The key findings of this study are the following: (1) early (3 days and 2 weeks) PP period is associated with a significant inhibition of maternal mesenteric artery reactivity to PE that returned to pre-pregnancy levels at 4 and 12 weeks PP. (2) Inhibition of NO production resulted in the restoration of postpartum PE reactivity to pre-conception levels. (3) Vasodilator reactivity of maternal mesenteric arteries to ACh was unaltered at any PP period, and was similar to reactivity found in virgin mice. (4) Early PP was associated with a persistent increase in arterial distensibility that regressed to pre-conception levels at 12 weeks PP and correlated with a reduction in collagen composition of arteries at early PP periods. (5) Mesenteric arteries from LP mice show a reduced reactivity to PE and increased passive distensibility compared to virgin controls. (6) Vessels from 2-week non-nursing PP mice demonstrate an increased PE reactivity, diminished responses to ACh, and reduced distensibility compared to 2-week PP breastfeeding mice.
Normal pregnancy is associated with significant functional changes in maternal mesenteric vasculature that include attenuation of constrictor responses to PE and Angiotensin II (AGTII), and enhanced flow-induced endothelium-mediated vasodilation [17, 28,29,30,31,32,33,34,35,36,37,38]. These changes may importantly contribute to increased mesenteric blood flow and reduced peripheral vascular resistance documented in uncomplicated pregnancies in women . Data on structural changes and mechanical behavior of mesenteric arteries in pregnancy is less conclusive. Increase, no change, or even reduction in passive arterial distensibility of mesenteric arteries from late pregnant rats and mice has been reported [39,40,41]. These discrepant observations may be related to species (e.g., mouse versus rat) or strain differences in the animals used to characterize the mechanics of mesenteric arteries in pregnancy. Such differences may be related to several factors, such as genetics, environment, hormone responsiveness, or immune regulation.
Much less is known about functional and structural changes in the postpartum maternal vasculature of animals and humans. In previous studies in rats, at 4 weeks PP, the reactivity of mesenteric vessels to vasoconstrictor (PE) or vasodilator (methacholine) was similar to those at pre-conception . The findings reported herein and our previous observations  agree with these studies.
However, the data from the current study demonstrates a significantly reduced constrictor responsiveness to PE in mesenteric arteries at 3 days and 2 weeks PP (Fig. 1), while reactivity to PE returned to pre-pregnancy levels by 4 and 12 weeks PP. At the same time, the vasoconstriction to graded depolarization with high K+ at 2 weeks PP was not different from vascular responses of virgin controls (Fig. 3a). This data indicates that the intrinsic ability of vascular smooth muscle cells (VSMs) to contract (contractility) is not affected during the PP period. Because the EC50 values for PE-induced vasoconstriction were not significantly different between studied groups (Fig. 5S), the data suggest that sensitivity of arteries to alpha-adrenoreceptor stimulation is not modified PP. We conclude that reduced downstream efficacy of vasoconstriction in response to stimulation of alpha-adrenoreceptors is a characteristic feature of LP and the early PP period. It has been shown that LP is associated with reduced mesenteric artery reactivity not only to PE but also to angiotensin II, vasopressin, and other vasoconstrictors. In rats, a decreased PE efficacy in late pregnancy returned to NP baseline by 1 week PP [32, 35, 41, 43,44,45,46]. Whether the vasoconstriction to different receptor-stimulated agonists is also attenuated in the PP period remains to be explored.
In resistance vasculature, the contraction of VSM is tightly controlled by numerous mediators and mechanisms generated by vascular endothelial cells [47, 48]. During late pregnancy, the basal production of NO and prostacyclin is significantly enhanced in the endothelium of the peripheral vasculature [49,50,51]. In addition, inhibition of vascular NO or prostacyclin production in late gestation results in significantly augmented systemic blood vessel reactivity to vasoconstrictors [43, 49,50,51]. In our study, we used ACh-induced vasodilation as an indicator of endothelial function in mesenteric arteries. In the peripheral vasculature, ACh produces its vasodilator effect via activation of muscarinic receptors on endothelial cells that triggers a cascade of intracellular events resulting in elevation of endothelial cell Ca2+, production, and release of several relaxing factors . Herein, we report that the vasodilator responses to ACh were not significantly different at both early and late PP periods compared with the preconception state (Fig. 2), indicating that endothelial function was not affected.
Intriguingly, when we tested the role of NO in inhibiting PE-induced constriction in the early PP period, the blockade of NO generation with L-NNA resulted in the restoration of vasoconstriction to PE to pre-pregnancy levels (Fig. 3e). At the same time, we found that eNOS RNA expression in whole mesenteric arteries of 2 weeks PP tended to be higher, but was not significantly different from that found in (Fig. 4S) virgin vessels. It is formally possible that local enzyme activity may be increased and this would need further study to confirm.
In mouse mesenteric arteries, alpha-adrenoreceptors are located exclusively on VSMs as a direct application of PE to endothelial cells produces no calcium response . In arteries, stimulation of alpha-adrenoreceptors results in generation of second messengers: inositol trisphophate (IP3) and diacylglycerol. These molecues are responsible for intracellular Ca2+ elevation and Ca2+ sensitization, respectively leading to contraction of VSMs (reviewed in ). In mesenteric arteries of mice, IP3 can diffuse from cytosol of VSMs to endothelial cells via myoendothelial gap junctions. This event in turn stimulates endothelial channels, NO release, and vasodilation that limits the PE-induced constriction [52, 54].
In contrast to adrenoreceptor stimulation, the major mechanism of VSM contraction in response to high K+ depolarization is influx of Ca2+ via voltage-gated Ca2+ channels with no IP3 generation . This may explain the lack of differences in vasoconstriction to high K+ in virgin and PP mice. Based on the published observations, we speculate that IP3-dependent PE-stimulated endothelial NO production might be a specific feedback mechanism that restricts vasoconstriction in the early PP period more efficiently than in pre-conception. The validation of this concept deserves further investigation.
Inhibition of NO production with L-NNA resulted in a marked reduction in ACh-induced vasodilation but residual vasodilator responses were similar in arteries from virgin and PP mice (Fig. 3f–h). Our findings are in agreement with published observations showing no change in flow-mediated vasodilation of women’s brachial artery before pregnancy and 1-year postpartum . In the context of our data, we speculate that PP-induced attenuation in adrenoreceptor-stimulated vasoconstriction may contribute to the increased mesenteric blood flow observed in 1-year PP women .
Our study demonstrates that the early PP period in mice is associated with significant remodeling and change in the mechanical behavior of mesenteric arteries. As shown in Fig. 4a, passive lumen diameters are increased in 2- and 4-week PP mice. In addition, arterial distensibility is significantly increased compared with pre-conception levels (Fig. 5). These changes in the structure and mechanical behavior of arteries may contribute to increased arterial compliance observed in vessels form PP women  and supports the role of mesenteric vasculature in the long-term beneficial cardiovascular effects of normal pregnancy.
Elastin and collagen are two major components of the arterial extracellular matrix responsible for extensibility and vessel wall strength, respectively . Collagen is a very stiff protein that limits blood vessel distensibility . We found a significant reduction in collagen levels in both media and adventitia that positively correlates with increased distensibility of mesenteric arteries from early PP mice (Fig. 6a and c). Therefore, reduction in collagen content likely is in part responsible for the increased passive distensibility observed in mesenteric arteries at early 2 weeks PP. These changes in arterial collagen are temporal, as no significant alterations were detected at 12 weeks PP as compared to pre-conception (Fig. 6b and d). At the same time, no changes were found in the elastin contents of the mesenteric arterial wall of PP vs. virgin mice (Fig. 6e–h). Although elastin and collagen contents determine blood vessel distensibility and compliance, other molecules and related interactions contribute to elastin or collagen-mediated vascular elasticity [56,57,58]. For example, a decrease in cross-linking and stability between collagen molecules occurs in mouse cervix cervical tissue in late pregnancy and leads to increased cervical distensibility , and we speculate that this mechanism may additionally contribute to the remodeling of vessels at PP. Structural reorganization of elastic fibers may be another mechanism involved in arterial tissue remodeling during pregnancy . Investigation of cross-linking proteins and elastin structure in the mesenteric vascular extracellular matrix in pregnancy or postpartum is an important focus for future studies.
Reduced PE responsiveness in early PP vessels reported herein may be a consequence of pregnancy-induced modulation of vascular function extended into the early PP period or may result from new coincident regulation to return the vascular system to its pre-pregnancy state. The former suggestion is in line with a significant reduction in PE-induced responses of mesenteric arteries from LP vs. virgin mice (Fig. 7a). Moreover, passive distensibility of mesenteric arteries from LP mice was significantly increased compared with that at pre-conception. Our data are in agreement with an idea that reduced sensitivity to PE and increased passive distensibility of mesenteric arteries in early PP is a consequence (at least in part) of changes induced in mesenteric vasculature during pregnancy.
The idea that new coincident regulation of the vasculature may occur PP is supported by our findings. Although we found no significant changes in passive lumen diameters and the wall thickness of arteries from LP vs. virgin mice (Fig. 7d and e), passive diameters were gradually increased early PP and reached significance at 2 and 4 weeks PP (Fig. 4a). Moreover, arterial wall thickness increased and then decreased PP, consistent with our previous observations .
There is experimental evidence that augmented blood flow in the mesenteric circulation results in a subsequent increase in the vessel diameters [60, 61]. Such outward remodeling of mesenteric arteries was preceded by significantly elevated mesenteric blood flow in type-2 diabetic mice . Our data together with that from clinical studies  support the hypothesis that outward remodeling of mesenteric arteries is mostly induced by PP as a consequence of increased mesenteric blood flow. There are at least two possibilities to explain PP increased blood flow in mesenteric circulation. The first is redistribution of blood volume from uterine to peripheral circulation after the termination of pregnancy. The second possibility is that increased food intake and gut metabolism required to obtain the nutrients needed for breastfeeding drives increased mesenteric blood flow.
The role of breastfeeding in the modulation of function and structure of mesenteric arteries was assessed via comparison of data from 2-week postpartum nursing vs. non-nursing mice. We found that reduced contractility to PE in nursing mice returned to pre-pregnancy levels in non-nursing mice by 2 weeks PP. In addition, vasodilation to ACh was significantly attenuated in non-nursing vs. nursing PP mice. The relationship between lack of nursing and decreased endothelial responsiveness to vasodilators has yet to be explored in this model. High levels of prolactin, an important hormone related to breastfeeding, are interestingly related to poor endothelial function in postmenopausal women , and perhaps there is an interaction between sex hormone stimulation and prolactin that preserves endothelial function in pregnancy and the PP period. In contrast, oxytocin may have direct therapeutic benefit, due to reduction in oxidative stress .
Finally, an increase in passive distensibility in early PP was not found in arteries from non-nursing mice (Fig. 8). These data all together demonstrate the importance of breastfeeding in supporting the extension of the vascular effects of pregnancy into the early PP period. Mesenteric vasculature contributes to the regulation of peripheral vascular resistance and systemic blood pressure [21, 22]. Therefore, the reduced contractility and increased compliance of these vessels we observed at early PP only in breastfeeding mice may suggest an important beneficial mechanism that contributes to reduced blood pressure in lactating women. Consistent with this, clinical observations present ample evidence that lactating women show significantly reduced levels of systolic and diastolic blood pressure after normal or complicated pregnancies [18, 19, 65].
Our studies provide a timeframe for mesenteric vascular adaptations that occur during pregnancy and extend into the PP period but respond to modification by PP events such as nursing. Functional changes (NO-dependent attenuation of vasoconstriction) appear to have a different arc (e.g., shorter) than structural changes and appear to be modified by PP events. We could speculate then that specific stressors—emotional, physical, and environmental—may lead to different effects whether they occur during pregnancy or PP. Our studies also suggest potential final common mechanisms by which these vascular changes occur. Future studies will focus on the determination of upstream elements feeding into these mechanisms. Particularly important would be those immediately preceding return to pre-pregnancy biology. Further examination of these elements as they interact with other drivers of postpartum physiology will be important for animal models and clinical care.