In Utero Thirdhand Smoke Modulates Platelet Gene Expression in Exposed Mice

Abstract

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 uteroTHS 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 uteroTHS 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 uteroTHS 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 uteroexposed mice, which was accompanied by an enhanced clot retraction response. Consistent with these findings, our in vivo studies showed that in uteroTHS 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 uteroTHS exposure is detrimental to human health and should increase public awareness and inform policy for managing exposure to this type of smoke.