Maternal Engineered Nanomaterial Inhalation During Gestation Disrupts Vascular Kisspeptin Reactivity

Abstract

Maternal engineered nanomaterial (ENM) inhalation is associated with uterine vascular impairments and endocrine disruption that may lead to altered gestational outcomes. We have shown that nano-titanium dioxide (nano-TiO2) inhalation impairs endothelium-dependent uterine arteriolar dilation in pregnant rats. However, the mechanism underlying this dysfunction is unknown. Due to its role as a potent vasoconstrictor and essential reproductive hormone, we examined how kisspeptin is involved in nano-TiO2-induced vascular dysfunction and placental efficiency. Pregnant Sprague Dawley rats were exposed (gestational day [GD] 10) to nano-TiO2 aerosols (cumulative dose = 525 ± 16 μg; n = 8) or sham exposed (n = 6) and sacrificed on GD 20. Plasma was collected to evaluate estrogen (E2), progesterone (P4), prolactin (PRL), corticosterone (CORT), and kisspeptin. Pup and placental weights were measured to calculate placental efficiency (grams fetus/gram placental). Additionally, pressure myography was used to determine uterine artery vascular reactivity. Contractile responses were assessed via cumulative additions of kisspeptin (1 × 10-9 to 1 × 10-4 M). Estrogen was decreased at GD 20 in exposed (11.08 ± 3 pg/ml) versus sham-control rats (66.97 ± 3 pg/ml), whereas there were no differences in P4, PRL, CORT, or kisspeptin. Placental weights were increased in exposed (0.99 ± 0.03 g) versus sham-control rats (0.70 ± 0.04 g), whereas pup weights (4.01 ± 0.47 g vs 4.15 ± 0.15 g) and placental efficiency (4.5 ± 0.2 vs 6.4 ± 0.5) were decreased in exposed rats. Maternal ENM inhalation exposure augmented uterine artery vasoconstrictor responses to kisspeptin (91.2%±2.0 vs 98.6%±0.10). These studies represent initial evidence that pulmonary maternal ENM exposure perturbs the normal gestational endocrine vascular axis via a kisspeptin-dependent mechanism, and decreased placental, which may adversely affect health outcomes.

Keywords: engineered nanomaterials; kisspeptin; microcirculation; placenta; titanium dioxide.

Figure 1.

Characterization of nano-TiO₂. A, Real-time mass concentration measurements of the nano-TiO₂ aerosol during a typical inhalation exposure. The red line represents the target concentration, ∼12 mg/m³. B, Size distribution of the nano-TiO₂ aerosol (aerodynamic diameter) using a high-resolution electrical low-pressure impactor (ELPI+). The red line represents a log-normal fit of the histogram (count median diameter = 188 ± 0.36 nm). C, Size distribution of the nano-TiO₂ aerosol (mobility diameter) sampled from the exposure chamber using a scanning mobility particle sizer (SMPS—light gray) and an aerodynamic particle sizer (APS—dark gray, negligible values). The red line is representative of a log-normal fit of the histogram (count median diameter = 190 nm). D, Transmission electron microscope (TEM) image of a typical nano-TiO₂ agglomerate.

Figure 2.

Kisspeptin induces constriction in uterine arteries exposed to nano-TiO₂. Kiss-dependent reactivity of uterine arteries from control and exposed animals was determined using pressure myography (N = 7–8). Statistics were analyzed with 1-way ANOVA (p ≤ .05). *Sham-control group versus nano-TiO₂-exposed groups.

Figure 3.

Plasma hormone analysis of steroid hormones at GD 20. ELISAs were performed on plasma from sham-control (N = 6) or nano-TiO₂ inhalation-exposed (N = 6) dams for (A) estrogen (E2), and (B) corticosterone (CORT), and (C) progesterone (P4). Statistics were analyzed with 1-way ANOVA (p ≤ .05). *Sham-control group versus nano-TiO₂-exposed groups.

Figure 4.

Plasma hormone analysis of peptide hormones at GD 20. ELISAs were performed on plasma from sham-control (N = 6) or nano-TiO₂ inhalation-exposed (N = 6) dams for (A) kisspeptin (E2), and (B) follicle-stimulating hormone (FSH), (C) luteinizing hormone (LH), and (D) prolactin (PRL). Statistics were analyzed with 1-way ANOVA.

Figure 5.

Kiss1R localization within the uterine and placental vasculature. Kiss1R (green) colocalization with SMA (red) in the mesometrial triangle (A), and within the placental vasculature (blood vessels indicated with BV) (B). Scale bar represents 100 µm for both panels; white arrow indicates positive staining for SMA, green arrow indicates positive staining for Kiss1R, and the yellow arrow indicates colocalization of Kiss1R and SMA.

Figure 6.

Kiss localization in the uterine and placental vasculature. Kiss (green) colocalization with α-smooth muscle actin (SMA, red) in placental microvessel (as seen in yellow), nuclear DAPI staining in blue in a whole placental slice (A), and magnification of an arteriole showing colocalization with SMA and Kiss (B; white arrow indicates positive staining for SMA, green arrow indicates positive staining for Kiss, and the yellow arrow indicates colocalization of Kiss and SMA). Kiss (green) trophoblast cells surrounding microvessels in the placenta indicated by SMA (red), however there is no colocalization between Kiss and SMA (C, D). Scale bar represents 1mm for panel A and 100 μm for panels B, C, D.

Figure 7.

Proposed mechanism of action of kisspeptin within the uteroplacental unit in the rat during gestation. Kiss, which is synthesized by the trophoblast cells of the placenta, bind to Kiss1R in the arteries within the uterine wall. Exposure to ENM during gestation alters uterine vascular reactivity to Kiss, and potentially decreases blood flow to the placental units to decrease pup weight and increase placental weight.