Modulation of gene expression by senescence in human endothelial cells cultured from atherosclerotic patients

Nada Farhat, Nathalie Thorin-Trescases, Louis P. Perrault & Eric Thorin. Montreal Heart Institute, Faculty of Medicine, Department of Surgery, Montreal H1T 1C8, Quebec, Canada.

Endothelial cellular replicative senescence, i.e. the limited ability of primary cultured cells to divide, is associated with specific changes in gene expression (Wagner et al., 2001). It normally occurs in vivo during the ageing process, associated or not with cardiovascular risks factors (Minamino et al., 2002). To characterise the modulation of gene expression associated with senescence and atherosclerosis in endothelial cells (EC), we induced replicative senescence in cultured EC isolated from atherosclerotic patients.

ECs were isolated from segments of internal mammary artery (hIMA, n=5 to 23) isolated from patients undergoing coronary artery bypass. Aortic endothelial cells (hAoEC, purchased from Clonetics) isolated from young and healthy donors were used as control cells. Cells were serially passaged and maintained in culture until replicative senescence (growth arrest) was reached. Total RNA was isolated using a Qiagen kit at each passage. RNA was then reverse-transcribed into first-strand complementary DNA. Real-time polymerase chain reaction (PCR) was carried out using the DNA-binding dye SYBER Green I for the detection of PCR products. In order to quantify preproEndothelin-1 (ppET), ETB receptor, eNOS, Cox-1 and Cox-2 genes expression, the mRNA levels in each sample was calculated relative to GAPDH, which did not vary with senescence and between cell types. Data are expressed as the ratio of mRNA level of the gene considered to GAPDH.

Serial passage-induced replicative senescence resulted in a decrease (P<0.05) in mRNA expressions of ppET by 45% (n=6) and Cox-1 by 43% (n=5), no change in mRNA expression of eNOS (n=4) and increase (P<0.05) in mRNA expression of the ETB receptor by 54% (n=5) and Cox-2 by 267% (n=6). When compared to control hAoEC at an intermediate passage 4 (n=4), hIMA EC expressed similar levels of ppET mRNA (1.3±0.2 versus 2.1±0.9; P=0.06) but highly reduced levels of eNOS mRNA (9x10-5±5x10-5 versus 2.3±0.01; P<0.05) and lower levels of Cox-1 mRNA (0.03±0.01 versus 0.62±0.01; P<0.05). In contrast, hIMA EC expressed higher levels of Cox-2 mRNA (0.5±0.1 versus 0.20±0.01; P<0.05) and ETB receptor mRNA (1.8±0.4 versus 0.70±0.01; P<0.05).

In summary, replicative senescence is associated with a down-regulation of the expression of gene coding for enzymes involved in the production of NO and PGI2, two dilatory and cytoprotective endothelium-derived factors. In contrast, replicative senescence is associated with a rise in the expression of inflammatory markers such as the Cox-2. ET-1, known to regulate EC proliferation, is reduced while the expression of the mRNA for the ETB receptor increases possibly as an attempt to stimulate ET-1 production. In conclusion, replicative senescence of EC isolated from patients with coronary artery disease modulates the expression of genes in favours of a pro-atherogenic genotype.

Minamino T, et al. (2002). Circulation. 105 , 1541-1544.
Wagner M, et al. (2001). Exp. Gerontol. 36 , 1327-1347.