7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells

Abstract

Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 µg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.

Keywords: Gerotarget; apoptosis; atherosclerosis; cytotoxicity; endothelial cells; inflammation.

Figure 1. Cytotoxicity of 7-KC (10-40 μg/ml) to endothelial cells after 3 days of exposure

A. Morphologic changes of EAHY endothelial cells after exposure to different concentrations of 7-KC, B. Cyotoxicity of 7-KC to endothelial cells as analyzed by MTT. Results were expressed as Mean ± SE (n = 6). *denotes statistically significant difference (p < 0.05) when compared with solvent control.

Figure 2. Effect of 7-KC (10-50 μg/ml) on cell cycle progression and apoptosis of endothelial cells

A. Effect of 7-KC on cell cycle distribution of endothelial cells as analyzed by Modifit Software, B. Effect of 7-KC on sub-G0/G1 population of endothelial cells was analyzed by Cell Quest program. Results were expressed as Mean ± SE (n = 3).

Figure 3. Effect of 7-KC (5-40 μg/ml) on apoptosis of endothelial cells as analyzed by PI and annexin V dual fluorescent flow cytometry

A. One representative flow cytometry picture was shown. LL (lower left): viable cells, UL (upper left): necrotic cells, LR (lower right): pro-apoptotic cells, UR (upper right): apoptotic cells, B. Quantitative analysis of PI + annexin V flow cytometric analysis. Results were expressed as Mean ± SE (n = 3).

Figure 4. Effect of 24-h exposure to 7-KC on cell cycle-related Cdk1 and cyclin B1 mRNA and protein expression of endothelial cells

A. mRNA expression of Cdk1 and cyclin B1 as analyzed by PCR. Beta-actin expression was used as control. MW (molecular weight - base pairs [bp]) B. Cdk1 and cyclin B1 protein expression as analyzed by western blotting. MW (molecular weight, KD), Expression of beta-actin and GAPDH was used as control for PCR and western blot, respectively. One representative RT-PCR and western blotting result was shown.

Figure 5. Stimulation of p-ATM, p-ATR, p-Chk1, p-Chk2 and p-p53 expression by 7-KC (20 μg/ml) to endothelial cells

EAHY endothelial cells were exposed to solvent control and 20 μg/ml of 7-KC for 24 hours. Immunofluorescent (IF) microscopic observation was done to evaluate the expression of A. p-ATM, B. p-ATR, C. p-Chk1, D. p-Chk2 and E. p-p53 in endothelial cells. One representative IF picture was shown. (blue - DAPI, red or green - target proteins, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p53)

Figure 6. Effect of 7-KC on IL-8 expression and production

EAHY endothelial cells were exposed to 7-KC. A. Total RNA was isolated and subjected to RT-PCR analysis of IL-8 expression, B. Culture medium was collected and used for measurement of IL-8 secretion in endothelial cells. Results were expressed as Mean ± SE (n = 5).

Figure 7. Effect of 7-KC on cellular ROS levels and Akt activation

A. 7-KC (10-40 μg/ml) induced ROS production as shown by increase in cellular DCF fluorescence. Results were expressed as Mean ± SE (% of control, n = 3), B. 7-KC stimulate Akt phosphorylation as shown by increase in cellular p-Akt fluorescence (blue - DAPI, red - p-Akt). One representative immunofluorescent study was shown. C. LY294002 attenuated the 7-KC-induced apoptosis of endothelial cells as analyzed by PI+Annexin V dual staining flow cytometry. Results were expressed as percentage (% of control, n = 6) of cell residing in UL, LL, UR and LR area of flow cytometry chart. D. LY294002 prevented the 7-KC-induced IL-8 mRNA expression of endothelial cells.