Mechanisms of whole smoke conditioned media induced cytotoxicity to human aortic endothelial cells. (August 2019)

• Record Type: Journal Article
• Authors: Makwana, Om; Flockton, Hannah; Smith, Gina A.; Watters, Gary P.; Nisar, Rizwan; Fields, Wanda
• Is Part Of: Toxicology in vitro. Volume 58(2019)
• Page Start: 239
• Page End: 244
• Publication Date: 2019-08
• Subjects: Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2019.03.011
• Languages: English
• ISSNs: 0887-2333

Abstract

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. Highlights: Whole smoke conditioned media is cytotoxic to primary Human Aortic Endothelial Cells at doses exceeding 4000 ng/mL nicotine equivalence. Whole smoke conditioned media cytotoxicity was conserved in four cell donors in the NRU and MTT in vitro assays Mechanism of whole smoke conditioned media cytotoxicity appeared dose-dependent in Human Aortic Endothelial Cells mediated primarily by apoptosis. Human Aortic Endothelial Cells may be a useful primary cell model in determining the cytotoxicity of cigarette smoke exposure.