| Objective Part 1:To explore the change of circulating endothelial cells (CECs) in active and passive smokers, and to observe the effects of inflammation on microvascular injuries. Part 2:To explore the molecular mechanisms of cigarette smoke on microvascular injuries via treating endothelial cells with cigarette smoke extract (CSE) in vitro.Methods Part 1:The number of CECs were evaluated in 24 healthy couples, among whom 12 were composed of active smoking husbands and never smoking wives, who were recognized as passive smokers. The other 12 non-smoking couples were recruited as controls. CECs were quantified by flow cytometry as CD45lowCD133-CD146+. Plasma levels of tumour necrosis factorα(TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), von Willebrand factor (vWF), soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble thrombomodulin (sTM) were determined by ELISA. Part 2:HUVECs were treated with different concentrations of CSE for cell proliferation assay (MTT assay). The permeability of HUVEC monolayers was measured using Transwell system. To determine the migration and angiogenesis of HUVECs, the "scratch assay" and capillary-like tube formation assay were employed. Immunofluorescence studies of occludin, claudin-5 and ZO-1 were used to observe endothelial barrier. The expression of occludin, claudin-5, ZO-1 and NF-κB p65 were detected by western blot.Results Part 1:The number of CECs was significantly higher in male active smokers than in male non-smoking controls (365-3528 cells/mL, median 1078 cells/mL versus 143-1827 cells/mL, median 536 cells/mL, p<0.05), and significantly higher in female passive smokers than in female non-smoking controls (261-3673 cells/mL, median 1597 cells/mL versus 69-1834 cells/mL, median 455 cells/mL, p<0.01). Plasma levels of TNF-α, IL-6, CRP, vWF, sE-selectin and sTM were significantly higher in male active smokers compared with male controls (p<0.05 or p<0.01) and significantly higher in female passive smokers than in female controls (p<0.05 or p<0.01). Plasma level of sICAM-1 was significantly higher in male active smokers compared with male controls (p<0.05). Part 2:No toxic effect of 10% CSE to HUVECs was found since viability was consistently determined to be>85%. CSE significantly increased endothelial permeability compared with untreated cells. Pretreatment of HUVEC monolayer with 15d-PGJ2 (10μM) could significantly inhibit CSE-induced permeability increase. CSE significantly inhibited the migration of HUVECs. Cell number of wound closure in one visual field decreased from 629±28 in control to 364±17 in CSE group (P< 0.01). 15d-PGJ2 treatment increased the cell number of wound closure from 364±17 in CSE group to 546±20 in 15d-PGJ2+CSE group (P<0.01). CSE inhibited the angiogenesis of HUVECs.15d-PGJ2 could attenuate CSE-induced inhibition of angiogenesis on HUVECs. Immunofluorescence and Western blot analysis showed that 15d-PGJ2 attenuated CSE-mediated down-regulation of endothelial tight junction proteins occludin, claudin-5, and ZO-1.15d-PGJ2 reduced CSE-induced NF-κB activation.Conclusion Part 1:Active and passive smoking are associated with a marked increase in CECs as well as increase in markers of inflammation-induced endothelial damage. The low-grade systemic inflammation present in the active and passive smokers is likely to be the most important factor behind the increase of CECs. Part 2:15d-PGJ2 exerts anti-inflammatory effects that attenuate CSE-mediated endothelial dysfunction through the NF-κB signal transduction pathway.
|
PDF Full Text Request