Toxicogenomic analysis of mainstream tobacco smoke-exposed mice reveals repression of plasminogen activator inhibitor-1 gene in heart

Abstract

Tobacco smoking is associated with cardiovascular pathology. However, the molecular mechanisms of tobacco smoke exposure that lead to initiation or exacerbation of cardiovascular disease are unclear. In this study, the effects of mainstream tobacco smoke (MTS) on global transcription in the heart were investigated. Male C57B1/CBA mice were exposed to MTS from 2 cigarettes daily, 5 days/wk for 6 or 12 wk. Mice were sacrificed immediately, or 6 wk following the last cigarette. High-density DNA microarrays were used to characterize global gene expression changes in whole heart. Fifteen genes were significantly differentially expressed following exposure to MTS. Among these genes, cytochrome P-450 1A1 (Cyp1A1) was upregulated by 12-fold, and Serpine-1 (plasminogen activator inhibitor-1, PAI-1) was downregulated by 1.7-fold. Concomitant increase in Cyp1A1 protein levels and decrease in total and active PAI-1 protein was observed in tissue extracts by Western blot assay and enzyme-linked immunosorbent assay (ELISA), respectively. Observed changes were transient and were partially reversed during break periods. Thus, gene expression profiling of heart tissue revealed a novel cardiovascular mechanism operating in response to MTS. Our results suggest a potential role for PAI-1 in MTS-induced cardiovascular pathology.

FIG. 1

Validation of microarray results. Data are presented as fold change over control values (n = 5 mice/group, ± SEM): 1, 6 wk smoke; 2, 6 wk smoke + 6 wk break; 3, 12 wk smoke; and 4, 12 wk smoke + 6 wk break. Asterisk indicates significant at p < .05, t-test conducted on RT-PCR values.

FIG. 2

Cyp1A1 protein abundance in heart microsome extract (n = 5). MW: molecular weight marker; +ve control: BaP-treated lung microsome extract. Lanes 1–4: treatment groups, 6 wk smoke, 12 wk smoke, 6 wk smoke + 6 wk break, and 12 wk smoke + 6 wk break; lanes 5–8: matched controls.

FIG. 3

(A) Total immunoreactive PAI-1 in heart tissue extracts. (B) PAI-1 activity in heart tissue extracts. Data represent fold change over matched controls (n = 5, ± SEM): 1, 6 wk smoke; 2, 6 wk smoke + 6 wk break; 3, 12 wk smoke; 4, 12 wk + smoke 6 wk break.

FIG. 4

(A) Western blot of immunoreactive tPA in heart tissue extracts. Lanes 1, 3, 5, and 7 represent 6 wk smoke, 6 wk smoke + 6 wk break, 12 wk smoke, and 12 wk smoke + 6 wk smoke. Lanes 2, 4, 6, and 8 represent matched controls. (B) Total tPA activity present in heart tissue extracts. Data shown as fold change over matched controls:1, 6 wk smoke; 2, 6 wk smoke + 6 wk break; 3, 12 wk smoke; 4, 12 wk smoke + 6 wk break.