Vascular Endothelial Growth Factor Delivery to Placental Basal Plate Promotes Uterine Artery Remodeling in the Primate

Abstract

Extravillous trophoblast (EVT) uterine artery remodeling (UAR) promotes placental blood flow, but UAR regulation is unproven. Elevating estradiol (E₂) in early baboon pregnancy suppressed UAR and EVT vascular endothelial growth factor (VEGF) expression, but this did not prove that VEGF mediated this process. Therefore, our primate model of prematurely elevating E₂ and contrast-enhanced ultrasound cavitation of microbubble (MB) carriers was used to deliver VEGF DNA to the placental basal plate (PBP) to establish the role of VEGF in UAR. Baboons were treated on days 25 to 59 of gestation (term, 184 days) with E₂ alone or with E₂ plus VEGF DNA-conjugated MBs briefly infused via a maternal peripheral vein on days 25, 35, 45, and 55. At each of these times an ultrasound beam was directed to the PBP to collapse the MBs and release VEGF DNA. VEGF DNA-labeled MBs per contrast agent was localized in the PBP but not the fetus. Remodeling of uterine arteries >25 µm in diameter on day 60 was 75% lower (P < 0.001) in E₂-treated (7% ± 2%) than in untreated baboons (30% ± 4%) and was restored to normal by E₂/VEGF. VEGF protein levels (signals/nuclear area) within the PBP were twofold lower (P < 0.01) in E₂-treated (4.2 ± 0.9) than in untreated (9.8 ± 2.8) baboons and restored to normal by E₂/VEGF (11.9 ± 1.6), substantiating VEGF transfection. Thus, VEGF gene delivery selectively to the PBP prevented the decrease in UAR elicited by prematurely elevating E₂ levels, establishing the role of VEGF in regulating UAR in vivo during primate pregnancy.

During early human and nonhuman primate pregnancy, placental extravillous trophoblasts (EVTs) migrate to, invade, and replace the smooth muscle and vascular endothelial cells within the uterine spiral arteries, thereby remodeling and increasing lumenal area of these vessels (1–5). Consequently, spiral arteries change from high-resistance/low-capacity to low-resistance/high-capacity vessels, which increases uterine artery blood flow and promotes fetal development. Defective uterine artery remodeling (UAR) underpins the etiology of pregnancy disorders that comprise the syndrome of ischemic placental disease (6–10)—for example, preeclampsia, fetal growth restriction, and preterm birth (11–18)—which is associated with uterine artery blood flow impedance, maternal systemic vascular dysfunction, and maternal and neonatal morbidity/mortality (19–26). However, despite the absolute importance of UAR to successful pregnancy, the regulation of UAR has not been clearly established.

In vitro studies have shown that numerous growth factors, including vascular endothelial growth factor (VEGF)-A, IGF-II, epidermal growth factor (EGF), heparin-binding EGF, TNF-α, and others, can stimulate primary, immortalized, and malignant trophoblasts to migrate across synthetic membrane or form endothelial-like tubes (27–34). However, in other in vitro studies several of these factors, including VEGF (35–37), did not stimulate EVT migration or tube formation. Thus, which, if any, of these growth factors has a physiologically essential role in vivo in regulating UAR is unknown. Most importantly, considering the highly complex interplay of different cell types and molecular events that occur in vivo during UAR, it is unclear whether EVT migration and tube formation as assessed in vitro validly mirrors the process of UAR as it occurs in vivo. This represents a fundamental gap in knowledge, considering the essential role that UAR has in normal and abnormal pregnancy.

Clinical studies have shown that maternal serum levels of the truncated receptor sFlt-1 that binds to and suppresses bioavailability of VEGF were increased, and those of placental growth factor decreased preceding onset of the complications, for example, maternal vascular dysfunction, of preeclampsia (38–47). However, the clinical utility of screening for the latter and other biomarkers to predict preeclampsia has met with limited success (44). Moreover, placental expression and serum levels of sFlt-1, VEGF, IGF-I, EGF, heparin-binding EGF, and other peptides are either decreased, increased, or unaltered at term in pregnancies compromised by preeclampsia and fetal growth restriction (27, 30, 38, 39, 41, 45–51), which may simply be a consequence, not cause, of the pathophysiology elicited by these conditions. Importantly, because UAR was not simultaneously examined in these studies, the regulatory role of these factors on UAR has not been established in human pregnancy.

Systemic adenoviral delivery of sFlt-1 induced (41, 52), and adenoviral delivery of VEGF₁₂₁ in BPH/5 (53), sFlt-1 (54–56), and placental ischemia (57–59) rodent and sheep models prevented, maternal vascular dysfunction and fetal demise typical of preeclampsia, but UAR was not determined. Systemic administration to mice of an antibody that paradoxically neutralized both Flt-1 and sFlt-1 decreased uterine artery length and blood flow but not fetal weight (60). Induction of uteroplacental ischemia by aortic or uterine artery ligation increased maternal sFlt-1 levels and elicited renal vascular dysfunction in rodents (61) and baboons (62), but UAR was not examined. Thus, animal studies have focused on the clinical manifestations of adverse pregnancy but not UAR (63–65). Moreover, there are significant differences in placental morphology, the process of UAR, uterine vascular anatomy, and the maternal–placental–fetal endocrine interrelationships between rodents and humans (33, 66–69), making translation to the human uncertain. In sum, the regulation of UAR, particularly the role of VEGF, has not been established.

Our laboratory has been instrumental in establishing the baboon as a primate model for the study of human placental and fetal development. Humans and baboons exhibit similar anatomy, physiology, and ontogeny of the fetal–placental unit (67, 70) and share >96% DNA/genetic homology (71, 72). Using the baboon as a translational model, we have shown that the low level of ovarian estradiol (E₂) during the first trimester of normal pregnancy is essential for promoting UAR (73, 74). Thus, simply shifting the normal rise in E₂ from the second third to the first third of pregnancy suppressed UAR, and this was associated with a decrease in EVT VEGF expression, although this did not prove that VEGF mediated this process. Therefore, the early E₂-treated baboon provides a novel primate experimental model in which the temporal rise in levels of an endogenous hormone fundamental to pregnancy is simply advanced to investigate the regulation of UAR. Contrast-enhanced ultrasound (CEU)–mediated cavitation of acoustically active microbubble (MB) carriers (CEU/MB) has emerged as an innovative strategy to target delivery of genes in vivo to specific tissues (75–79). In the current study, therefore, our nonhuman primate model of prematurely elevating E₂ and CEU/MB was used to selectively deliver VEGF DNA to the placental basal plate (PBP) during early baboon pregnancy to establish the role of VEGF in regulating UAR.

Materials and Methods

Animals

Female baboons (Papio anubis) were obtained from the Southwest National Primate Research Center (San Antonio, TX), housed individually in large primate cages, and received standard primate chow (Teklad Primate Diet 2050; Envigo, Frederick, MD) and fresh fruit twice daily, and water ad libitum. Females were paired with male baboons for 5 days at the anticipated time of ovulation as estimated by menstrual cycle history and the pattern of external sex skin turgescence. Day 1 of pregnancy was designated as the day preceding perineal deturgescence. Baboons were cared for and used strictly in accordance with the United States Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th ed.). The present experimental protocol was approved by the Institutional Animal Care and Use Committees of the University of Maryland School of Medicine and Eastern Virginia Medical School.

Maternal baboons were untreated (n = 11), treated daily on days 25 to 59 of gestation (term, 184 days) with E₂ benzoate [25 µg/kg body weight/d subcutaneously (SC) in 1.0 mL of sesame oil, n = 11], or treated daily on days 25 to 59 with E₂ benzoate (25 µg/kg body weight/d SC) plus VEGF DNA and enhanced green fluorescent protein (GFP) DNA (n = 6) delivered to the PBP via CEU/MB as detailed below. At least eight randomly selected areas (5 mm³) of PBP tissue (which includes anchoring villi, trophoblastic shell, and decidua basalis) were collected following cesarean section performed under isoflurane anesthetization on day 60 of gestation. Blood samples (2 to 4 mL) were obtained from a maternal peripheral saphenous vein at 1- to 2-day intervals throughout the study period after sedation with ketamine HCl (10 mg/kg body weight, intramuscularly) and from the uterine veins at cesarean section for E₂, progesterone, cortisol, and VEGF assay.

CEU/MB VEGF delivery

Cationic lipid-encapsulated/decafluorobutane-filled MBs (∼2 µm diameter, zeta potential of 40 to 60 mV in 1 mM KCl) were charge conjugated with a bicistronic plasmid vector encoding VEGF₁₂₁ (Fig. 1) and GFP (synthesized by Nature Technology, Lincoln, NE) at a saturation ratio of 20 µg/10⁸ MBs (79) and 0.5, 2.4, or 4.5 × 10⁹ MBs with attached VEGF/GFP DNA/kg body weight/h infused (1.0 mL saline/min) into a maternal saphenous vein of ketamine-propofol–sedated baboons for 10 minutes on days 25, 35, 45, and 55 of gestation as illustrated in Fig. 2. VEGF₁₂₁ is a VEGF-A isoform that is abundantly expressed in the placenta, lacks a membrane-bound motif, and thus is readily secreted/freely diffusible and induces association of the type 2 (Flk-1) VEGF receptor with heparin sulfate, which potentiates the magnitude and duration of VEGF signaling (80). A 2.0- to 6.0-MHz 6C2 transducer (Siemens Acuson Sequoia 512; Siemens, Malvern, PATeklad Primate Diet 2050; Envigo, Frederick, MD) with high B-mode frequency and a resting mechanical index (that is, negative acoustic pressure/square root of frequency) of 0.2 was positioned on the abdomen during MB/VEGF DNA infusion, and the ultrasound beam was directed to the PBP, identified by two-dimensional images of the placental chorionic villi, gestational sac, and decidua. The mechanical index was then increased to 1.9 with repeated 5-second burst pulses (three bursts per minute) during the 10-minute delivery period to collapse the MBs and thus detach/release the VEGF DNA within the PBP region (Fig. 2). The ultrasound waves used to cavitate the MBs induce sonoporation (i.e., transient pores in the cell membrane and active cell uptake), thereby facilitating DNA uptake into cells (81–83). Between days 25 and 55 of baboon pregnancy the placental disc/basal plate diameter increases from ∼2.0 to 3.5 cm, which corresponds to the ultrasound sector width at the acoustic focus.

Figure 1.

Vector map of the NTC8685–human (h)VEGF₁₂₁–IRES–EGFP. CMV, cytomegalovirus; EGFP, enhanced GFP; HTLV-1, human T-cell leukemia virus type 1; IRES (ECMV), internal ribosomal entry site (from the encephalomyocarditis virus); PAS, primosomal assembly site; PAS-BH, primosomal assembly site sequence on the pBR322 H strand; PAS-BL, primosomal assembly site sequence on the pBR322 L strand; pUC, plasmid University of California; SV, simian virus; trpA, tryptophan A; VA RNAI, virus-associated RNA I, a type of noncoding RNA that plays a role in regulating translation.

Figure 2.

Illustration of CEU/MB-targeted delivery of the VEGF gene to the PBP of E₂-treated baboons during early pregnancy.

Quantification of UAR

PBP samples were fixed in 10% formalin, embedded in paraffin, sectioned (5 µm), and processed for hematoxylin and eosin histology and cytotrophoblast/epithelial cell–specific cytokeratin immunocytochemistry. Light microscopy (Nikon Eclipse E 1000 M; Nikon, Tokyo, Japan) and an image analysis system (IP Laboratory, version 3.63; Scanalytics, Fairfax, VA) were used to analyze all arteries in each tissue sample and categorize them into groups with diameters of <25, 26 to 50, 51 to 100, and >100 µm as performed in our laboratory (73). Arterial diameter was measured via an eyepiece micrometer as the smallest distance across the center of the vessel lumen from the inside edge of the surrounding smooth muscle (not invaded) or cytotrophoblast (invaded) layers. Each observed artery/arteriole was considered a separate vessel, although any single vessel may have appeared more than once as it traversed a given tissue section. The number of arterioles/arteries exhibiting EVT invasion in hematoxylin and eosin sections was quantified by identifying spiral arterioles/arteries in which the vessel wall was extensively occupied by trophoblasts. Vessel invasion was confirmed by demonstration of positive cytokeratin immunostaining in corresponding serial sections. The level of UAR is expressed as the number of vessels exhibiting EVT invasion divided by the total number of vessels counted.

Cytokeratin immunocytochemistry

PBP tissue sections were microwaved in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval, incubated in H₂O₂ to inhibit endogenous peroxidase, and blocked with 5% normal horse serum (Millipore Sigma, St. Louis, MO). Tissues were incubated overnight at 4°C with mouse monoclonal antibody to cytokeratin [12.5 mg/mL dilution, 345779, CAM 5.2; BD Biosciences, San Jose, CA (84)] and then incubated for 1 hour at room temperature with biotinylated anti-mouse IgG [Vector Laboratories, Burlingame, CA (85)] and for 1 hour at room temperature with avidin–biotin–peroxidase complex (Vectastain Elite ABC horseradish peroxidase, Vector Laboratories). Tissue sections were developed using diaminobenzidine and Harris hematoxylin. Negative control for immunocytochemistry was the substitution of nonimmune mouse serum (Invitrogen/Thermo Fisher Scientific, Waltham, MA) for primary antibody.

GFP immunofluorescence

Placental plate tissue sections were microwaved in 0.01 M sodium citrate buffer (pH 6.0), incubated overnight at 4°C with polyclonal rabbit antibody to GFP [1:1000 dilution; A11122; Invitrogen/Thermo Fisher Scientific (86)], and then incubated for 1 hour at room temperature with Alexa Fluor donkey anti-rabbit secondary antibody [Invitrogen/Thermo Fisher Scientific (87)]. The sections were coverslipped with mounting media containing 4′,6-diamidino-2-phenylindole. Negative controls for immunofluorescence included tissue sections from animals not infused with plasmid VEGF/GFP and substitution of rabbit IgG isotype [Invitrogen/Thermo Fisher Scientific (88)] for primary antibody.

Proximity ligation assay of VEGF

Paraffin-embedded PBP tissue sections (5 µm thick) were subjected to 0.01 M sodium citrate and serum block and incubated overnight at 4°C with rabbit polyclonal primary VEGF antibody [1:250 dilution, RB 9031, P1611F; Thermo Fisher (89)]. Tissue was then incubated for 90 minutes at 37°C in a humidity chamber with proximity ligation assay (PLA) probes consisting of two secondary anti-rabbit antibodies each tagged with an oligonucleotide [diluted 1:5 in antibody diluent supplied in the Duolink In Situ PLA kit (Millipore Sigma) as described previously (74, 90)]. A ligation solution (supplied in the PLA kit and diluted 1:5 in water) consisting of two oligonucleotide linkers, complementary to each PLA probe and ligase (diluted 1:40 in the ligation solution), was added to tissue and incubated for 30 minutes at 37°C in a humidity chamber. Tissue sections were washed and incubated for 100 minutes with amplification solution (supplied in the PLA kit and diluted 1:5 in H₂O) consisting of nucleotides and fluorescently labeled oligonucleotides and polymerase (diluted 1:80 in the amplification solution). The ligation step connects the hybridization linkers to the PLA probe oligonucleotides, forming a complete circle connecting both antibodies, and the polymerization step uses rolling circle DNA amplification to generate a concatemeric oligonucleotide product linked to the antibody complex. The fluorescently labeled oligonucleotides hybridize to the rolling circle amplification product, and the resultant signal is visible as a fluorescent dot. The tissues were then washed in decreasing concentrations of Tris-based buffers (PLA kit wash buffers A and B). Following the last wash, the PLA sections were incubated overnight at 4°C with cytokeratin (for cell-specific localization of VEGF) followed the next day by a 1-hour incubation with Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen/Thermo Fisher Scientific). The sections were then washed, air dried, and coverslipped with mounting media containing 4′,6-diamidino-2-phenylindole for detection of the nuclei. VEGF protein red PLA signals were visualized by fluorescence microscopy (Nikon Eclipse E 1000) and image analysis software (IP Laboratory). PLA signals were quantified and expressed per pixelated nuclear area using MetaMorph software (version 7.8.0.0; Molecular Devices, San Jose, CA) in a minimum of five randomly selected areas (32,350 µm²) of the distal region of anchoring villi, trophoblastic shell, and trophoblasts within spiral arteries. Negative controls for PLA included substitution of rabbit IgG isotype for the primary antibody or omission of one PLA probe.

Serum steroids and VEGF

Serum E₂, progesterone, and cortisol levels were quantified by an Immulite competitive chemiluminescent enzyme immunoassay (91–93) and VEGF (free, bioavailable) levels by Quantikine ELISA [R&D Systems, Minneapolis, MN (94)]. For the Immulite immunoassays, the intra-assay and interassay CVs were 6.9% and 7.3%, respectively, for E₂, 7.6% and 7.9% for progesterone, and 6.3% and 10.5% for cortisol. For the Quantikine assay, the intra-assay CV was 5.4% for VEGF.

Statistical analysis

Data were expressed as the mean ± SE and analyzed by ANOVA with post hoc comparison of the means by a Newman–Keuls multiple comparison test or the Kruskal–Wallis test and a Dunn multiple comparison test.

Results

Serum steroid and VEGF levels and weights

Mean (±SE) maternal peripheral serum E₂ levels on days 25 to 60 of gestation were fivefold greater (P < 0.01) in E₂-treated (0.75 ± 0.06 ng/mL) and E₂ plus VEGF DNA–treated (0.81 ± 0.07 ng/mL) baboons than in untreated animals (0.15 ± 0.01 ng/mL). Maternal saphenous vein and uterine vein serum E₂ levels on day 60 were approximately fourfold and twofold greater (P < 0.01), respectively, in E₂-treated and E₂ plus VEGF–treated baboons than in untreated animals (Table 1). However, serum E₂ levels in the first trimester in E₂-treated baboons were twofold lower than in the second trimester of normal pregnancy (∼2.0 ng/mL).

Table 1.

Maternal Serum E₂ levels (ng/mL) and Placental, Maternal, and Fetal Body Weights in Baboons

Treatment	N	Saphenous Vein	Uterine Vein	Placental Weight, g	Fetal Weight, g	Maternal Weight, kg
Untreated	11	0.21 ± 0.03a	0.59 ± 0.08a	32.0 ± 1.8	12.6 ± 0.5	16.5 ± 0.7
E2	11	0.82 ± 0.05b	1.15 ± 0.05b	29.0 ± 1.1	11.3 ± 0.5	15.7 ± 0.5
E2 + VEGF	6	0.87 ± 0.07b	0.98 ± 0.06b	30.4 ± 2.1	13.6 ± 0.9	15.8 ± 0.5

Mean ± SE maternal saphenous and uterine vein serum E₂ levels and placental, maternal, and fetal body weights on day 60 of gestation in baboons untreated or treated daily on days 25 to 59 (term, 184 d) with E₂ (25 µg/kg body weight/d SC) or on days 25 to 59 with E₂ (25 µg/kg body weight/d) plus VEGF DNA delivered by CEU/MB to PBP on days 25, 35, 45, and 55.

^a,bValues marked with different letters are statistically different at P < 0.01.

Mean (±SE) maternal serum progesterone levels in untreated and E₂-treated baboons were 12.2 ± 0.7 and 14.5 ± 1.2 ng/mL, respectively, in the saphenous vein on days 25 to 60 and 48.2 ± 3.9 and 47.6 ± 2.7 ng/mL, respectively, in the uterine vein on day 60. Maternal peripheral serum cortisol levels on days 25 to 60 were 32.8 ± 2.0 and 33.4 ± 1.8 µg/dL, respectively, in untreated and E₂-treated animals. VEGF was not detectable (limit of detection ≤2 pg/mL) in either maternal saphenous or uterine vein serum or plasma in untreated, E₂-treated, or E₂ plus VEGF DNA–treated baboons.

Placental and maternal and fetal body weights on day 60 of gestation were similar in untreated, E₂-treated, and E₂ plus VEGF–treated baboons (Table 1).

CEU/MB imaging and UAR

Figure 3 includes a grayscale Doppler image of the endometrium, placenta, and fetus (Fig. 3A) and CEU images showing the presence of VEGF DNA–labeled MBs/contrast agent in the endometrium and PBP but not the fetus before (Fig. 3B) and absence at the end (Fig. 3C) of a 5-second burst and collapse of the MBs to release VEGF DNA on day 45 of gestation. This visually confirms MB cavitation, which was repeated every 20 seconds to allow fresh MB wash-in.

Figure 3.

(A) Grayscale Doppler image of the endometrium (E), placenta (P), and fetus (F), and CEU images (B) before and (C) after collapse of the MBs/uncoupling of VEGF DNA on day 45 of gestation in an E₂-treated baboon.

Figure 4 shows representative histology of the chorionic floating villi, anchoring villi (which are the principal source of EVTs), trophoblastic shell, and decidua basalis. Illustrated in this figure are spiral arteries invaded by EVTs in an untreated baboon (Fig. 4A), arteries not remodeled in an E₂-treated baboon (Fig. 4B), and arteries invaded in an E₂ and VEGF DNA–treated baboon (Fig. 4C). The marked structural modification of the vessel wall after EVT invasion in the untreated baboon contrasts with the highly coiled structure of the noninvaded arteries in the E₂-treated animal. Importantly, spiral artery remodeling appeared to be restored by VEGF DNA delivery to E₂-treated animals.

Figure 4.

(A–C) Photomicrographs of hematoxylin and eosin histology of the basal plate (scale bars, 100 µm) and (D) percentage remodeling of uterine spiral arteries and arterioles (i.e., the number of vessels exhibiting EVT invasion divided by total number of vessels counted) classified by vessel diameter in baboons untreated or treated with E₂ daily on days 25 to 59 of gestation or with E₂ daily on days 25 to 59 plus VEGF DNA targeted by CEU to the PBP on days 25, 35, 45, and 55. Values with different lettering are statistically different (P < 0.05 to P < 0.01) from one other. AV, anchoring villi; DB, decidua basalis; FV, floating villi; SA, spiral artery; TS, trophoblastic shell.

When quantified by image analysis as shown in Fig. 4D, the mean (±SE) number of uterine spiral arteries invaded and remodeled by EVTs, expressed as a ratio of the total number of arteries counted, in untreated baboons for vessels with diameters of <25 µm (5.0% ± 2.3%), 26 to 50 µm (5.5% ± 1.4%), 51 to 100 µm (21.8% ± 7.1%), and >100 µm (63.5% ± 5.7%) was lower (P < 0.05 to P < 0.001) in E₂-treated animals (0.2% ± 0.1%, 0.7% ± 0.2%, 3.0% ± 1.1%, and 18.0% ± 4.3%, respectively). Importantly, the concomitant administration of E₂ and targeted delivery of VEGF DNA to the PBP by CEU/MB restored the level of UAR to values (6.2% ± 4.9%, 6.8% ± 3.5%, 15.2% ± 7.1%, and 43.2% ± 6.3%, respectively; Fig. 4D) greater (P < 0.05 to P < 0.01) than in animals treated with E₂ alone and similar to those in the untreated baboons. Thus, as shown in Fig. 5 the overall level of remodeling collectively of uterine arteries >25 µm in diameter was ∼75% lower (P < 0.001) in E₂-treated (7% ± 2%) than in untreated baboons (30% ± 4%) and restored by E₂ plus VEGF treatment to a level (22% ± 5%) greater (P < 0.05) than in animals treated only with E₂ and similar to that exhibited in the untreated animals. The overall level of vessel remodeling appeared to be similar when 0.5, 2.4, or 4.5 × 10⁹ VEGF-conjugated MBs/kg body weight/h were infused in E₂-treated baboons, indicating a maximal response at the lowest dose of VEGF administered. In contrast to the changes in vessel remodeling, we have previously shown that the total number of spiral arteries of different sizes within the decidua basalis was similar in untreated and E₂-treated baboons (95).

Figure 5.

Percentage remodeling of uterine spiral arteries collectively >25 µm in diameter on day 60 of gestation for the baboons in which artery invasion is shown in Fig. 4. Values with different lettering are statistically different (P < 0.05 to P < 0.001) from each other.

VEGF quantification by PLA

GFP was localized as green fluorescent dots in trophoblasts within uterine spiral arteries after VEGF/GFP delivery to E₂-treated baboons (Fig. 6A), but they were not present in uterine arteries of untreated baboons that did not receive VEGF/GFP (Fig. 6B). To confirm the efficacy of in vivo transfection and treatment effects, VEGF protein levels were quantified by PLA in the PBP. VEGF protein was detected by PLA as small red immunofluorescent signals in cells of the distal anchoring villi (Fig. 7A), trophoblastic shell (Fig. 7B), and trophoblasts within vessels, that is, uterine spiral arteries (Fig. 7C). In contrast to the abundant expression of VEGF in untreated baboons, VEGF expression in E₂-treated animals appeared to be lower in the distal region of the anchoring villi (Fig. 7D), trophoblastic shell (Fig. 7E), and trophoblasts that have migrated to and invaded the spiral arteries (Fig. 7F). Importantly, VEGF protein appeared to be restored in each of these locations in E₂ plus VEGF–treated animals (Fig. 7G–7I). Thus, when quantified by PLA as the number of immunofluorescent signals per pixelated nuclear area × 10⁴ collectively within the distal anchoring villi, trophoblastic shell, and trophoblasts within vessels, VEGF protein levels were more than twofold lower (P < 0.01) in E₂-treated (4.2 ± 0.9) than in untreated baboons (9.8 ± 2.8) and restored to normal after VEGF gene delivery to E₂-treated animals (11.9 ± 1.6, Fig. 8). When rabbit IgG isotype was substituted for the primary VEGF antibody, the mean (±SE) number of PLA signals per nuclear area × 10⁴ was 0.1 ± 0.0 in the PBP tissue, confirming specificity of the PLA method and VEGF antibody.

Figure 6.

Photomicrographs of GFP localization (green dots) in trophoblasts within uterine spiral arteries on day 60 of gestation in (A) baboons treated with E2 plus VEGF/GFP DNA and (B) untreated animals. Nuclei are labeled blue. Scale bars, 25 µm.

Figure 7.

Photomicrographs of the localization of red PLA VEGF immunofluorescent signals in the distal anchoring villi (DAV), trophoblastic shell (TS), and trophoblasts in vessels (TIV) on day 60 of gestation in (A–C) untreated baboons and baboons (D–F) treated with E₂ or (G–I) treated with E₂ plus VEGF DNA. Each red PLA signal represents a single VEGF protein molecule. EVTs are labeled green (cytokeratin). Nuclei are labeled blue. Scale bars, 25 µm.

Figure 8.

VEGF protein levels quantified by PLA as analyzed collectively in the distal anchoring villi, trophoblastic shell, and trophoblast within uterine spiral arteries of the PBP on day 60 of gestation in baboons untreated, treated with E₂, or treated with E₂ plus VEGF DNA. Values with different lettering are statistically different (P < 0.05 to P < 0.01) from each other.

Discussion

The current study shows that delivery of the VEGF gene selectively to the PBP prevented the decrease in UAR induced by prematurely elevating E₂ in early baboon pregnancy. Moreover, the current study showed that levels of VEGF were reduced in the PBP of baboons in which UAR was suppressed by early E₂ treatment when compared with untreated animals in which endogenous E₂ levels are very low, whereas VEGF levels were restored to normal in baboons concomitantly treated with E₂ and the VEGF gene. Therefore, the current study establishes that VEGF has a pivotal role in vivo in regulating EVT invasion and remodeling of the uterine spiral arteries in nonhuman primate pregnancy.

A multitude of growth and related factors have been shown in cell culture to stimulate primary or immortalized trophoblasts or cancerous chorionic villous cells to migrate across semipermeable synthetic membrane or aggregate into tube-like structures (27–34). However, because of the difficulty in replicating in vitro the complex physiological, paracrine, and stereospecific interplay of different cell types and cellular remodeling processes that occur in vivo, it has not been established which, if any, of the many factors studied in vitro has a physiologically relevant role in vivo in early pregnancy in regulating EVT migration, invasion, and remodeling of the uterine spiral arteries. In the current study, VEGF delivery alone prevented the striking decline in uterine spiral artery transformation experimentally induced in early baboon pregnancy, thereby establishing its fundamentally important role in the latter process. Because VEGF promotes cell migration, remodeling, and proliferation mechanisms, including integrin expression (96), it is likely that the observed impact of VEGF on vessel transformation is mediated by integrin, matrix metalloproteinase, cell adhesion, and related cell remodeling molecules. Indeed, we have shown that expression of α₁β₁ and α₅β₁ integrins (74) and metalloproteinase-9 (97) within the PBP of E₂-treated baboons was reduced, coinciding with a decrease in VEGF expression.

Demonstration of a regulatory role for VEGF on UAR in vivo in a well-established nonhuman primate model is highly significant from a basic science perspective considering the importance of UAR in promoting uterine artery blood flow and fetal development. Thus, we have shown that compared with untreated controls, uterine artery and fetal umbilical artery downstream flow impedance was increased, uterine artery volume flow was reduced (98), and fetal body weight was ∼10% lower (97) near term in vasochallenged baboons in which UAR had been suppressed by prematurely elevating E₂ in early pregnancy. The results of the current study are also significant from a clinical perspective considering the impact that defective spiral artery remodeling has in underpinning the devastating consequences of adverse conditions of human pregnancy, notably preeclampsia, fetal growth restriction, and preterm birth (19–26). The current study is foundational for future investigation of the potential ability of VEGF delivery to prevent the appearance of the maternal vascular impairments associated with defective UAR and placental perfusion.

A limitation of the current study is the absence of administration of CEU/blank plasmid–labeled MBs to E₂-treated baboons to assess potential effects of CEU acoustic pressure on spiral artery remodeling. However, because CEU/VEGF-conjugated MB delivery reversed and did not further alter the reduction in UAR observed in E₂-treated baboons, it seems unlikely that negative acoustic pressure alone elicited by CEU accounts for the restoration of vessel remodeling achieved by CEU VEGF delivery. Moreover, the CEU procedure to collapse the MBs and deliver VEGF was performed for a short period of time and on only 4 days of gestation. Additionally, CEU/MB burst at the same level of mechanical index/acoustic pressure employed in baboons of the current study did not alter the constant level of maternal blood flow/flux rate into the intervillous space between 6 and 12 weeks of human pregnancy (99) or the level of spiral artery perfusion/flux rate induced by maternal protein restriction in rhesus macaques (100). Ultrasound acoustic pressure bursts as employed in the current study also did not cause maternal complement activation or placental apoptosis, inflammation, or oxidative stress during middle and late macaque gestation or placental microvascular hemorrhage or syncytiotrophoblast microvilli disruption during the first trimester of human pregnancy (101). In addition, extensive data showed no evidence of petechiae, fibrosis, or proangiogenic effects in rodents exposed to MB reporter control plasmid delivery and CEU acoustic pressure bursts (76, 78, 79, 102, 103). We propose that early noninvasive real-time detection of defective UAR in early abnormal pregnancy when combined with CEU/MB VEGF delivery specifically to the PBP point to the potential therapeutic value of CEU/MB gene delivery in adverse conditions of human pregnancy.

UAR is initiated early in gestation and proceeds rapidly during the first trimester of pregnancy, coinciding with low levels of E₂ that are of ovarian origin. The rate of UAR then diminishes thereafter, coinciding with a progressive increase in E₂ levels, which are now synthesized by the placenta. In the current study, shifting the rise in E₂ from the second to the first trimester of baboon pregnancy repressed PBP VEGF expression and UAR, effects reversed by VEGF gene delivery. We further propose, therefore, that UAR is temporally coordinated by E₂ and that: (i) the low level of E₂ in the first trimester of pregnancy promotes placental EVT VEGF expression and thus a rapid rate of spiral artery remodeling (Fig. 9A and 9B), and (ii) the increase in E₂ with advancing pregnancy, as experimentally elicited by prematurely elevating E₂ in the first trimester, has a physiologically important role in repressing EVT VEGF formation and thus the extent to which the uterine arteries are remodeled (Fig. 9C and 9D). A restraint in the rate of UAR is physiologically important because extensive invasion and remodeling of the uterine arteries upstream, for example, in placenta acreta (104–106), impairs uterine artery vasomotor tone after delivery, a pathophysiological condition that causes excessive bleeding at the time of delivery.

Figure 9.

Proposed role of VEGF (A and B) in promoting EVT migration and UAR during normal pregnancy and (C and D) in mediating the repression of EVT migration and UAR induced by prematurely elevating estrogen in early primate pregnancy.

The experimental paradigm of prematurely elevating maternal serum E₂ levels to ∼1 ng/mL in early baboon pregnancy was employed as an approach to investigate the regulation of UAR. However, maternal serum E₂ levels are elevated to a similar nanogram range (i.e., ∼3 ng/mL) by superovulation during human in vitro fertilization (107–109), a procedure associated with an increased incidence of preeclampsia and fetal growth restriction (107–112), adverse conditions arising from improper placental perfusion. Freezing and implanting superovulated ova in a subsequent normal menstrual cycle in the presence of much lower levels of E₂ diminished the risk of abnormal pregnancy (108). Moreover, maternal exposure to endocrine disruptors that mimic E₂ action at the estrogen receptor site (113) and that are present in maternal serum in high levels during human pregnancy (114, 115) are thought to disrupt reproductive function and cause fetal growth restriction (116). Indeed, treatment of mice in early gestation with the endocrine disruptor bisphenol A at 50 µg/kg body weight, a level comparable to the daily human intake exposure (117), impaired spiral artery remodeling (118). Although an increase, decrease, or no change in E₂ levels has been shown at mid-gestation and term in human preeclampsia (119–121), this may secondarily reflect aberrant placental function elicited by this adverse condition of pregnancy.

VEGF DNA arriving in the uterine arteries during infusion of baboons was expected to be detached from the MBs by the increase in acoustic pressure and transcribed/translated into RNA/protein within tissues fed by the arterial effluent. The abundant expression of VEGF protein observed in trophoblasts within the spiral arteries, distal ends of the anchoring villi, and trophoblastic shell of E₂ plus VEGF DNA–treated baboons is consistent with the latter expectation and confirms VEGF transfection. VEGF transfection in tissues other than within the PBP is unlikely because MBs do not cross the syncytial border into the villous placenta or fetus [Fig. 3 and Hua et al. (122) and Arthuis et al. (123)], cell sonoporation was targeted to the PBP by the CEU beam, and free plasmid DNA is rapidly degraded by DNAase within the bloodstream (124, 125). Although cells in addition to EVTs may well have been transfected by VEGF—for example, fibroblasts and immune cells within the PBP and endothelial cells lining the spiral arteries—this would not confound the results because endogenous VEGF expressed by such cells as well as by EVTs may have a role in UAR.

We have previously shown that the volumes (i.e., mass) of the placental villous cytotrophoblasts and syncytiotrophoblast, as well as the level of placental villous cytotrophoblast 5-bromo-2′-deoxyuridine and Ki67 immunostaining reflecting proliferation, were unaltered by E₂ treatment in early baboon pregnancy (126). Additionally, maternal serum levels of progesterone, which is produced primarily by the villous placenta after days 20 to 25 of baboon pregnancy (127, 128), and cortisol produced by the adrenal (129) were similar in untreated and E₂/VEGF-treated baboons of the current study. Because the E₂ receptor is also expressed in the latter tissues, these findings suggest that the regulation of uterine spiral artery remodeling by E₂ reflects a direct action of E₂ on VEGF expression within the PBP and does not secondarily involve changes in placental villous trophoblast or adrenal structure or function.

A major advantage of CEU/MB gene delivery is that the VEGF-conjugated MBs did not cross the syncytia border and enter the villous placenta and fetus [Fig. 3B and Hua et al. (122) and Arthuis et al. (123)]. However, because the syncytiotrophoblast is bathed in maternal blood, once detached from the collapsed MBs, VEGF may enter the villous placenta. This would not confound the results of the current study, as we have previously shown that in contrast to the E₂-induced decline in VEGF expression by the EVTs, expression of VEGF is increased in placental villous trophoblasts by prematurely elevating E₂ levels in early baboon pregnancy (130, 131). The divergent effects of E₂ on VEGF expression by the extravillous and villous compartments are also exhibited in other estrogen-responsive tissues, including the breast and prostate (132). The differential regulation of VEGF by E₂ in the extravillous and villous placenta may reflect the cell-specific presence of estrogen receptor subtypes, as well as functional enhancers, coactivators and/or corepressors that modulate E₂-regulated target gene transcription (133–135).

In summary, VEGF gene delivery selectively to the PBP by CEU/MB prevented the decrease in UAR elicited by prematurely elevating E₂ levels in early baboon pregnancy, indicating that VEGF has a major role in vivo in regulating EVT invasion and remodeling of the uterine spiral arteries during nonhuman primate pregnancy. We propose that the low level of ovarian E₂ in the first trimester of pregnancy promotes placental EVT VEGF expression and consequently a rapid rate of UAR and the progressive rise in placental E₂ levels during the second and third trimesters, as experimentally induced by prematurely elevating E₂ in the first trimester, and has an important role in repressing EVT VEGF formation and thus the extent of UAR to promote a physiologically normal level of uteroplacental perfusion.

Glossary

Abbreviations:

CEU

contrast-enhanced ultrasound

CEU/MB

contrast-enhanced ultrasound–mediated cavitation of acoustically active microbubble carriers

E₂

estradiol

EGF

epidermal growth factor

EVT

extravillous trophoblast

GFP

green fluorescent protein

MB

microbubble

PBP

placental basal plate

PLA

proximity ligation assay

SC

subcutaneously

UAR

uterine artery remodeling

VEGF

vascular endothelial growth factor