Effects of maternal smoking on inflammation, autophagy/mitophagy, and miRNAs in endothelial cells: Influence of newborn sex

Maternal smoking (MS) during pregnancy is linked to well-documented adverse health effects for the mother and foetus, however the role of fetal sex was largely overlooked. Primary cultures of male and female human umbilical vein endothelial cells (MHUVECs and FHUVECs, respectively) were used. IL-6, IL-8, and TNF-alpha levels were measured in HUVECs supernatant. The expression of genes and proteins (oestrogen receptors (ERs), Hsp90, Beclin-1, p62, LC3, LAMP-1 and Parkin), as well as the expression of miR-29a-3p, miR-29b-3p, miR-126-3p, miR-133a-3p, and miR-146a-5p were analysed in cells obtained from foetuses born to non-smoking and smoking mothers. In HUVECs from foetuses born to non-smoking mothers, Beclin-1 protein was higher in MHUVECs (1.8 fold increase), while Parkin, Hsp90 proteins, and miR-146a-5p were elevated in FHUVECs (2.2, 2.6, and 2.1 fold increase, respectively), with no other significant differences. MS amplified these sex differences, with specific effects based on foetus sex. FHUVECs obtained from foetus born to smoking mothers showed higher levels of IL-8 (1399.36 +/- 123.96 pg/ml for FHUVECs vs 655.11 +/- 215.94; pg/ml for MHUVECs; P < 0.001), Hsp90 gene and protein (3.3 and 2.6 fold increase), and ER beta protein and Beclin-1 gene (2.1, and 4.9 fold increase), and lower levels of miR-29b-3p, miR-133a-3p, and miR-146a-5p than MHUVECs (0.27, 0.68, and 0.1 fold change, respectively). This study shows that primary HUVECs from fetuses born to smoking mothers retain a memory of smoking effects, with sex differences in gene expression, miRNA profiles, and autophagic responses, suggesting that maternal smoking impacts endothelial cell physiology in a sex-dependent manner.