Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells

Abstract

Endothelial dysfunction caused by the combined action of disturbed flow, inflammatory mediators and oxidants derived from cigarette smoke is known to promote coronary atherosclerosis and increase the likelihood of myocardial infarctions and strokes. Conversely, laminar flow protects against endothelial dysfunction, at least in the initial phases of atherogenesis. We studied the effects of TNFα and cigarette smoke extract on human coronary artery endothelial cells under oscillatory, normal laminar and elevated laminar shear stress for a period of 72 hours. We found, firstly, that laminar flow fails to overcome the inflammatory effects of TNFα under these conditions but that cigarette smoke induces an anti-oxidant response that appears to reduce endothelial inflammation. Elevated laminar flow, TNFα and cigarette smoke extract synergise to induce expression of the transcriptional regulator activating transcription factor 3 (ATF3), which we show by adenovirus driven overexpression, decreases inflammatory gene expression independently of activation of nuclear factor-κB. Our results illustrate the importance of studying endothelial dysfunction in vitro over prolonged periods. They also identify ATF3 as an important protective factor against endothelial dysfunction. Modulation of ATF3 expression may represent a novel approach to modulate proinflammatory gene expression and open new therapeutic avenues to treat proinflammatory diseases.

Figure 1. Effects of flow and cigarette smoke extract (CSE) on TNFα induced gene expression.

The mRNA expression of (A) VCAM1, (C) ICAM1, (D) E-selectin, (E) CCL2, (F) CX3CL1 and (G) IκBα, (n = 6 separate batches of HCAECs); or (B) protein expression of VCAM-1 (n = 3 separate batches of HCAECs). HCAEC were exposed to 5 ng/ml TNFα, 10% CSE or TNFα and CSE together (T + C) under OSS, LSS or ESS conditions, as illustrated schematically (H). Changes are expressed as mean fold change against LSS control ± S.E.M. *p < 0.05; **p < 0.01, ***p < 0.001 significant vs control for that shear stress, or other indicated comparison; ^#P < 0.05 vs OSS control

Figure 2. TNFα, but not CSE, suppress the expression of shear-responsive genes.

Changes in mRNA expression (n = 6 batches) of (A) KLF2, (B) KLF4, (C) THBD, (E) NOS3, (F) NOV or (D) THBD protein (n = 3 batches) were measured in HCAECs exposed to 5 ng/ml TNFα, 10% CSE or TNFα and CSE together (T + C) under OSS, LSS or ESS, expressed as mean fold change against control ± S.E.M. *p < 0.05; **p < 0.01, ***p < 0.001 significant vs control for that shear stress, or indicated comparison; ~~~P < 0.001 vs LSS and ESS (all conditions).

Figure 3. CSE increases the expression of Nrf2 responsive genes.

Changes in mRNA expression of (A) HMOX1, (C) SRXN1, (D) GCLM, (E) OSGIN1, (F) NQO1 (n = 6 batches) or (B) HMOX1 protein (n = 3 batches) in HCAECs exposed to 5 ng/ml TNFα, 10% CSE or TNFα and CSE together (T + C) under OSS, LSS or ESS conditions. Changes are expressed as mean fold change against LSS control ± S.E.M. *p < 0.05; **p < 0.01, ***p < 0.001 significant vs control for that shear stress, or indicated comparison; ^#P < 0.05 vs OSS control.

Figure 4. Shear stress, TNFα and CSE all increase ATF3 expression.

Changes in (A) ATF3 mRNA expression in HCAECs exposed to 5 ng/ml TNFα, 10% CSE or TNFα and CSE together (T + C) under OSS, LSS or ESS, expressed as mean fold change against control ± S.E.M. *p < 0.05; **p < 0.01, ***p < 0.001 significant vs control for that shear stress, or indicated comparison; ^#P < 0.05 vs OSS control; n = 6 batches. (B) Regulation of ATF3 mRNA expression in HCAECs following transduction with adenoviral vector overexpressing Nrf2, treatment with 5 ng/ml TNFα, or the combination in static culture. **P < 0.01 vs Untreated Control; ⁺⁺⁺P < 0.001 vs β-gal adenoviral vector control.

Figure 5. ATF3 overexpression reduces VCAM1 upregulation.

(A) Adenoviral-mediated ATF3 overexpression reduced TNFα-stimulated VCAM1 upregulation in HCAEC. (B) In NFκB reporter endothelial cell line NF-EA.hy926, ATF3 overexpression reduced TNFα-stimulated VCAM1 mRNA upregulation; however did not inhibit NFκB reporter activity (C), measured by changes in luciferase mRNA expression. *P < 0.05 vs untreated control; ^†††P < 0.001 vs no virus + TNFα or β-gal viral control + TNFα; n = 3.