The present study demonstrates that Ang II–induced ROS production leads to DNA damage and the accelerated biological aging of hVSMCs via 2 mechanisms: (1) SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with telomere attrition.
A common paradigm for the induction of cell senescence is loss of telomere integrity or critical shortening of telomeres, resulting in a “DNA damage response” and cell cycle arrest via activation of CDKIs.32
Telomere shortening occurs progressively with every cell division due the “end-replication problem”, eventually leading to critical shortening of telomeres which triggers replicative senescence. This process is accelerated by exposure of cells to oxidant stress and DNA damage.10,33
The telomere region of DNA is especially vulnerable to oxidative damage.34,35
Moreover, the repair of DNA within the telomere is inefficient relative to the rest of the genome.36
We show that the average loss of TRF length in hVSMCs in culture is ≈1.0% per population doubling, consistent with our previous data on human vascular endothelial cells27
and studies using cultured VSMCs.24,37
We also show that treatment with Ang II markedly accelerated this loss by 2.5-fold, thereby predisposing hVSMCs to accelerated replicative senescence.
Cell senescence may also arise more rapidly, in a manner independent of the slower, replication-dependent attrition of telomere DNA. This mechanism, termed “premature senescence” or SIPS, involves activation of identical CDKIs to those activated in replicative senescence.7
Induction of SIPS typically arises from treatment of cells with subcytotoxic levels of agents that generate ROS, eg, UV-irradiation, peroxides.38,39
To date, 2 possible mechanisms have been proposed to account for the induction of SIPS by ROS: (1) oxidant-induced p38 phosphorylation,40
and (2) critical but sublethal DNA damage with transient activation of p53 and subsequent expression of p2141
; although the dependence of p21 expression on p53 has recently been disputed.42
We show that Ang II rapidly induces oxidative DNA damage in hVSMCs, associated with an increase in p53 and rapid onset of senescence. These observations demonstrate the capacity of Ang II to induce SIPS via a mechanism dependent on the associated DNA damage. Importantly, our data using hTERT-expressing VSMCs confirm that SIPS is independent of telomere attrition—as similarly observed in other cell types.43,44
Our data are consistent with the hypothesis that the fate of the hVSMCs exposed to Ang II is dependent on the magnitude of the resulting DNA damage. We propose that severe and lethal DNA damage would result in apoptosis, consistent with reports of Ang II–induced apoptosis in some cell types.45-48
Sublethal but critical levels of DNA damage would rapidly induce p53-dependent SIPS. Lower levels of DNA damage sustained over several population doublings would target and accelerate the gradual erosion of telomeres, promoting premature replicative senescence. It is likely that all 3 responses would occur in the same cell population, the individual cells’ response to Ang II depending on the biological age of the cell, their individual sensitivity to Ang II, and the efficiency of their oxidant defense and DNA repair mechanisms. Further work is required to evaluate this hypothesis.
The development of vascular cell senescence is increasingly recognized as an important mechanism in the pathogenesis of vascular disease.14,49
Senescent vascular cells are not benign and may undergo a phenotypic shift to a more inflammatory and atherogenic phenotype.8,50
Cells with a senescence phenotype have been identified in human and experimental animal arteries at sites prone to atherosclerosis.8,17-20
In rabbits, denuded arteries show evidence of extensive SA-β
-gal staining has been detected in diseased coronary arteries but not in healthier internal mammary arteries in humans.8
It has also been shown that the senescent cells persist in the vascular wall14
and so may inhibit natural repair by inhibiting recruitment of progenitor cells. The importance of vascular cell senescence in determining the function of an aging vascular system has stimulated interest in identifying the molecular mediators and pathways for its induction. Our data strongly support the hypothesis that Ang II may play a key role in the development of human vascular cell senescence.
Telomere length is also used as a marker of vascular cell aging in vivo. When senescence is observed in the vascular wall, those cells often show evidence of telomere attrition. For example, VSMCs with shortened telomeres have been identified in established atherosclerotic plaques51
and at sites of enhanced vascular wall stress in humans, ie, those sites predisposed to atheroma.52
Evidence for the clinical significance of shortened telomeres is now emerging albeit fuelled by literature using the leukocyte telomere as a surrogate marker for vascular wall aging.49
Cawthon and colleagues showed strong associations between reduced telomere DNA length in adults and premature mortality from cardiovascular disease.21
Furthermore, people with shorter telomeres have a 3-fold higher risk of premature MI22
and patients with coronary artery disease have shorter leukocyte telomeres compared with disease-free controls.22,53
Moreover, a recent report links shorter telomere DNA with CVD risk factors and subsequent risk of MI or stroke.54
The link between oxidative damage, telomeres, and vascular senescence has been further strengthened by studies detailing telomere-dependent senescence of VSMCs within human atherosclerotic plaques via a mechanism dependent on oxidative DNA damage and cell cycle arrest.9
Our data support and extend this concept by suggesting a pathway from oxidative DNA damage through to senescence, with the hypothesis that the magnitude of DNA damage determines whether cells enter senescence through a telomere-independent SIPS pathway, or a replication and telomere-dependent pathway.
Our data demonstrate Ang II–dependent induction of senescence in hVSMCs via the induction of ROS from NADPH oxidase and subsequent DNA damage. These findings are supported by a recent report suggesting that Ang II induced premature senescence in human aortic smooth muscle cells over a period of 3 days.30
These authors suggested that telomere attrition was not involved because telomere length did not change over 3 days in culture. Our current observations, using hTERT-expressing cells provide much more conclusive evidence that Ang II induces SIPS via a telomere-independent pathway which is ROS and DNA damage dependent. Both studies support the importance of the p53-p21 pathway in the development of Ang II–induced SIPS. Consistent with our findings, Ang II was recently reported to accelerate endothelial progenitor cell senescence via ROS generation and downregulation of telomerase.31
In contrast, our data suggest that Ang II–induced DNA damage, rather than its impaired repair, is the primary mechanism for accelerated telomere loss in adult human cells. This is not surprising mindful of the much greater telomerase activity in progenitor cells. Nevertheless, the senescence of both resident mature vascular cells and progenitor cells may play a role in the biological aging and associated pathology of vascular tissues.
Our hypothesis that Ang II may play a direct role in accelerating vascular aging processes is also supported by reports showing that treatment of experimental animals with drugs that inhibit the renin-angiotensin system, markedly increases survival and prevents many of the pathological changes associated with cardiovascular aging.55
Our studies extend these observations and provide the first direct evidence that Ang II acting via its AT1
receptor can induce DNA damage and senescence, either with or without accelerated telomere attrition, in adult hVSMCs.
Our study has some limitations. Firstly, our model uses human saphenous vein SMCs. Although ROS production by Ang II has been studied in both human and murine VSMCs taken from different vascular sites,1,2,56
ours is the first data on induction of DNA damage and senescence by Ang II using saphenous vein cells. However, it is likely that these data are applicable to other sources of VSMCs that respond to Ang II. Secondly, this study uses cultured cells in vitro to investigate mechanisms of senescence rather than a direct examination of senescence in vivo. However, the evidence cited above suggests that the mechanisms of senescence are likely to be similar in both settings. Finally, Dikalov et al57
recently critically reviewed the sensitivity and applicability of lucigenin chemiluminescence in the detection of ROS in vascular cells. Potential artifacts arising from lucigenin auto-oxidation were thought, on balance of the evidence in the literature, to be insignificant. Although this remains a possibility, in many cases lucigenin detection of ROS in vascular cells has been corroborated by alternate assays and our data are consistent with these findings.
In conclusion, our data show that Ang II causes DNA damage in hVSMCs via NADPH oxidase-derived ROS and that this results in SIPS or replicative senescence. These findings suggest novel mechanisms to directly implicate Ang II in the pathogenesis of human vascular cell aging and vascular disease.