In utero exposure to ultrafine particles promotes placental stress-induced programming of renin-angiotensin system-related elements in the offspring results in altered blood pressure in adult mice

Abstract

Background: Exposure to particulate matter (PM) is associated with an adverse intrauterine environment, which can promote adult cardiovascular disease (CVD) risk. Ultrafine particles (UFP) (small size and large surface area/mass ratio) are systemically distributed, induce inflammation and oxidative stress, and have been associated with vascular endothelial dysfunction and arterial vasoconstriction, increasing hypertension risk. Placental stress and alterations in methylation of promoter regions of renin-angiotensin system (RAS)-related elements could be involved in UFP exposure-related programming of hypertension. We investigated whether in utero UFP exposure promotes placental stress by inflammation and oxidative stress, alterations in hydroxysteroid dehydrogenase 11b-type 2 (HSD11B2) and programming of RAS-related elements, and result in altered blood pressure in adult offspring. UFP were collected from ambient air using an aerosol concentrator and physicochemically characterized. Pregnant C57BL/6J p^un/p^un female mice were exposed to collected UFP (400 μg/kg accumulated dose) by intratracheal instillation and compared to control (nonexposed) and sterile H₂O (vehicle) exposed mice. Embryo reabsorption and placental stress by measurement of the uterus, placental and fetal weights, dam serum and fetal cortisol, placental HSD11B2 DNA methylation and protein levels, were evaluated. Polycyclic aromatic hydrocarbon (PAH) biotransformation (CYP1A1 and NQO1 (NAD(P)H dehydrogenase (quinone)1)) enzymes, inflammation and oxidative stress in placentas and fetuses were measured. Postnatal day (PND) 50 in male offspring blood pressure was measured. Methylation and protein expression of (RAS)-related elements, angiotensin II receptor type 1 (AT₁R) and angiotensin I-converting enzyme (ACE) in fetuses and lungs of PND 50 male offspring were also assessed.

Results: In utero UFP exposure induced placental stress as indicated by an increase in embryo reabsorption, decreases in the uterus, placental, and fetal weights, and HSD11B2 hypermethylation and protein downregulation. In utero UFP exposure induced increases in the PAH-biotransforming enzymes, intrauterine oxidative damage and inflammation and stimulated programming and activation of AT₁R and ACE, which resulted in increased blood pressure in the PND 50 male offspring.

Conclusions: In utero UFP exposure promotes placental stress through inflammation and oxidative stress, and programs RAS-related elements that result in altered blood pressure in the offspring. Exposure to UFP during fetal development could influence susceptibility to CVD in adulthood.

Keywords: Hypertension; Placental stress; Programming disease; Ultrafine particles.

Fig. 1

Experimental design of animal UFP exposure and sample collection. GD, Gestation day (pregnancy day); PND, post natal day (days after birth). Time mated mice were exposed by intratracheal instillation to H₂O and UFP. The instilled dose was 12 μg/dam was distributed over six instillations on GD 6.5, 8.5,10.5, 12.5,14.5 and 16.5

Fig. 2

Physicochemical characterization of UFP. The geometric size was determined by a) TEM imaging, b) the frequency of the size distribution of UFP, c) SEM imaging, and d) fractional composition of the UFP collected

Fig. 3

In utero exposure to UFP induces placental dysfunction and promotes activation of hypothalamic pituitary adrenal axis (HPA). a Protein levels and representative image of Western blot of placenta HSD11B2. n = 6/group; * p < 0.05. One-way ANOVA. b DNA methylation of the HSD11B2 promoter in placenta determined by pyrosequencing. n = 4/group; * p < 0.05. Two-way ANOVA. c Dam serum cortisol and d fetus cortisol were determined by ELISA. n = 4/group. Data are expressed as mean ± SEM. *p < 0.05 and ** p < 0.01. One-way ANOVA

Fig. 4

In utero exposure to UFP induces oxidant damage by metabolism of PAHs. Levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured by ELISA in a) placenta and b) fetus. n = 4/group; * p < 0.05 and ** p < 0.01. One-way ANOVA. c Protein levels and representative image of Western blot of dam lung CYP1A1 and NQO1, and d) placental CYP1A1 and NQO1. n = 4/group. Data are expressed as mean ± SEM. * p < 0.05 and ** p < 0.01. One-way ANOVA

Fig. 5

In utero exposure to UFP induces systemic and intrauterine inflammation. Concentrations of pro-inflammatory cytokine: interleukin-β (IL-β), IL-6 and tumor necrosis factor-α (TNF-α). The anti-inflammatory cytokines IL-10 and IL-4. Chemokines: neutrophil chemoattractant (KC), macrophage inflammatory protein 2 (MIP-2) and monocyte chemoattractant protein 1 (MCP-1). Vascular endothelial growth factor (VEGF) ware measured with multiplex magnetic beads in a) dam serum collected, b) dam lung, c) placenta and d) fetus total protein. n = 4/group. Data are expressed as mean ± SEM. *p < 0.05, ** p < 0.01 and *** p < 0.001. One-way ANOVA

Fig. 6

In utero exposure to UFP induces cardiovascular programming by DNA hypomethylation of the Agtr1a, Agtr1b and ACE promoter in fetus and adult lung. Percentage of methylated 5 CpG sites in Agtr1a, Agtr1b and ACE promoter in the a, c and e) fetus and b, d and f) lung. n = 4/group; *p < 0.05, **p < 0.01 and *** p < 0.001. Two-way ANOVA. c Protein levels and representative image of Western blot of G) fetus AT₁R and H) ACE, and I) lung AT₁ and ACE. n = 4/group. Data are expressed as mean ± SEM. * p < 0.05 and ** p < 0.01. One-way ANOVA