GESTATIONAL MODIFICATION OF MURINE SPIRAL ARTERIES DOES NOT REDUCE THEIR DRUG-INDUCED VASOCONSTRICTIVE RESPONSES IN VIVO

Abstract

Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery–like structures to dilated vein-like structures between gestation days (gd)8 and12 in mice and weeks 12–16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 pre-spiral arterial remodeling and gd12 (post-remodeling) to a range of concentrations of adenosine (ADO, 10⁻⁸–10⁻⁶M), acetylcholine (ACh, 10⁻⁷–10⁻⁵M), phenylephrine (PHE, 10⁻⁷–10⁻⁵M) and angiotensin II (AngII, 10⁻⁸–10⁻⁶M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to ADO was attenuated in uterine, arcuate and basal arteries while constrictor activity to AII was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and be relevant to ongoing human pregnancies.

INTRODUCTION

Pregnancy presents unique cardiovascular challenges. One such challenge is development of vascular support for the growing placenta, the organ responsible for exchange of nutrients and waste products between fetal and maternal circulations. Adequate blood flow and placental development are critical to fetal and maternal health. For example, 3 to 5% of pregnant women develop the hypertensive syndrome pre-eclampsia (PE) [1] and require emergency, often pre-term, delivery of the placenta and its associated fetus. A key step suspected in development of PE is restriction of blood flow to the placenta associated with incomplete spiral arterial (SA) remodeling [2]. Although blood flow to the human placenta can be measured [3], the determinants controlling blood flow to the placenta are undefined.

Control of vascular perfusion within tissue is generally attributed to small arteries and arteries [4, 5]. In skeletal muscle, control of capillary recruitment and tissue perfusion at rest and during contraction is attributed to the terminal arteriolar microvessels [6–10]. Arteriolar vascular diameters are environmentally influenced by neural, humoral and locally released tissue factors. It is hypothesized that local tissue influences increase when skeletal muscle increases its metabolism, thus, linking tissue metabolism and blood flow (for review see [11, 12]).

Maternal blood flow to the placenta travels through the uterine artery terminal microvasculature that includes the uterine, arcuate, radial and spiral arteries (SA) which feed the conceptus and basal arteries which feed the endometrium [2, 13, 14] (Figure 1A). Maternal SA undergo significant structural modification and enlargement about midgestation. Modifications appear to be initiated by uterine Natural Killer (uNK) cells [15–19] and completed by intramural and intravascular trophoblast cells in both humans and mice [2, 20, 21]. Because of the unique cyclic rebuilding of the terminal microvasculature of the uterine artery due to menstrual and estrous cycles and following pregnancy, it cannot be assumed that regulatory mechanisms defined in skeletal muscle will apply to the uterine microvasculature during pregnancy.

Figure 1. Schematic representation and photomontages of the uterine vasculature.

A) Schematic representation of the gd12 uterine arterial tree. Nomenclature follows the convention of Ramsey and Donner (1980; human)[14], Adamson et al. (2002; mouse)[13] and Osol and Mandela (2009; human and mouse)[62]. Bold arrow indicates direction of blood flow. DB: decidua basalis; MLAp: mesometrial lymphoid aggregate of pregnancy. B) Photomontage of the uterine vasculature at gd8 using intravital microscopy. Arterial endothelium was stained with Alexa Fluor 488 conjugated to Isolectin from Griffonia simplicifolia. Bar=100μm. C) (i): Typical appearance of the mesometrial side of a gd8 implant site dually stained with DBA lectin and PAS. Uterine artery (UA) entry is positioned uppermost; representative coils are marked by arrowheads. Smaller diameter arteries (cut in cross section) near the 2 layered myometrium (Myo) include arcuate and radial arteries. SA (examples are boxed) occur in the decidua basalis (DB) closer towards ectoplacental cone trophoblasts (T). Embryo cavity (EC), residual uterine lumen and DBA+ yolk sac endothelium are identified. Two high power inserts of SA (insert1 and 2) show the thickness of the walls (outlined) relative to the lumen and the proximity of numerous PAS+DBA− (purple only) and PAS+DBA+ (purple plus brown) uNK cells. Diameters of uNK cells in DB are known to be greater than diameters of uNK cells nearer the myometrium [57]. Bars: (i) 100um (insert 1and 2) 20um. D) Photomontage of the uterine vasculature at gd12 using intravital. Arterial endothelium were stained as in (B). Bar =100μm. E) (i): Typical appearance of the mesometrial side of a gd12 implant site dually stained with DBA lectin and PAS. Entry of the now dilated uterine artery is positioned uppermost. Differentiation of the uNK cell-enriched MLAp at each implant site separates the two layers of myometrium and rings the uterine artery. The DB and the mature placenta (PL) are identified. SA in the DB are boxed, red arrows indicate SA. High power image (1) shows the uterine artery in the MLAp and its association with PAS+DBA− (purple only) and PAS+DBA+ (purple plus brown) uNK cells. The uterine artery is outlined to highlight the thickness of wall artery, relative to the lumen. High power image (2) shows the remodeled SA from the center of DB and its associated PAS+DBA+ (purple plus brown) uNK cells. PAS+DBA− uNK cells are rare in this region by gd12.5. The SA ratio wall/lumen ratios are reduced compared to gd8 (images are at the same magnification). Bars: (i) 100um (insert 1 and 2) 20um.

Most studies addressing regulation of the uterine vasculature use explanted vessels tested as sliced rings or by wire myography. For the latter studies, surrogate vessels such as omentum are frequently used. At present no methodology studies maternal uterine microvasculature reactivity in their native environment. Here, we report the development of a model to visualize intact, in vivo, blood perfused, decidual, terminal arteriolar microvessels using intravital microscopy. We determined the reactivity of each level of the uterine microvasculature to agonists at gestation day (gd) 8, prior to SA remodeling (Figure 1C) and gd12, post-remodeling (Figure 1E). The results in intact animals are discordant with those from arterial explant studies. We show that the radical modification of spiral arteries necessary to enhance blood flow to the placenta during fetal development does not sacrifice contractile function of this vessel.

MATERIALS AND METHODS

Animals

C57BL/6/NCrl (B6) wildtype mice (n=30) from Charles River (St. Constant, QU) were housed under normal husbandry at the University of Guelph. Nulliparous female mice aged 8–14 weeks were paired overnight with syngenic males. Presence of a copulatory plug in the morning was considered gd0 and mated females were housed individually until used for experiments. Food and water were provided ad libidum. All experimental protocols were approved by the University of Guelph’s Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.

Surgical preparation for in vivo intravital microscopy of the endometrial vasculature

Mice (gd8 or gd12) were anaesthetized with 70mg/kg sodium pentobarbital ip. Mice underwent a tracheotomy and catheterization whereby polyethelene catheters (internal diameter ~25–35μm) were inserted into the right jugular vein for supplemental anaesthetic (70mg/kg), the left common carotid artery to monitor mean arterial pressure (MAP) and the left femoral vein for administration of fluorescent dye (see below). Mice were placed supine on a lucite board fitted with a heated water coil at 42°C to maintain oesophageal temperature at 37°C. A 2cm incision was made ventrally to gain access to the uterus, originating from the caudal-most nipples and bisecting the abdomen. One uterine horn was selected and exteriorized. The mouse was turned onto the right side and the uterine horn secured to a custom-made silicone (Siligard, WPI, Sarasota, Florida) pedestal at both ends with insect pins. Gd8 preparations were oriented such that the anti-mesometrial surface was laid against the silicone and pinned so the vasculature entered the implantation site from the ‘top’, this orientation that allowed for unobstructed blood flow and ideal visualization of the vasculature. For gd12, the fetus had to be separated from the uterine tissue (due to its size) in order to orient the preparation as described above. To remove the fetus, an incision was made along the anti-mesometrial side of the uterine horn. The anti-mesometrial tissue was retracted to expose three amniotic sacs. The embryo proper and amniotic membranes were excised from these implantation sites to facilitate the necessary tissue orientation of the center site of interest on the silicone pedestal. The uterine tissue was secured in place with insect pins through the anti-mesometrial uterine tissue remaining.

The exteriorized uterus was continuously superfused with a bicarbonate-buffered physiological salt solution (PSS) containing (mmol/L) NaCl 131.9, KCl 4.7, CaCl₂ 2.0, MgSO₄ 1.2 and NaHCO₃ 30, maintained at 37°C and equilibrated with a 5% CO₂-95% N₂ gas mixture to a pH of 7.4 ± 0.05. The superfusate was supplemented with 10⁻⁹M isoproterenol (Sigma-Aldrich, St. Louis, MO, USA) to decrease uterine smooth muscle motility [22].

Once the surgical preparation was complete, the mouse was transferred to the microscope and the vasculature was allowed to acclimate for 45–60mins prior to experimental protocols.

Experimental protocols

15mins prior to the experimental protocols, a 0.4ml bolus injection of fluorescent dye Alexa Fluor 488 conjugated to Isolectin GS-IB₄ from Griffonia simplicifolia (166μg/ml; Invitrogen, CA) to label the endothelial cells of the microvasculature was administered via the femoral catheter. The full volume was injected over 10–15secs. A slight increase in MAP was typical following the bolus injection but MAP was quickly reestablished to normal.

For all experimental protocols, the fluorophore was excited with a 100 Watt mercury-arc lamp using a 480±20nm narrow band pass filter and emissions recorded using a 535±30nm narrow bandpass filter. Fluorescent emissions were collected with a linear 12-bit black and white cooled CCD camera (Roper Coolsnap HQ, Photometrics, Tuscon, AZ, USA) mounted on a modified Olympus BX51WI microscope (Olympus Corp., Tokyo, Japan) using either a 4X objective (numerical aperture (na) 0.13) or a 10X (na 0.30) long working distance objective with a 1.6X magnification changer. Final magnification of the vasculature was ~1000x.

Prior to drug application, a set of low magnification images was taken of the vascular network (similar to those in Figure 1B and D) to ensure consistency in the classification of branch order. Images were captured at high magnification of each site of interest for measurement of baseline diameter. Images were captured using Image Pro Plus software (Media Cybernetics, Bethesda, MD) and saved for offline analysis. The endometrial tissue was then topically exposed to the lowest concentration of a randomly selected agonist via the superfusate. The agonists used were: AngII (10⁻⁸mol/L-10⁻⁶mol/L) and PHE (10⁻⁷mol/L-10⁻⁵mol/L) as vasoconstrictors, ACh (10⁻⁷mol/L-10⁻⁵mol/L) and ADO (10⁻⁸mol/L-10⁻⁶mol/L) as vasodilators. Each preparation was tested with one or 2 agonists; for the latter case, one vasodilator and one vasoconstrictor were randomly selected, as was agonist application order. The initial concentration of agonist was applied via the superfusate (agonist + PSS) for 2mins (4mins for AngII) and then images were captured at each vessel segment of interest in the continued presence of the agonist. The subsequent concentration was immediately applied for 2mins (4mins for AngII) and again images were captured at each vessel of interest. This was repeated once more for the final concentration. The agonist was then washed out with PSS for one hour prior to administration of the second agonist, when applicable. At the completion of the protocols, maximum vasodilations were induced with 10⁻⁴mol/L SNP and images of each vessel segment of interest captured.

Histology

Archived, paraffin-embedded implantation sites (4% paraformaldehyde fixed by perfusion) from gd8 and gd12 B6 mice previously reported as typical [23] were sectioned at 6μm, dually stained with DBA lectin and periodic acid Schiff’s (PAS) reagent to identify uNK cells as described elsewhere [24], or with TRITC-conjugated DBA lectin (Vector Laboratories Inc., Burlington, ON, Canada) and Alexa-fluor 488-conjugated anti-mouse actin, (eBioscience, Inc, San Diego). For immunofluorescence, sections (n=2 pregnancies and 4 total implantation sites/gd) were deparaffinizated, hydrated, washed with PBS, blocked 30 mins with Bovine Serum Albumin (BioShop) 1% diluted in PBS, washed and incubated overnight (4°C) with anti-mouse actin (10ug/ml). Sections were then washed and incubated with TRITC-DBA lectin (5ug/ml) for 40 min at room temperature. Nuclei were visualized by DAPI staining using ProLong Gold anti-fade reagent containing DAPI (Invitrogen Life Technology, Burlington, ON, Canada).Tissues were studied, photographed and annotated using an AxioImager M.1 microscope and Axiovision software (Carl Zeiss, Oberkochen, Germany).

Data and statistical analysis

Vessel diameters were measured using calibrated Image Pro software. All data are expressed as mean±SEM. Arterial diameters are expressed as change in diameter from baseline in micrometers (μm). Only one artery of each branch order per endometrial preparation was used and n indicates the number of arteries observed. Group means were compared first with a 2 way ANOVA to determine interactions between drug concentrations and gd, where applicable. Subsequently a repeated measures ANOVA was performed for each variable. When significance reached p≤0.05, a protected least squares difference test was used post hoc to test group means. Due to differences observed in baseline diameters (Table 1), linear regression analysis was used to determine correlations between either the initial baseline diameter or the dilatory potential (maximum diameter – baseline diameter) of the arteries and the response to the lowest dose of each drug.

Table 1. Average baseline and maximum diameters for all branch orders of the uterine microvasculature.

Diameters are expressed as mean ± SEM.

Branch	gd8				gd12
	Baseline (μm)	Baseline as % Max	Maximum (μm)	n	Baseline (μm)	Baseline as % Max	Maximum (μm)	n
Uterine	67.7 ± 2.6	79.6 ± 2.5	85.2 ± 2.5@	29	72.2 ± 3.2	88.9 ± 2.9*	81.6 ± 3.4@	39
Arcuate	60.1 ± 3.4	79.8 ± 3.8	76.0 ± 3.8@	29	76.0 ± 3.7*	93.8 ± 3.5*	81.3 ± 3.1	39
Radial	58.2 ± 3.0	80.4 ± 2.6	72.5 ± 3.0@	29	73.2 ± 4.4*	92.8 ± 2.4*	79.0 ± 4.5@	39
Spiral	63.2 ± 3.8	81.5 ± 3.3	77.8 ± 3.8@	29	126.8 ± 3.3*	95.2 ± 3.0*	134.2 ± 3.9*	39
Basal	28.0 ± 2.5	75.4 ± 3.7	37.6 ± 3.2@	29	36.1 ± 2.5*	82.5 ± 5.1	42.8 ± 1.8@	39

^@ denotes significantly different from baseline diameter at gd8 or gd12.

^*denotes significantly different from gd8.

Note: Basal artery baseline and maximum diameters were always significantly smaller than all other branch orders within each gd. At gd12, SA baseline and maximum diameters were always significantly larger than all other branch orders.

RESULTS

Visualization and branch order classification of the endometrial vasculature

Isolectin GS-IB₄ from Griffonia simplicifolia (specific for terminal α-D-galactosyl moieties) conjugated to AlexaFluor 488 brightly visualized B6 endothelium of the mesometrial uterine and decidual vasculature permitting identification of vessel branch order and diameter measurements (Fig. 1B and 1D). In skeletal muscle vasculature (cremaster), preliminary studies showed that isolectin GS-IB₄ AlexaFluor 488 dye had no effects on baseline diameters or on agonist-induced responses.

Fig. 1B and 1D depict photo montages of myometrial vessels from of gd8 and gd12 implant sites. The uterus is supplied by the main uterine artery loop within the mesenteric connective tissue; branches from this main supply segmentally enter the myometrium and supply microvascular units to mucosa and, during pregnancy, to implant sites. These branches are termed uterine arteries; within the uterus they branch transversely into arcuate arteries (Fig. 1A). The arcuate arteries supply daughter branches, radial arteries that penetrate deeper into the myometrium. Near the boundary of the decidua basalis, radial arteries branch into both SA which supply the fetus, and basal arteries which provide flow to endometrial tissue.

Localized areas of constriction were commonly observed at many levels of the microvasculature (Fig. 2) at gd12 but rarely at gd8. These appear similar to the radial artery circumferential bands noted in gd14.5 CD1 mice by Adamson et al., (2002) using vascular casting. We observed that the focal constrictions were static in position and decreased the diameter of the vessel by 21.3±2.4%. The focal constrictions were reactive, decreasing diameter in response to 10⁻⁶M PHE (19.5±3.1%) to the same degree as the vessel diameter near the focal constriction (21.3±2.4%). The focal constrictions vasodilated in response to 10⁻⁴M sodium nitroprusside (SNP, a nitric oxide donor) to produce maximal dilation but only dilated to 79.2±3.4% of the diameter of its resident vessel.

Figure 2. Representative images of the localized constrictions observed at different levels of the endometrial vasculature at gd12.

White arrows indicate the localized areas of constriction in an A) uterine artery, B) radial artery and C) basal artery. Bars = 100μm.

Vascular baseline and maximum diameters

At gd8, baseline diameters across all branch orders were similar except the basal arteries were significantly smaller (Table 1). At gd8, maximum diameters following SNP administration were all significantly greater for all branch orders than the respective baseline diameters. At gd8, all levels of the uterine arterial tree rested at ~80% of maximum diameter.

At gd12, baseline diameters of all branch orders were significantly greater than at gd8 except the uterine artery. Maximum diameters for all branch orders were not significantly different between gd12 and gd8 except the SA. At gd12, resting arteries were more dilated/less vasoconstricted than at gd8 as the resting diameters represented a significantly greater percentage of maximum diameters for each branch order (calculated as ~10% increase; Table 1). In stark contrast to the other branch orders, SA had ~2 fold increase in both baseline and maximum diameters at gd12 compared to gd8. This large change indicates extensive SA growth and/or remodeling and confirms the correct classification of branch orders. Of note, baseline diameter of the SA at gd12 was less than maximal (as determined by comparing baseline diameter to diameter in the presence of the maximal dilating concentrations of SNP).

Significant differences in baseline diameters within each branch order between gd8 and gd12 may suggest that the smooth muscle of the vessel wall is at a different length and may possibly affect the response to agonists. The lack of correlations between baseline diameters and the measured agonist-induced responses (average r² values for gd8 were 0.32 ± 0.07 and for gd12 were 0.21 ± 0.06) indicated that differences in baseline diameters (i.e. smooth muscle length) within branch orders between gd8 and gd12 were not important determinants of the measured agonist-induced responses.

Uterine vascular responses to vasoactive agonists at gd8 and gd12

Vasodilatory reactivities of the uterine vasculature were assessed with ACh and ADO. Given the resting baseline diameter of gd12 vessels (Table 1), very little dilatory potential (maximum diameter-baseline diameter) was available (maximum diameter was determined using 10⁻⁴M SNP). Exposure to the highest concentration of either dilator caused the vessels to reach maximal diameter. ACh, an endothelial cell-dependent vasodilator, induced significant vasodilations (Fig. 3A–3E) with no significant difference between gd8 and gd12 for any arterial branch order. Conversely, ADO reactivity tended to decrease in uterine, arcuate and radial arteriearteries at gd12 compared to gd8, while SA and basal arterial reactivities to ADO remained constant (Fig. 3F–3J).

Figure 3. Comparison of gd8 and gd12 changes in endometrial artery diameters in response to topically applied ACh (A–E) and ADO (F–J).

The arterial response to the application of ACh (10⁻⁷mol/L – 10⁻⁵mol/L; gd8: n=7, gd12: n=8) in 2 minute intervals on A) Uterine artery, B) Arcuate artery, C) Radial artery, D) Basal artery and E) Spiral artery. The arterial response to the application of ADO (10⁻⁷mol/L – 10⁻⁵mol/L; gd8: n=8, gd12: n=9) in 2 minute intervals on F) Uterine artery, G) Arcuate artery, H) Radial artery, I) Basal artery and J) Spiral artery. All responses are significantly different from baseline unless marked by a +. α denotes significantly different (p<0.05) from gd8 at the same concentration. Error bars represent standard error.

AngII induced significant vasoconstrictions in all branch orders observed at both gds. Fig. 4 displays high magnification images of gd12 AngII responsive radial, basal and SA. Gd12 SA showed similar, potent reactivity to all concentrations of AngII, as did radial and basal arteries while AngII reactivity within gd12 uterine and arcuate arteries was significantly increased compared to gd8 (Fig. 5A and 5E). Reactivities to increasing concentrations of PHE were generally similar at gd8 and gd12 (Fig. 5F–5J), except for arcuate arteries that showed increased sensitivity. Again, the gd12 SA remained similarly and significantly reactive to all concentrations of PHE tested.

Figure 4. Representative images of different levels of the endometrial vasculature at baseline and constricting to 10⁻⁵M PHE at gd12.

A) and B) show a radial artery before and after constriction with 10⁻⁵M PHE respectively. C) and D) show a SA before and after constriction with 10⁻⁵M PHE respectively. E) and F) show a basal artery before and after constriction with 10⁻⁵M PHE respectively. Bars = 100μm.

Figure 5. Comparison of gd8 and gd12 changes in endometrial artery diameters in response to topically applied AII (A–E) and PHE (F–J).

The arterial response to the application of AII (10⁻⁸mol/L – 10⁻⁶mol/L; gd8: n=7, gd12: n=8) in 2 minute intervals on A) Uterine artery, B) Arcuate artery, C) Radial artery, D) Basal artery and E) Spiral artery. The arterial response to the application of PHE (10⁻⁷mol/L – 10⁻⁵mol/L; gd8: n=7, gd12: n=14) in 2 minute intervals on F) Uterine artery, G) Arcuate artery, H) Radial artery, I) Basal artery and J) Spiral artery. All responses are significantly different from baseline unless marked by a +. a denotes significantly different (p<0.05) from gd8 at the same concentration. Error bars represent standard error.

Gd12 spiral arteries lack detectable smooth muscle actin

Due to the unexpected vasoreactive responsiveness of modified spiral arteries, immunohistochemical studies were undertaken to determine whether vascular smooth muscle expressing cells still persisted at gd12. Strong actin expression was detected in the myometrium of all specimens and served as a positive control. At gd8 and 12, walls of the uterine artery were smooth muscle actin reactive (Fig. 6A and 6B). The walls of uterine artery branches just inside of the myometrium (putatively identified as radial arteries by size and location) contained some actinreactive smooth muscle cells at gd8 and gd12 (Fig. 6C and 6D). Substantial numbers of uNK cells were associated with these vessels on gd12. Spiral arteries identified by location at gd8 expressed actin; however, gd12 spiral arteries did not (Figure 6E–H). At both, gd8 and 12, uNK cells appeared to interact with cells of the spiral arterial walls and lumens.

Figure 6. Double Immunofluorescence using Alexa-fluor 488- anti- mouse actin (green) and TRITC-DBA lectin (red) on the gd8 (A,C,E,G) and gd12 (B,D,F,H) implantation sites.

In all images, the mesometrium is towards the top. Uterine arterial smooth muscle cells at gd8 and gd12, (A and B respectively), were strongly reactivity with anti-actin. Additionally in (B), reactivity was present in the myometrium (lower left). Myometrium is not present in (A) and the upper brightness is a tissue fold. In A) three uterine artery segments are shown, while in B), only one segment is present. Putative radial arteries (*; C and D), at gd8 and gd12 respectively are shown in the MLAp, just below the anti-actin stained myometrium (white arrows). Radial arteries displayed less anti-actin reactivity (arrowhead) than uterine arteries. Much greater numbers of uNK cells (red) are present in the gd12 MLAp (representative cells are marked with yellow arrows) than in this nascent region at gd8 (C) when uNK cells are more common in deciduas basalis. In E–H, spiral arteries (#) are shown in decidua basalis at gd8 or gd12. Actin is detected in the spiral artery smooth muscle cells (arrowhead (E, G)), at gd8, but was undetectable at gd12 (F,H). (G and H) are higher magnification images of different vessels at gd8 (G) and gd12 (H). At both times, uNK cells (yellow arrows) are observed interacting with arterial wall cells and on gd12.5 with endothelium. Erythrocytes within the arterial lumens are autofluorescent. DAPI stained nuclei in all of the tissue sections are blue. Bars A–F= 50μm, G–H= 20μm. Vein- Vn.

DISCUSSION

The process of gestational structural modification of human and mouse spiral arteries has been addressed extensively using ultrasonography, histopathology and explant studies but an understanding of the integrated functional capacity of the uterine arterial tree has not previously been available. We successfully developed a pregnant mouse preparation for in vivo viewing of the uterine vasculature that permits functional comparisons between segments of this microvascular tree. We characterized the baseline features of this circulatory unit and the responses of its subcomponents (uterine, arcuate, radial, basal and SA) to agonists at times prior to histological detection of SA remodeling and after histological evidence for completion of SA remodeling [19, 25]. A strength of this intravital microscopy preparation is that all native physiological regulators of vessel diameter remain intact, including humoral, neuronal and local mediators. For example, arterial diameters were characterized within the MLAp, an area highly enriched in uNK cells that have perivascular, intramural and intravascular locations [26] (Figure 1C and 1E). The uNK lineage includes angiogenic subsets with potential vasoregulatory roles producing NO (mouse [27]), ANP (mouse [28]), vascular endothelial and placental growth factors in mouse and humans (for example see [29–32]), and vasodilatory cytokines (TNFa: in rat [33], and mouse [34]; interferon gamma: in mouse [35]). Another major advantage of this preparation is that agents of interest can be applied to the uterine circulation rather than systemically, to mimic local in vivo events that are more complex than reflected in culture.

Retention of physiological relationships between cells is important because baseline arterial diameters are established by complex interactions of multiple stimuli: signals arising from adjacent “in contact” cells, local signals arising within the tissue such as shear stress and paracrine mediators, and signals derived external to the site (such as circulating hormones, cytokines, chemokines, antigens, epigenetic regulators and neurotransmitters). Tone was observed in all segments of the uterine arterial tree before and after SA remodeling. The transition from early to mid-pregnancy increased baseline but not maximal diameters in all segments except the SA. The latter showed increased maximal diameters after modification. These observations indicate a systematic vasodilation at each level of the microvasculature which would decrease uterine vascular resistance, data consistent with the known endometrial hemodynamics during this period [36].

Our data show that each level of the uterine microvasculature was responsive to ACh at both gd8 and gd12 with no significant change in sensitivity between gestation days of any branch order of the uterine microvasculature. An increase in sensitivity to ACh has been shown between the non-pregnant state and the pregnant state in the mesenteric uterine artery of different species using different vascular preparations [37–41] and there is conflicting data as to what occurs throughout pregnancy. Pulgar et al. (2011) [42] using wire myography did not see a change in reactivity of uterine arteries to ACh between mid and late gestation in mice while Bell (1973) [43], using perfused uterine artery vessels observed an increased sensitivity throughout gestation in guinea pigs. Our data would indicate that if there are changes in endothelial cell NO-dependent vasodilation that they are not due to events occurring around spiral artery modification.

The reactivity of the endometrial microvasculature to ADO in pregnancy has not been well defined. Studies suggest that ADO is a potent vasodilator of the mesenteric uterine artery in the non-pregnant state [44] in an endothelial-independent manner [45] although there are reports of ADO vasodilation through NO dependent mechanisms [46]. We show that the uterine, arcuate and radial arteries significantly decrease their reactivity to ADO by gd12. In skeletal muscle, ADO is an important mediator of local metabolic control of arterial diameter (for review see [47], with the source of ADO being skeletal muscle cells themselves. The source of ADO in the uterus is not known although, during pregnancy, maternal plasma ADO is known to increase [48]. The importance of the change in reactivity to ADO will depend on sources and abundance of ADO throughout conceptus development.

We observed that each level of the uterine microvasculature constricted in the presence of PHE at both gd8 and gd12 and the arcuate and radial arteries became more responsive to PHE from gd8 to gd12. Mesenteric uterine and branches from the mesenteric uterine artery have been shown to be more sensitive to PHE in pregnancy than in the non-pregnant state [49–53] although this observation is controversial [37, 41, 54–56]. Both the arcuate and radial arteries are located deeper in the MLap region which may be locally affected differently than vessels higher in the MLap (uterine artery) region or predominantly in the decidua basalis (such as basal arteries and spiral arteries). These changes in reactivities of vessels in the MLAp coincide with high uNK localization in this region and may represent uNK cell-regulated effects (see Fig. 1C and 1E). Both the arcuate and radial arteries are located more towards the longitudinal uterine muscle layer and visceral peritoneum. This location may be a distinctive environment to that of vessels (uterine artery) higher in the MLAp, a region enriched in immature, proliferative uNK cells or predominantly in decidua basalis (such as basal and SA), a region enriched in mature, post mitotic uNK cells [57]. Thus, changes in blood vessel reactivity may depend on the specific locations of the vessels within the endometrium and its resident influences.

In line with reports from mice [42] and other species (human: [58]; rat: [51, 52, 59]), we detected a significant increase in AngII reactivity within the endometrial microvasculature. The uterine and arcuate arteries increased their sensitivity to AII between gd8 and gd12. As mentioned above, hese changes in reactivities of vessels in the MLAp coincide with high uNK localization in this region and may represent uNK cell-regulated effects. Since NK cells possess a functional renin-angiotensin system (RAS) [60] uNK cells may be potential mediators of paracrine communication with the uterine microvasculature. RAS signaling promotes chemotactic effects on lymphocytes [60, 61], which may represent a means of promoting maintenance of uNK cells in close proximity to these arteries, and maintaining paracrine communication within the MLAp.

We observed focal constrictions at multiple levels of the uterine vasculature at gd12 and have further shown that focal constrictions are vasoactive. The development of focal constrictions from gd8 to gd12 could represent the beginning of a fundamental shift in blood flow control specific to implantation sites. Supporting this fundamental paradigm shift in control of blood flow, we found that gd12 SA remained reactive (despite confirmed extensive remodeling and loss of smooth muscle actin between gd8 to gd12) and gained reactivity to AII following modification. Therefore, further investigation into both focal constrictions and SA reactivity post-modification will be required in order to understand their importance to blood flow control throughout fetal development.

Perspectives

The major contributions reported herein are: 1) the development of an intravital microscopy technique specifically adapted to visualize the in vivo reactivity of multiple levels of the endometrial microvasculature during mouse gestation; 2) the uterine vasculature becomes less vasoconstricted under baseline conditions between gd8 to gd12; 3) the arteries located within the MLAp show increases in AngII sensitivity and decreases in ADO sensitivity between gd8 to gd12 and 4) uNK cell-induced SA remodeling does not affect SA reactivities to any agonist tested. These data provide unique insights into the physiological vasoregulatory patterns of the uterine vasculature during mouse mid-gestation in regions that are dramatically modified over pregnancy and under the unique paracrine influences of uNK cells and trophoblasts. These findings challenge our current understanding of the functions of physiologically modified SA and should be considered when assessing modified human SA.