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.

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.

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.

(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.

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.

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.

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.