Sustained Maternal Smoking Triggers Endothelial-Mediated Oxidative Stress in the Umbilical Cord Vessels, Resulting in Vascular Dysfunction

Abstract

Nitric oxide (NO) bioavailability is fundamental in the regulation of redox balance and functionality of the endothelium, especially in the case of the umbilical cord (UC), which has no innervation. The analysis of UC vessel-related complications could serve as a useful tool in the understanding of the pathophysiological mechanisms leading to neonatal cardiovascular disorders. Therefore, the aim of this study was to characterize the mechanisms that rule the severity of prenatal endothelial dysfunction, induced by the long-term effect of maternal smoking. Our analysis describes the initiation and the consequences of endothelial nitric oxide synthase (NOS3) deactivation, along with the up-regulation of possible compensatory pathways, using structural, molecular and biochemical approaches. This study was carried out on both the UC arteries and veins originated from neonates born to non-smoking and heavy-smoking mothers. The alterations stimulated by maternal smoking are vessel-specific and proportional to the level of exposure to harmful materials passed through the placenta. Typically, in the primarily exposed veins, an increased formation of reactive oxygen species and an up-regulation of the highly-efficient NOS2-NO producing pathway were detected. Despite all the extensive structural and functional damages, the ex vivo heat and cadmium ion-treated UC vein pieces still support the potential for stress response.

Keywords: arginase; endothelial dysfunction; maternal smoking; metal exposure; nitric oxide synthase; xanthine oxidoreductase.

Figure 1

Morphological changes, viability and reactivity in Ctrl and Sm umbilical cord (UC) vessels. On the representative electron micrographs focusing on the endothelial layer, significant changes can be observed compared to the Ctrl (a) endothelial cells (white asterisks) with the Sm artery (b) and Sm vein (c) endothelial cells (black arrows). In addition to losing their intercellular contacts, cells also show damaged/dying phenotypic features in the cytoplasm and nuclei (N). The most remarkable is the nuclear fragmentation and the occupancy of intra- and intercellular vacuoles (red asterisks) at the expense of the cytoplasm. Scale bars: 2 µm (a); 2 µm (b); 5 µm (c). (Lu = vessel lumen). (d) Graphical presentation of the viability assay data set on isolated Ctrl- and Sm-derived human umbilical vein endothelial cell (HUVEC) populations. Flow-cytometer-quantified data are ordered on the interleaved bar graph, where the percentage distribution of healthy, early and late apoptotic cells are expressed within the whole isolated Ctrl and Sm populations, and shown as mean ± SD. Total cell numbers were the following: Ctrl (n = 237500 ); Sm (n = 129600 ), from three and two individual samples, respectively. Statistics: grouped analysis with two-way ANOVA, followed by Holm–Sidak’s multiple comparisons test. * p ≤ 0.05, *** p ≤ 0.001 and **** p ≤ 0.0001. (e) Bar graph showing the changes in HSP90 mRNA expression, upon heat and combined (heat + Cd²⁺) treatment in Ctrl- and Sm-derived UC veins. Ctrl (n = 2) and Sm (n = 2) samples tested in triplicates. Statistics: grouped analysis with two-way ANOVA, followed by Holm–Sidak’s multiple comparisons test. ns. = non – significant, * p ≤ 0.05, and ** p ≤ 0.01.

Figure 2

Changes in oxidative stress markers among Sm-derived UC vessels. Bar graphs showing the semiquantified results of 4-HNE, 3-NT specific immunolabeling and the intercalated redox indicator dihydroethidium staining (DHE) levels in Sm arteries (a) and in Sm veins (b). Red dots symbolize the overall mean fluorescence intensity (MFI) values from individual Sm samples, while green dots refer to summarized data from the Ctrl group, applied as the baseline of comparison (mean ± SEM). Sample numbers: CtrloxDHE (n = 304 artery and 929 vein regions of interest (ROI) from 12 individual samples); SmoxDHE (n = 961 artery and 1229 vein ROI from 12 individual samples), Ctrl4-HNE (n = 233 artery and 589 vein ROI from 11 individual samples); Sm4-HNE (n = 232 artery and 448 vein ROI from 9 individual samples); Ctrl3-NT (n = 1033 artery and 44 vein ROI from 11 individual samples); Sm3-NT (n = 277 artery and 39 vein ROI from 9 individual samples). Statistics: unpaired t-test followed by Mann–Whitney test to compare ranks * p ≤ 0.05, **** p ≤ 0.0001.

Figure 3

Changes of NO synthesis determining factors in Sm-derived UC vessels. Bar graphs showing the semiquantified results of NOS3-, P-NOS3-, ARG1- and NOS2-specific immunolabeling in Sm arteries (a,c) and in Sm veins (b,d). Red dots symbolize the overall MFI values from individual Sm samples, while green dots refer to the summarized data from the Ctrl group, applied as the baseline of comparison (mean ± SEM). Sample numbers: CtrlNOS3 (n = 95 artery and 112 vein ROI from 12 individual samples); SmNOS3 (n = 53 artery and 102 vein ROI from 11 individual samples), CtrlARG1 (n = 77 artery and 781 vein ROI from 10 individual samples); SmARG1 (n = 64 artery and 732 vein ROI from 9 individual samples); CtrlNOS2 (n = 211 artery and 130 vein ROI from 11 individual samples); SmNOS2 (n = 100 artery and 243 vein ROI from 9 individual samples). Statistics: unpaired t-test followed by Mann–Whitney test to compare ranks ns. = non-significant, **** p ≤ 0.0001.

Figure 4

Changes in NOS-independent NO production in Sm-derived UC vessels. Bar graphs show the semiquantified results of xanthine oxidoreductase (XOR)-specific immunolabeling and the tNOx concentrations determined by a Griess microassay in Sm arteries (a) and in Sm veins (b). Red dots symbolize the overall MFI or tNOx concentration values from individual Sm samples, while green dots refer to the summarized data from the Ctrl group, applied as the baseline of comparison (mean ± SEM). Sample numbers: CtrlXOR (n = 44 artery and 83 vein ROI from 8 individual samples); SmXOR (n = 33 artery and 19 vein ROI from 8 individual samples). In case of CtrltNOx (n = 8 individual samples) and SmtNOx (n = 8 individual samples) measurements carried out in triplicates. Statistics: unpaired t-test followed by Mann–Whitney test to compare ranks, ** p ≤ 0.01, **** p ≤ 0.0001.