mean telomere length (as measured by terminal restriction fragment length) of 5 kilobases is associated with entry into senescence. These data demonstrate the strong relationship between telomere attrition and cell senescence in HUVECs. They suggest that DNA damage and subsequent telomere attrition are likely to be key mechanisms driving the development of endothelial senescence in the pathogenesis of vascular disease.