Telomere uncapping and vascular aging

Abstract

Although most telomere biology research continues to focus on telomere shortening, there is increasing evidence that telomere deprotection, or “uncapping,” is more biologically and possibly clinically important. Telomeres form t-loops to prevent the chromosome ends from appearing as a double-stranded DNA break and initiating a DNA damage response. Breakdown of the t-loop structure, referred to as uncapping, can lead to cellular senescence, increased oxidative stress, and inflammation in tissues. In this review, we describe how telomere uncapping potentially leads to age-related vascular dysfunction and increased cellular senescence, oxidative stress, and inflammation. Importantly, we present evidence to argue that telomere uncapping is more biologically relevant than telomere shortening and a better marker of vascular aging and target for antiaging interventions.

INTRODUCTION

Advancing age is the greatest risk factor for development of cardiovascular disease (CVD). Changes to the vasculature with age, such as impaired endothelial function and increased large artery stiffness, are key contributors to this increased CVD risk (16). As such, it is important to identify age-related vascular changes, as these represent therapeutic targets for the prevention or treatment of CVD. Although it is clear that increased oxidative stress and inflammation are key contributors to the vascular aging phenotype [as reviewed in depth by Seals et al. (28)], a comprehensive understanding of the age-associated processes that promote and sustain increased vascular oxidative stress and inflammation remains elusive. In this review, we describe how telomere dysfunction potentially leads to age-related vascular dysfunction and increased cellular senescence, oxidative stress, and inflammation. Importantly, we present evidence to argue that telomere uncapping is more biologically relevant than telomere shortening and a better marker of vascular aging and target for antiaging interventions.

TELOMERE STRUCTURE AND FUNCTION

Telomeres are composed of TTAGGG repeats that form the natural ends of chromosomes (20). When DNA is replicated, a section of the 5′ sequence cannot be copied and is therefore lost, a situation known as the “end replication problem.” As telomeres are at the ends of chromosomes, telomeric DNA is lost with each replication, thus preserving coding DNA (29). In stem and germ cells, telomerase adds sequence back to telomeres, ensuring minimal telomere shortening over many cell divisions. However, in somatic cells, including vascular endothelial and smooth muscle cells, telomerase activity is low, and telomere length is shortened over time with each cell division (29).

Another vital function of the telomere is the formation and maintenance of the t-loop at the chromosome end. If the chromosome end was present within the nucleus as a linear DNA strand with an abrupt end, this end would be recognized as a double-stranded DNA (dsDNA) break by the DNA damage repair machinery (11). To combat this, the single-stranded overhanging portion of the telomere invades into the preceding double-stranded telomere region, forming the t-loop (10, 31). A telomere that forms this t-loop is termed “capped.” A specialized complex of six proteins, knows as the shelterin complex, is responsible for forming and maintaining the t-loop. Breakdown of the t-loop structure, referred to as “uncapping,” results from telomeres being critically short or damaged, such as by oxidative damage or from impairment of the shelterin complex (Fig. 1A) (3, 10). Uncapping of as few as five telomeres will initiate a rapid dsDNA break response and subsequent cellular senescence (7, 10, 34).

Fig. 1.

Telomere uncapping induced cellular senescence. A: age-related shortening or damage to telomeric DNA may lead to uncapping of the t-loop and initiation of a double-stranded DNA break (DSB) response. The first step in the DSB response is phosphorylation of histone 2A.X (p-H2A.X) molecules within telomeric chromatin, which in turn leads to tumor suppressor protein-p53 (p53) activation. B: activated p53 localizes to its target gene promoters and mediates RNA polymerase (RNA Pol)-dependent expression of the corresponding genes, such as cyclin-dependent kinase inhibitor 1 (p21). p21 is a potent cell cycle inhibitor that physically binds and inhibits cyclin-dependent kinase complexes (CDKs), such as CDK2, to halt the cell cycle. Cellular senescence is a permanent halting of the cell cycle, which leads to secretion of proinflammatory and prooxidant factors.

CELLULAR SENESCENCE AND AGING

Cellular senescence is the permanent halting of the cell cycle and is a biological response to prevent the replication of DNA with irreparable damage. In response to DNA damage as well as telomere uncapping, tumor suppressor protein p53 is activated, leading to increased expression of cyclin-dependent kinase inhibitor 1A (p21), which prevents the progression of the cell cycle (Fig. 1B) (7, 10). If p21 signaling is constitutively activated, then the cell becomes senescent. A key feature of senescent cells is the release of reactive oxygen species and proinflammatory factors, known as the senescence-associated secretory phenotype. This increase in local inflammation reinforces senescence signaling in an autocrine/paracrine manner and encourages immune cell recruitment to clear damaged (senescent) cells (9). However, this mechanism becomes pathological with aging, as senescent cells accumulate in tissues, leading to chronic oxidative stress, inflammation, and dysfunction. The importance of cellular senescence to aging has been reviewed in depth by others (6, 15).

Senescent cells accumulate in vascular tissue with aging, as demonstrated by increased p53 bound to the p21 promoter and p21 expression with age in resistance arteries collected from human subjects (18). Similarly, p53, p21, and p16 expression increase with aging in vascular endothelial cells collected from healthy human subjects (26). Furthermore, the expression of senescence markers in the endothelial cell is inversely correlated with in vivo vascular endothelial function in humans (26). This relation has been proven empirically in mice, in which the clearance of senescent cells by genetic manipulation or senolytic treatment leads to improved vascular endothelial function in aged mice (25). In addition, in vitro studies in human vascular endothelial cells or smooth muscle cells passaged to replicative senescence have elevated oxidative stress and inflammatory signaling, and senescent endothelial cells release less of the key vasodilator nitric oxide (12, 13, 27). Therefore, in the context of normal aging, senescent cells that accumulate in vascular tissue may lead to a cascade of widespread inflammation and oxidative stress, resulting in impaired vascular function. Thus, it is critically important to understand the role that telomere uncapping plays in vascular aging, as this may be a key source of cellular senescence and age-associated functional impairment.

VASCULAR TELOMERE SHORTENING WITH AGING

In epidemiological studies, age-related telomere shortening has been observed in human arterial tissue (2, 8, 30), and shorter leukocyte mean telomere length is associated with CVD risk (14, 21). In addition, arterial telomere length is inversely correlated with atherosclerotic plaque severity (22, 23). Furthermore, short telomeres can cause vascular endothelial dysfunction, as demonstrated in telomerase-deficient mice after multiple generations (5). However, the beneficial effects of telomerase on vascular function may be related to reducing mitochondrial reactive oxygen species rather than effects on telomere length (1, 4). While vascular aging phenotypes are present and fairly consistent across species, the definitive role of telomere length is not clear. For example, mouse telomeres are up to five times longer than human telomeres and unlikely to reach a critical length during an animal’s lifetime (24). Furthermore, it is not telomere length per se that is a signal for cellular senescence but rather the uncapping of the chromosome end, which can occur when the telomere reaches a critical shortness or is damaged, and subsequent DNA damage signaling.

VASCULAR TELOMERE UNCAPPING WITH AGING

Telomere uncapping can be measured by phosphorylation of histone γ-H2A.X at serine 139 (p-H2A.X) in telomeric chromatin, a marker of DNA damage and an initial signal for the dsDNA damage response. In resistance arteries collected from human subjects, telomere uncapping increases with age and hypertension (18, 19). This age- and hypertension-associated telomere uncapping occurs independent of mean telomere length (18, 19). Importantly, markers of p21-induced senescence are related to telomere uncapping but not to mean telomere length (18, 19). These results indicate that telomere shortening is not necessary for telomere uncapping and subsequent senescence signaling in the context of normal vascular aging. This suggests that oxidative damage to telomeres or shelterin complex dysfunction may play a key role in age-related telomere uncapping in the vasculature.

Vascular telomere uncapping may not occur similarly to all populations with age. Women have a greater increase in arterial telomere uncapping with age compared with men (32), indicating a possible interaction of aging and menopause. Fasting blood glucose positively correlates with vascular telomere uncapping in women but not in men, whereas systolic blood pressure and pulse pressure positively correlate with vascular telomere uncapping in men but not in women (32). Thus, the rate and causes of vascular telomere uncapping may differ between the sexes.

Experimental studies in rodents have implicated a role for telomere uncapping in vascular dysfunction. A recent study by Wang et al. (33) reported that loss of shelterin protein telomeric repeat binding factor 2 function in vascular smooth muscle cells leads to increased atherosclerotic plaque formation and markers of senescence in atherosclerosis-prone mice. In addition, senolytic treatment in old or atherosclerosis-prone mice leads to a reduction in telomere uncapping, as indicated by fewer telomere-associated foci (p-H2A.X colocalized with telomeres) and improved vascular function (25). However, the causal relationship among telomere uncapping, p21-induced senescence, and age-associated vascular functional impairment has not been demonstrated.

VASCULAR TELOMERE UNCAPPING VERSUS SHORTENING

The above evidence casts doubt on the relevance of telomere shortening but highlights the potential importance of telomere uncapping to vascular aging and dysfunction. Although telomere shortening certainly occurs with aging in vascular tissues, the only known pathway by which critically short telomeres can impart cellular dysfunction is through telomere uncapping and initiation of a dsDNA damage response. This is supported by the finding that markers of cellular senescence are related to telomere uncapping but not telomere shortening in vascular tissue (18, 19). Thus, it is our position that telomere uncapping and cellular senescence represent more biologically relevant markers of vascular aging and CVD risk and potentially more effective targets for vascular-directed interventions than telomere length. Future studies are needed to establish a link between telomere uncapping and CVD. In addition, definitive studies with experimental models of telomere uncapping, such as the cre-inducible Trf2 deletion model developed by Titia de Lange’s laboratory (17), will provide urgently needed insights into the roles of telomere uncapping and cellular senescence in age-associated vascular phenotypes.

POTENTIAL THERAPEUTICS TO TARGET TELOMERE UNCAPPING

Given the evidence for increased vascular telomere uncapping with advancing age, this pathway may provide a therapeutic target for treating age-related vascular dysfunction and CVD (Fig. 2). However, it is important to remember that telomere uncapping and cellular senescence have critical physiological roles. Recapping telomeres has never been demonstrated experimentally and may lead to the division of cells with damaged DNA. In contrast, age-related telomere uncapping may result directly from a dysfunctional shelterin complex, in which case this complex could be manipulated to improve telomere capping. Therapeutic targets may also be found in the upstream causes, or in the downstream consequences, of telomere uncapping. Recently discovered senolytic compounds clear senescent cells and can lead to improvements in vascular function (25); however, translation to humans has yet to be attempted. Thus, given the lack of plausible therapeutic targets, it is critically important that the causes of and signaling pathways involved in age-related vascular telomere uncapping be better elucidated.

Fig. 2.

Potential role of telomere uncapping in vascular aging and cardiovascular disease (CVD). Vascular telomere uncapping can lead to senescent cell accumulation and the senescence-associated secretory phenotype (SASP). Senescent cell accumulation leads to chronic SASP-mediated inflammation/oxidative stress, whereas chronically elevated inflammation/oxidative stress will promote further accumulation of senescent cells, as indicated by the bidirectional arrow. Vascular telomere uncapping increases with age (represented by blue line), and individuals with higher (pathological) amounts of vascular telomere uncapping at a given age may potentially have a greater CVD risk (represented by red line). The purple X indicates general targets of present therapeutic strategies for CVD. Green Xs indicate that clearance of senescent cells and attenuation of telomere uncapping represent hypothetical therapeutic targets that could improve CVD outcomes.

CONCLUSIONS

Although most telomere biology research continues to focus on telomere shortening, there is increasing evidence that telomere uncapping is more biologically relevant. Recent studies in humans have demonstrated that telomere uncapping increases with age and hypertension in vascular tissue. Importantly, vascular markers of cellular senescence are related to telomere uncapping but not to mean telomere length in vascular tissue. Likewise, vascular telomere uncapping and associated markers of senescence have been linked with elevated blood pressure in men and increased fasting glucose in women. However, evidence is still needed to confirm that telomere uncapping causes dysfunction and an aging phenotype in the vasculature. Furthermore, it will be important to identify the causes and consequences of vascular telomere uncapping with advancing age. Intervening with the upstream or downstream pathways related to telomere uncapping could potentially mitigate vascular dysfunction with aging and reduce cardiovascular disease risk.

GRANTS

This work was supported by National Institute on Aging Grants AG-040297, AG-043952, AG-046326, AG-044339, and AG-050238, Department of Veterans Affairs Grant 1I01BX002151, and a University of Utah Center on Aging pilot grant.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.G.M., A.J.D., and A.E.W. prepared figures; R.G.M., A.J.D., and A.E.W. drafted manuscript; R.G.M., A.J.D., and A.E.W. edited and revised manuscript; R.G.M., A.J.D., and A.E.W. approved final version of manuscript.