Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors

Abstract

Risk factors for cardiovascular diseases (CVD) increase oxidative stress, and they are proposed to hasten endothelial cell (EC) damage and dysfunction. Our objective was to elucidate the impact of chronic exposure to risk factors for CVD on senescence of EC isolated and cultured from internal mammary arterial segments of patients with severe coronary artery disease. Senescence induced by serial passages resulted in progressive telomere shortening, and short initial telomeres predicted early appearance of senescence in culture. Neither time course of senescence nor telomere length was age-dependent, suggesting that biological age, rather than chronological age, determined the dynamics. Senescence appeared earlier in patients with longer history of risk factor for CVD, and multivariate analysis suggested that hypertension hastened the onset of senescence. Risk factors for CVD override the effects of chronological aging likely by generating stress-dependent damage: senescent EC exhibited oxidative stress (increase in lipid peroxydation and caveolin-1 gene expression) and cell damage markers (loss of eNOS expression and increase in Cox2 mRNA, lower TRF1 protein level). Thus, cell senescence was triggered both by telomere-dependent and -independent pathways. In conclusion, chronic exposure to risk factors for CVD accelerated the development of endothelial senescence that could contribute to the pathogenesis of CVD.

Introduction

Vascular aging is associated with a decrease in endothelial dilatory and antithrombotic functions (Cooper et al., 1994, Lakatta, 2002, Donato et al., 2007). This typical endothelial dysfunction, however, is also present in younger patients with cardiovascular diseases (CVD) (Cohen, 1995). At the cellular level, aging of healthy vascular endothelial cells (EC) leads to senescence, a state of permanent growth arrest in which cells remain alive and metabolically active for months but refractory to mitogenic stimuli (Ben-Porath and Weinberg, 2005, Chen and Goligorsky, 2006). Senescence can be triggered by cell divisions, leading to cumulative telomere attrition down to a threshold length at which cells enter the so-called replicative senescence (Allsopp et al., 1995). In healthy humans, telomere shortening is age-dependent (Slagboom et al., 1994, Chang and Harley, 1995, Brouilette et al., 2007) and likely the consequence of life-long reparative cell divisions. Telomere length has been proposed to contribute and to be a predictor of mortality in many aged-related diseases (Cawthon et al., 2003). Cellular senescence can occur prematurely, independent of replicative age, following exposure to multiple types of stress (stress-induced senescence), such as oxidative stress (Toussaint et al., 2000), DNA damage and mitogenic stress (Ben-Porath and Weinberg, 2005).

Senescence is characterized by specific changes in cell morphology and gene expression, which reduce EC function (Benetos et al., 2001, Wagner et al., 2001) and thus are proposed to be pro-atherogenic (Cohen, 1995, Serrano and Andres, 2004, Edo and Andres, 2005). Although mitotically inactive, senescent cells are not physiologically inert: they secrete degradative enzymes, inflammatory cytokines and growth factors (Chen and Goligorsky, 2006) and this could promote or contribute to the pathogenesis of human atherosclerosis (Minamino and Komuro, 2007). While the concept of senescence of EC is established, its origin is debated. Life-long cell repairs likely promotes instability and one consequence is an imbalance in the regulation of the redox environment towards pro-oxidation, a process accelerated by risk factors for CVD (Csiszar et al., 2002). The ensuing oxidative stress induces cellular damage (Lorenz et al., 2001, von Zglinicki et al., 2005) and further accelerates reparative cell division, promoting telomere instability (Kurz et al., 2004). Excessive telomere shortening in circulating white blood cells has been reported from patients with hypertension (Jeanclos et al., 2000), coronary atherosclerosis (Samani et al., 2001, Fitzpatrick et al., 2007, Brouilette et al., 2007), premature myocardial infarction (Brouilette et al., 2003), heart disease (Starr et al., 2007) and diabetes (Jeanclos et al., 1998). There is therefore a strong rationale for the hypothesis that risk factors for CVD accelerate the normal aging process as recently proposed by Chen and Goligorsky (2006). It is not known, however, if the association of short telomere length with the risk of coronary heart disease applies to other cell types, especially vascular tissue. The telomere-independent caveolin/p53 pathway has been reported to be activated by oxidative stress and lead to senescence (Galbiati et al., 2001, Volonte et al., 2002). In replicative senescence, however, telomere attrition following activation of ATM/p53-p21 pathway has been observed (Pandita, 2002, Herbig et al., 2004, Herbig and Sedivy, 2006).

The objective of the present study was therefore to elucidate the impact of chronic exposure to risk factors for CVD on the senescence of EC isolated and cultured from patients with severe coronary artery disease (CAD) and to find the pathway of choice leading to senescence. We demonstrate that the duration of exposure to risk factors for CVD, especially hypertension, positively correlates with the propensity of EC senescence, independent of the age of the donor. Thus, stress associated cell damages accelerated replicative senescence.