Mechanisms of whole smoke conditioned media induced cytotoxicity to human aortic endothelial cells

Abstract

Chronic exposure to cigarette smoke can lead to endothelial dysfunction and potentially endothelial cell death. Here, we exposed Human Aortic Endothelial Cells (HAECs) to whole smoke conditioned media (WSCM) over a range of nicotine equivalence (n.e.) concentrations (0-8000 ng/mL n.e.). After 24 h, Neutral Red Uptake (NRU) and reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan was determined for each exposure concentration and compared to control. IC₅₀ values in the NRU assay were: 4582 ng/mL n.e. ± 1074, 4587 ng/mL n.e. ± 951, 4993 ng/mL n.e. ± 1239 and 4691 ng/mL n.e. ± 402 for four HAEC donors. IC₅₀ values in the MTT assay were: 4885 ng/mL n.e. ± 1341, 4584 ng/mL n.e. ± 806, 5749 ng/mL n.e. ± 783 and 5228 ng/mL n.e. ± 593 for the four donors. To examine the mechanism responsible for WSCM-induced cytotoxicity in HAECs, flow cytometry using necrosis (Propidium Iodide) and apoptosis (Annexin V) markers were used. Annexin V-positive cell populations increased in a dose dependent manner while increases in PI-positive cell populations occurred at the highest doses of WSCM (5000-8000 ng/mL n.e.). Western blotting for cleaved caspase-3 confirmed that apoptosis occurs at >5000 ng/mL n.e. WSCM, coinciding with reduced HAEC survival.