Objective
The continued development and use of engineered nanomaterials (ENM) has given rise to concerns over the potential for human health effects. Although the understanding of cardiovascular ENM toxicity is improving, one of the most complex and acutely demanding “special” circulations is the enhanced maternal system to support fetal development. The Barker hypothesis proposes that fetal development within a hostile gestational environment may predispose/program future sensitivity. Therefore, the objective of this study was 2-fold: (1) to determine whether maternal ENM exposure alters uterine and/or fetal microvascular function and (2) test the Barker hypothesis at the microvascular level.
Study Design
Pregnant (gestation day 10) Sprague-Dawley rats were exposed to nano-titanium dioxide aerosols (11.3 ± 0.039 mg/m 3 /hr, 5 hr/d, 8.2 ± 0.85 days) to evaluate the maternal and fetal microvascular consequences of maternal exposure. Microvascular tissue isolation (gestation day 20) and arteriolar reactivity studies (<150 μm passive diameter) of the uterine premyometrial and fetal tail arteries were conducted.
Results
ENM exposures led to significant maternal and fetal microvascular dysfunction, which was seen as robustly compromised endothelium-dependent and -independent reactivity to pharmacologic and mechanical stimuli. Isolated maternal uterine arteriolar reactivity was consistent with a metabolically impaired profile and hostile gestational environment that impacted fetal weight. The fetal microvessels that were isolated from exposed dams demonstrated significant impairments to signals of vasodilation specific to mechanistic signaling and shear stress.
Conclusion
To our knowledge, this is the first report to provide evidence that maternal ENM inhalation is capable of influencing fetal health and that the Barker hypothesis is applicable at the microvascular level.
Anthropogenic engineered nanomaterials (ENMs) are manufactured specifically for their unique properties at the nanometer scale (<100 nm in 1 dimension). Although their applicability may appear infinite, significant resources have been committed to focus ENM development on engineering and biomedical applications. ENMs have already impacted public health through diverse daily uses (eg, surface coatings, cosmetics, food, drug delivery systems, and implantable medical devices). In many of these applications, adult toxicities have been observed; however, the fetal consequences of maternal exposure to ENM are essentially unknown.
Fetal toxicity and the genetic basis of adult disease are an initiative within the National Institute of Environmental Health and Safety. The general understanding of adult cardiovascular ENM toxicity is modest to good ; yet, the maternal and fetal consequences of maternal ENM exposures during gestation are unknown. The “Barker hypothesis” proposes that the association between retarded growth and cardiovascular disease is due to chronic physiologic and metabolic effects that are imposed on a fetus by a hostile gestational environment. Limited animal and in vitro studies suggest that maternal ENM exposure has direct consequences on the uterus, placenta, and fetus. Nanomaterial influence on any of these tissues can have dire consequences on maternal and/or fetal health. Long et al evaluated the influence that direct nano-sized titanium dioxide (TiO 2 ) exposure would have on rat neuronal cell cultures and revealed rapid damage to neurons at low concentrations. Blum et al found cadmium oxide within the uterine and placental tissue after maternal inhalation that resulted in associated maternal weight gain and impaired fetal and neonatal growth. Injected ENMs have been shown to reach the uterus easily and stimulate uterine atrophy. Similarly, intravenous maternal ENM exposure also compromises gestation by causing early miscarriage, placental vascular lesions, and fetal malformations that are linked to reactive oxygen species production. Taken together, these findings have a common component, uterine microvascular function that, if altered, may contribute to the generation of a hostile gestational environment.
The most striking observations are structural and functional fetal abnormalities after maternal ENM exposure: impaired implanted fetal resorption (principally in the late gestation), reduction in the number of live fetuses delivered, impaired postnatal growth and weight gain, and generalized neurophysical deficits or neurobehavioral alterations in the offspring. These proof-of-principle studies have shed light on an extremely important issue by injecting high ENM doses to produce significant repeatable results. The functional microvascular ramifications from the maternal or fetal perspective have never been studied in these regards. Furthermore, few studies have focused on the most likely route of ENM exposure: inhalation. It remains to be determined whether ENMs exert a biologic influence through targeting maternal tissues or cross the maternal-fetal barrier to stimulate direct effects on the fetus. Animal studies that evaluate the mechanisms that affect microvascular outcomes must be explored.
The microcirculation is the principal level of the vasculature for a host of physiologic parameters that include growth, metabolism, peripheral resistance, tissue perfusion, nutrient/waste exchange, permeability, and leukocyte trafficking. Virtually every pathologic development has a microvascular origin and/or consequence. Our research program has advanced a generally good understanding of the systemic consequences of inhaled ENMs in the coronary and skeletal muscle microcirculations ; however, no work has been done in regards to pregnancy. For example, only recently has environmental particulate matter (air pollution) inhalation been associated with maternal blood pressure disturbances and low birthweights. ENMs differ significantly from particulate matter because of their homogeneous composition, unique properties that are associated with their small size, and potential toxicities that are associated with intentional exposures.
Functionally, the microcirculation acts to regulate blood flow distribution while protecting downstream tissues from high arterial pressures and blood flow rates: roles that are crucial for fetal health and survival of a pregnancy. Precise maintenance of blood flow, within an environment of profound remodeling and growth, is paramount for maternal health and fetal development. ENM exposure has been shown to impair normal microvascular reactivity and function in a range of vascular beds, including heart and skeletal muscle. It is reasonable to speculate that maternal ENM exposure may also influence normal uterine function and lead to a hostile gestational environment, which is capable of impairing fetal microvascular reactivity. Therefore, the purpose of this study was to test the Barker hypothesis from a microvascular prospective.
Materials and Methods
Animal model
Sprague Dawley rats (female, 250-275 g; male, 300-325 g) were purchased from Hilltop Laboratories (Scottsdale, PA). Rats were housed at West Virginia University with food and water provided ad libitum and acclimated for at least 72 hours before use or mating. Females were monitored before breeding to ensure estrus, at which time each female was placed with an individual male. Female rats were then smeared every 12 hours to verify breeding by the presence of sperm. To ensure that all methods were performed humanely and with regard to alleviation of suffering, all procedures were approved by the Institutional Animal Care and Use Committee of the West Virginia University. To increase the likelihood of experimental success and acquisition of viable fetal tissue, rats were placed within the inhalation facility after gestational day 10; if there was maternal exposure before this point, there was a greater chance of reduced litter numbers and ischemic regions within the uterus.
ENM
Nano-TiO 2 powder (aeroxide TiO 2 ; Evonik Corporation, Parsippany, NJ) is a mixture composed of anatase (80%) and rutile (20%) TiO 2 , with a primary particle size of 21 nm and a surface area of 48.08 m 2 /g. We prepared the nano-TiO 2 for aerosolization by drying, sieving, and storing the powder.
Inhalation exposure
We previously reported and described the nano-aerosol generator and exposure system that we used for the current experiments (US patent no. 13/317,472). Briefly, the system was developed specifically for rodent particle inhalation exposures. The apparatus was developed with a vibrating fluidized bed, a Venturi disperser (Vaccon, Medway, MA), cyclone separator, impactor and mixing device, an animal housing chamber, and real-time monitoring devices with feedback control. Aerosols are generated by allowing an air stream to pass through the vibrating fluidized bed and into the Venturi vacuum pump, drawing air and the nano-TiO 2 as it passes. Aerosols enter the cyclone separator, which is gated to remove agglomerates of >400 nm at an input blood flow rate of 60 L/min of clean dry air before entering the exposure chamber.
Size distribution, mean aerodynamic diameter, and relative mass concentration of the aerosols were monitored in real time (Electrical Low Pressure Impactor; Dekati, Tempere, Finland); the particle size distribution was also measured in real time with a Scanning Mobility Particle Sizer device (TSI Inc, St. Paul, MN). Once the steady-state aerosol concentration was achieved, exposure duration was adjusted to achieve a daily calculated deposition of 45 ± 2 μg. Animals were placed within the inhalation chamber and exposed for 5 hours per day for an average of 8.2 ± 0.85 days at a final mass concentration of 11.3 ± 0.039 mg/m 3 or to filtered air (0 mg/m 3 ; control). Tremendous care was taken to achieve the same final mass concentration for each consecutive day of exposure. Our ability to reliably and repeatedly characterize the aerosols in real time (Electrical Low Pressure Impactor and Scanning Mobility Particle Sizer device) enabled us to achieve this day-to-day consistency.
Calculated total deposition was calculated based on mouse methods previously described and normalized to rat weight and to pregnant rat minute ventilation with the following equation: D = F × V × C × T, where F is the deposition fraction (10%), V is the minute ventilation based on body weight, C equals the mass concentration (mg/m 3 ), and T equals the exposure duration (minutes). 24 Our laboratory previously documented and quantified pulmonary TiO 2 deposition after inhalation exposure at similar aerosol concentrations.
Tissue preparation
Pregnant rats were anesthetized with isoflurane (5% induction, 2% maintenance) on gestational day 20, 24 hours after the last exposure. Rats were killed by exsanguination, after which the uterus was removed, flushed of excess blood, and placed in a dish of chilled (4°C) physiologic salt solution (PSS). The uterus was inspected visually for evidence of partial resorption, and fetal tissue was removed and weighed.
Microvessel isolation
Maternal
Despite the label, premyometrial radial uterine arteries are considered representative of the uterine microcirculation based on passive diameter (approximately 150 μm), anatomic position within the parenchyma (which represents the third to fourth order branching pattern), and functional vascular resistance (systemic pressure decreases of approximately 100 mm Hg). The uterus was pinned to visualize the vasculature, and a distal segment of the radial artery was isolated, removed, transferred to a vessel chamber (Living Systems Instrumentation, Burlington, VT) that contained fresh oxygenated PSS, cannulated with glass pipettes, and secured with nylon sutures (11-0 ophthalmic; Alcon UK, Surrey, England). Arterioles were extended to their in situ length, pressurized to 60 mm Hg with PSS, superfused with warmed (37°C) oxygenated (21% O 2 /5% CO 2 /74% N 2 ) PSS at a rate of 10 mL/min, and allowed to develop spontaneous tone. Internal and external arteriolar diameters were measured digitally with video calipers (Colorado Video, Boulder, CO) and recorded.
Fetal
Pups were pinned supine within a chilled (4°C) dissecting chamber that was filled with PSS. The tail artery was isolated, excised, and transferred to the vessel chamber described earlier. The tail artery was selected for study because (1) it is primarily responsible for thermal homeostasis and is fully functional at birth (2) it provides a significant pressure gradient from the proximal to the distal end of the tail that creates resistance, which is a representative characteristic of the microcirculation, and (3) other vascular beds within the pup were considered too short to cannulate successfully because of the excessive branching that is associated with tissue growth and development. To maintain consistency between experiments, fetal arteries were pressurized to 60 mm Hg.
Arteriolar reactivity
After equilibration, arteriolar reactivity was evaluated in random order to ensure that responses were neither interactive nor time-dependent over the course of the experiment in response to (1) blood flow–induced dilation (endothelial mechanotransduction to elicit a shear stress response by changes to luminal blood flow [reductions and increases in blood flow from 0 μL/min to 30 μL/min at 5 μL/min increments]), (2) acetylcholine (10 –9 -10 –4 mmol/L; an endothelium-dependent dilator that acts through the muscarinic receptor to activate endothelium-derived nitric oxide synthase and arachidonic acid pathways), (3) spermine NONOate (10 –9 -10 –4 mmol/L; an endothelium-independent dilator and spontaneous nitric oxide donor), and (4) phenylephrine (10 –9 -10 –4 mmol/L; a vascular smooth muscle constrictor that acts through the α 1 -adrenergic receptor). After assessments of arteriolar reactivity, the superfusate was replaced with Ca 2+ -free PSS until passive tone could be established (<150 μm maximum diameter).
Statistics and formulae
Data are expressed as means ± SE. Prudent sampling of multiple vessels per rat served to characterize inherent uterine microvascular heterogeneity and reduce the number of animals that were required to complete the datasets. Therefore, in this study, the experimental unit was the arteriole; 1-3 microvessels were studied per rat for the uterine studies and 1-3 fetal pups per litter for the fetal experiments. However, we can acquire only 1 tail arteriole from each pup.
Spontaneous tone was calculated by the following equation: [(D M – D I )/D M ] × 100, where D M is the maximal diameter recorded at 60 mm Hg under Ca 2+ -free PSS as described earlier, and D I is the initial steady-state diameter that was achieved before the experimental period. Vessels were used for experiments only if spontaneous tone of ≥20% was achieved.
The experimental responses to acetylcholine, NONOate, and phenylephrine are presented as percent relaxation from baseline diameter: [(D SS – D CON )/(D M – D CON )] × 100, where D SS remains the steady-state diameter that was achieved after each chemical bolus, and D CON is the control diameter that was measured immediately before the dose-response experiment. All experimental periods were at least 2 minutes, and all steady-state diameters were collected for at least 1 minute. Representing the responses in this manner allowed us to normalize for potential differences in baseline diameters before each dose-response curve.
Shear stress was calculated from volumetric blood flow (Q) according to the following equation: τ = 4ηQ/πr 3 , where η represents viscosity (0.8 centipoise), Q represents volumetric blood flow rate (measured with a calibrated blood flow indicator; Living Systems Instrumentation, Burlington, VT), and r equals the vessel radius.
Student t tests were used to compare sham and exposed animal and vessel characteristics. The t test was also used to evaluate differences between animals that were exposed for ≤7 days and those that were exposed for >7 days. Concentration-diameter (acetylcholine, phenylephrine, and NONOate) and blood flow–diameter curves were assessed by 2-way repeated measures analysis of variance to detect differences between and within groups. Pairwise comparisons post-hoc analysis comparisons (Bonferroni) were made if significance ( P ≤ .05) was reached (SigmaPlot 11.0; Systat Software Inc, San Jose, CA). Equations of first-order regression lines were developed to assess line slope relationships (Prism; GraphPad Software, Inc, La Jolla, CA).
Results
ENM exposure and uterine health
There were expected significant differences between the pregnant sham and exposed rats with respect to days of exposure, concentration, calculated total exposure, and calculated total deposition by experimental design ( Table 1 ). These differences did not affect litter size, litter weight, or the average pup weight. However, when the data for the exposed rats were broken down based on days of exposure, there was a significant difference in litter weight ( Table 1 ). These results indicate a longer duration of exposure during gestation, greater exposure concentration, or exposure earlier in a pregnancy has a greater impact on fetal development.
| Treatment | Rats/litters studied, n | Days of exposure, n | Aerosol concentration × time (mg/m 3 × hr) | Calculated total exposure, μg | Calculated total deposition, μg | Average litter size, n | Average litter weight, g | Average weight per pup, g |
|---|---|---|---|---|---|---|---|---|
| Sham | 6 | 0 | 0 | 0 | 0 | 13.25 ± 1.03 | 42.01 ± 11.86 | 3.73 ± 0.15 |
| Exposed (total) | 10 | 8.2 ± 0.85 a | 11.31 ± 0.39 a | 380 ± 60 a | 121 ± 19 a | 10.75 ± 1.64 | 48.01 ± 8.77 | 4.3 ± 0.49 a |
| Exposed (≤7 d) | 6 | 6.33 ± 0.33 | 10.53 ± 0.37 | 257 ± 18 | 82 ± 6 | 12.16 ± 1.19 | 58.53 ± 6.49 | 4.89 ± 0.41 |
| Exposed (>7 d) | 4 | 11 ± 0.91 b | 12.34 ± 0.19 | 556 ± 90 b | 178 ± 29 b | 6.5 ± 3.8 b | 16.45 ± 9.86 b | 2.51 ± 0.01 b |
Overall, shorter exposures (<7 days) were favored to increase experimental success because, with longer exposures (>7 days), whole litters were lost to fetal death or varying stages of fetal resorption. Therefore, for the remainder of the studies, analysis will remain exposed vs sham groups.
Microvascular characteristics
There were no significant differences between the pregnant rats with respect to age, weight, or passive uterine radial artery diameter ( Table 2 ). However, there were significant differences between sham and exposed uterine radial artery active diameter and spontaneous vessel tone before interventions. These results provide evidence that indicate ENM exposure leads to increased uterine vascular tone and smaller active diameter. If this were to persist in vivo, the increase in peripheral resistance may reduce uterine blood flow or alter the distribution of blood flow within or between uterine horns because of bidirectional blood flow. Unlike humans, within a rodent model of pregnancy, the arcading network of blood flow supplies a constant perfusion within the uterus that allows for multiple pup litters.
| Microvascular characteristic | Pregnant rats studied, n | Arterioles studied, n | Gestational age | Weight, g | Active diameter, μm | Passive diameter, μm | Vessel tone |
|---|---|---|---|---|---|---|---|
| Maternal uterine arteriole | |||||||
| Sham | 6 | 9 a | 16.2 ± 1.10 wk | 403 ± 17.8 | 85.89 ± 6.61 | 112.33 ± 6.20 | 26.91 ± 2.63 |
| Exposed | 9 | 16 a | 14.1 ± 0.58 wk | 355 ± 26 | 66.75 ± 3.20 b | 103.06 ± 4.74 | 35.47 ± 2.12 b |
| Fetal tail artery | |||||||
| Sham | 4 | 7 c | 20 d | 3.54 ± 0.17 | 93.28 ± 3.87 | 126.57 ± 5.92 | 26.94 ± 2.49 |
| Exposed | 6 | 11 c | 20 d | 4.95 ± 0.33 b | 106.36 ± 7.56 | 137.50 ± 6.45 | 25.75 ± 2.75 |
a The number of uterine arterioles that were evaluated
c The number of fetal pup arterioles that were evaluated (1 per pup).
With respect to the fetal tail arteries, there were no significant differences in age, spontaneous active diameter, passive diameter, or vascular tone ( Table 2 ). Unintentionally, the fetal tissue that was studied from exposed dams was significantly larger than the sham group ( Table 2 ).
Aerosolized ENM distribution
Figure 1 is a graphic representation of the nanoparticle diameter distribution. The primary diameter of nano-TiO 2 is 21 nm; however, these particles tend to agglomerate, which leads to an aerodynamic diameter of 149 ± 3.9 nm, which is consistent with other experiments within our research program. Therefore, when aerosolized powder is used, it is important to monitor particle characteristics in real time to maintain a consistent aerosol distribution within and between experiments.
Endothelium-dependent vasodilation
Maternal endothelium-dependent arteriolar dilation is significantly attenuated after ENM exposure ( Figure 2 , A). Reduced endothelium-dependent dilation through acetylcholine stimulation may be attributed to an alteration to nitric oxide signaling; however, acetylcholine also leads to signaling through pathways of arachidonic acid metabolism (prostacyclin, lipoxygenase). Therefore, although nitric oxide signaling may be significantly perturbed, residual endothelium-dependent dilation may be due to alternate signaling pathways.
