For this study, we assessed vascular reactivity in vessel segments from maternal, uterine, and fetoplacental circulations at vulnerable life stages. Alterations to vascular reactivity were identified on GD 20 after maternal exposure on GD 4, 12, and 17. Together, a single maternal exposure on GD 4 led to modification of vascular reactivity in each functional compartment (e.g., endothelium-dependent, -independent, and smooth muscle). Further, these variations were identified through serial assessments of the maternofetal circulation, with the greatest impairments identified in vascular smooth muscle reactivity, initiated through α-adrenergic signaling. Interestingly, fetal umbilical vein and fetal aorta reactivity were primarily modified in exposures initiated at GD 4. It is important to clarify that these single exposures took place 3, 8, and 16 days prior to assessment. Previous temporal studies of a non-pregnant occupational model identified epicardial arteriolar dysfunction for 7 days after multi-walled carbon nanotube inhalation [13]. Through these studies, we identified that a single maternal ENM inhalation exposure, even early in gestation, can have profound and long-lasting effects in maternal and fetal hemodynamic control.

Endothelium-dependent relaxation was assessed by generating cumulative concentration–response curves to MCH, following preconstriction with PHE. Reduced cholinergic activity was reported for uterine artery from animals exposed to ENM at early, mid, and late gestation. Earlier investigations by our group reported significantly blunted endothelium-dependent relaxation in isolated fetal tail arterioles following maternal exposure to nano-TiO 2 aerosols [16]. Collectively, these findings in combination with our published work confirm maternal exposure to ENM during gestation impairs fetal systemic cardiovascular function. Of note, we reported poor agonist stimulated relaxation of vascular segments from umbilical vein and thoracic fetal aorta. These observations are consistent with existing literature [23] and indicate that cholinergic responses may not be the primary signaling pathway for endothelium-dependent dilation within the umbilical vein or fetal aorta. Poor reactivity may be due to reduced cholinergic receptor populations in these understudied tissue beds, reduction in NO production or bioavailability, or reliance on compensatory signaling mechanisms. Further assessments should consider alternate pharmacological agents to assess the contribution of nitric oxide production or the generation of metabolites of arachidonic acid (e.g., the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester and the cyclooxygenase antagonist indomethacin). In addition, future assessments may include examining the response to physiological modifications, including changes in shear stress and transmural pressure to further evaluate endothelial health.

Interestingly, fetal tissues derived after ENM 4 and ENM 17 exposures demonstrated an improved relaxation to NO indicating the possibility of increased NO-sensitivity after early/late gestation maternal ENM inhalation. These responses would further emphasize the consideration of alternate EDHF agonists in future work. The local production of reactive oxygen species has also been implicated in the development of contractile and bioenergetic dysfunction and impairments in endothelium-dependent vasoreactivity associated with exposure to nano-TiO 2 [24, 25]. Accordingly, incubation with 2,2,6,6-tetramethylpiperidine-N-oxyl, a superoxide dismutase mimetic, and catalase, a hydrogen peroxide scavenger, would help to determine the influence of local reactive oxygen species production associated with nano-TiO 2 exposure during pregnancy. Smooth muscle contractility was assessed in response to the selective α-adrenergic agonist, PHE. We observed that a single exposure to nano-TiO 2 at mid (GD 12) and late (GD 17) gestation decreased maximum developed tension in the uterine artery. In contrast, smooth muscle contractility in the thoracic fetal aorta was significantly augmented in response to ENM exposure, an observation that was consistent at early, mid, and late gestation. Further, in ring segments from umbilical veins, contraction to 10−2M PHE was poor and variable. This observation is consistent with previous reports of agonist-induced contractile activity of murine umbilical arteries and veins [23]. In accordance with existing literature, exposure of umbilical vein segments to KPSS induced a maintained contraction. When compared with KPSS-induced contraction of uterine arteries, umbilical veins showed a significant decrease in maximal tension generation; similar findings have been reported in humans [26]. In isolated human umbilical vein rings, adrenaline was more potent than phenylephrine, 16% of the vessel rings did not demonstrate adrenergic responses to adrenaline or phenylephrine [27].

We observed differential responses between aortic, uterine, umbilical, and fetal aortic vessel segments with exposure to nano-TiO 2 at multiple days across gestation. However, we have previously focused on the microcirculatory level of the vasculature [14, 16]. Given this context, our current work aligns with the findings of existing studies of serial vascular assessment [﻿28﻿], that while macrocirculatory reactivity is blunted, the greatest impairments are identified in the microcirculation. Overall, we postulate heterogeneous responses in vascular reactivity may be related to differences in cholinergic and α-adrenergic receptor populations and/or reliance on compensatory mechanism signaling cascades. This may include a shift in the role of arachidonic acid metabolites in the regulation of vascular tone (i.e., a disturbance in the ratio of the vascular smooth muscle dilator prostacyclin and the potent vasoconstrictor thromboxane A 2 ) and/or increased reliance on Rho-kinase signaling [20, 22] after maternal ENM exposure.

Vascular remodeling during pregnancy, the time and pattern of which is specific to vessel size and location, is carefully orchestrated [29]. Pregnancy is accompanied by a progressive rise in uteroplacental blood flow (UPBF) in order to satisfy gestational demands of the developing fetus [1]. In rats, changes in total uterine blood flow are detected on or around the final third of gestation (~ GD 15) [30]. Increases in total UPBF are facilitated by a combination of circulatory adaptations including a rise in cardiac output and an expanded intravascular volume. Under normal circumstances, MAP decreases slightly or remains unchanged. Consistent with normal pregnancy, we observed no significant differences in MAP between naïve, sham control, and ENM-exposed groups. Accordingly, changes in cardiac output are accompanied by a reciprocal decrease in vascular resistance. Together, vessel-specific structural remodeling and a shift in uterine vascular reactivity (reduced vessel tone and enhanced vasodilation) determine pregnancy-related changes in UPBF [1]. Future studies may explore the influence of known pregnancy-related changes in the vascular micro-environment on vascular reactivity in pregnancy.

Technological advancements in nanomaterial science afford an abundance of opportunities for innovation. Nanotechnology has led to achievements in the accuracy and effectiveness of medical diagnostic equipment, vehicle fuel efficiency, sources of renewable energy, environmental remediation, and therapeutic efficiency of pharmaceuticals. While the unique physiochemical properties of engineered nanomaterials incite a wealth of enthusiasm, the pervasive impact of this technology also begets concerns that development may be outpacing our understanding of its safety. Exposure of the maternal environment to ENM during pregnancy has been shown to impact health of the dam, fetus, and adult offspring [31]; however, the consequences of specific temporal (gestational day) and spatial (vascular location) considerations are largely undetermined. In this respect, the current study is the first to examine how exposure to nano-TiO 2 at critical periods of gestation affects vascular responses during pregnancy, and whether the resulting changes differentially affect regionally discrete blood vessels of maternal and fetal circulations.