It is well known that exposure to smoking during pregnancy has been associated with cardiovascular disease (CVD) in the offspring. It is important to note that direct exposure to smoking is not the only means of tobacco exposure during pregnancy. Indeed, there is a new form, termed thirdhand smoke (THS) that has been gaining attention as of late. THS refers to the residual tobacco smoke contamination that remains after a cigarette is extinguished, and which persists for months. This leads to the accumulation of chemicals over time, which undergo chemical reactions and aging, thereby becoming more toxic. To date, the negative effects of in utero THS on CVD are understudied. Since platelets are a major player in the genesis of CVD, we employed a transcriptomic (mRNA and miRNA), in vitro and in vivo-based approaches to investigate the effects of in utero THS exposure on platelet gene expression and function, as well as the risk of developing thrombosis in the adult offspring. Thus, C57 female mice were exposed to THS or clean air (control) one week before mating and throughout gestation, using a validated exposure protocol. The exposure was stopped at birth, and the offspring males were used for experimentation once they have reached at 8 - 10 weeks of age. RNA-Seq next generation sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of the in utero THS exposed mice. Pathway enrichment analysis revealed differential gene-expression changes in pathways associated with metabolism, oxidative stress, and platelet activation. Moreover, using miRNA-seq, we found 35 differentially expressed miRNAs in the in utero THS exposed platelets compared with controls. The miRNA-mRNA integrated interactions analysis showed 107 validated interactions including interesting interaction between downregulated micorRNAs (miR-101a-3p, miR-144-3p, miR-16-1-3p and miR-542-5p) and upregulated Cxcl12 protein coding gene, a known platelet activation cytokine. We next sough to confirm that changes in the platelet gene expression in the in utero THS exposed mice are accompanied by changes in platelet function. To this end, separate experiments showed that activation of Akt and ERK, key biochemical markers of platelet activation, is enhanced in the in utero exposed mice, which was accompanied by an enhanced clot retraction response. Consistent with these findings, our in vivo studies showed that in utero THS exposed mice have significantly shortened (104 ± 10.21[sec]) survival in the pulmonary thromboembolism model in comparison to the clean air control (179 ± 20.6 [sec]). Collectively, our results show altered platelet gene expression, enhanced platelet function, and increased risk of thrombosis in mice that were subjected to the in utero THS exposure, relative to the control. These findings support the notion that in utero THS exposure is detrimental to human health and should increase public awareness and inform policy for managing exposure to this type of smoke.
This research was supported, in part, by the NIEHS and the NHLBI, of the National Institutes of Health under Awards Number R21ES029345 and R01HL145053.
Figure 1: A) Heat map of significantly differentially expressed platelet transcripts from in utero THS and healthy clean air . Red indicates increased relative expression, and blue indicates decreased relative expression. B) Volcano plot with significantly increased and decreased transcripts. C) Manhattan plot pathway enrichment analysis of differentially expressed. genes.; Figure 2: Chord plot depicting the miRNA and mRNA interaction. Thickness of the connection is corresponding to the number of involved miRNA.