Placental villous vascular endothelial growth factor expression and vascularization after estrogen suppression during the last two-thirds of baboon pregnancy

Abstract

We have recently shown that placental cytotrophoblast vascular endothelial growth factor (VEGF) expression and vessel density were increased by elevating estrogen and decreased by suppressing estrogen in early baboon pregnancy. The present study determined whether the elevation in estrogen which occurs in the last two-thirds of baboon pregnancy also has a role in the regulation of placental villous VEGF expression and angiogenesis. Placentas were obtained on day 170 of gestation (term, 184 days) from baboons untreated or treated with the aromatase inhibitor CGS 20267 or CGS 20267 plus estradiol daily on days 30-169. Serum estradiol levels in CGS 20267-treated baboons were decreased (P < 0.001) by 95%, however, placental cytotrophoblast VEGF mRNA levels (means +/- SE, attomoles/microg RNA) were similar in untreated (25,807 +/- 5,873), CGS 20267-treated (23,900 +/- 1,940) and CGS 20267 plus estradiol-treated (26,885 +/- 2,569) baboons. VEGF mRNA levels in the syncytiotrophoblast (2,008 +/- 405) and inner villous stromal cell (1,724 +/- 287) fractions of untreated baboons also were not altered by CGS 20267. However, whole villous VEGF mRNA levels in CGS 20267-treated baboons (18,590 +/- 2,315) were 4-fold greater (P < 0.001) than in untreated animals and restored to normal by estradiol. Percent vascularized area (15.88 +/- 0.88%) and vessel density (1,375 +/- 71/mm(2)) of the villous placenta in untreated animals were not altered by estrogen deprivation. We propose that villous cytotrophoblasts lose their responsivity to estrogen and that placental villous cytotrophoblast VEGF expression and angiogenesis are regulated by estrogen in a cell- and gestational age-specific manner, and that factors other than estrogen maintain VEGF expression in the last two-thirds of pregnancy.

Fig. 1

Maternal peripheral serum estradiol concentrations in baboons untreated (•, each data point is the mean of n = 4 animals) or treated daily with CGS 20267 (0.1–0.2 mg/day, sc, ○, n = 6) or CGS 20267 plus estradiol (0.1–0.2 mg/day, sc, Δ, n = 3) between days 30 (60) and 169 of gestation (term = 184 days)

Fig. 2

Representative competitive RT-PCR of VEGF in placental cytotrophoblasts of an untreated baboon on day 170 of gestation. Total RNA (75 ng) was mixed with 3-fold serial dilutions of VEGF CRS as depicted in Panel A. Samples were reverse transcribed and amplified for 26 cycles in the presence of specific primers. (A) The 323-bp target and 256-bp CRS products were separated via agarose gel electrophoresis and stained with ethidium bromide. (B) Intensities of the amplified products were analyzed by densitometry and the log of the ratios of VEGF CRS and target areas was plotted as a function of CRS added to each PCR reaction. Lines were constructed by linear regression analysis and VEGF mRNA levels determined from the equivalence points (i.e. intersection of the vertical with regression lines)

Fig. 3

VEGF mRNA levels in placental syncytiotrophoblast, inner villous core cells, and cytotrophoblasts isolated by Percoll gradient centrifugation and whole villous tissue on day 170 of gestation from baboons untreated or treated daily with CGS 20267 or CGS 20267 plus estradiol (E₂) benzoate between days 30 (60) and 169 of gestation (see footnote of Table 1). Values represent the means (± SE) of 3 to 6 baboons for each cell fraction and each treatment group (except syncytiotrophoblast from baboons treated with CGS 20267 plus E₂, n = 2). *Different at P < 0.001 from untreated control within respective tissue fraction (ANOVA and Newman–Keuls multiple comparisons test)

Fig. 4

Representative photomicrographs of VEGF immunocytochemistry (brown precipitate) in the villous placenta on day 170 of gestation in baboons untreated (A) or treated on days 30–169 with CGS 20267 (B). (C) VEGF immunocytochemistry when the primary VEGF antibody was preabsorbed with recombinant human VEGF. C, cytotrophoblast nucleus; S, syncytiotrophoblast nucleus; Ve, vascular endothelium. Magnification, ×200

Fig. 5

Percent vascularized area, i.e. ratio of total vessel area and total villous area examined, of placental villous tissue obtained on 170 of gestation from baboons untreated or treated daily with CGS 20267 or CGS 20267 plus estradiol (E₂) benzoate as detailed in the footnote of Table 1. Each data point represents the mean (± SE) determined on 20–40 different tissue sections from a single placenta

Fig. 6

Vascular density determined in the villous placenta of the same baboons on which placental vascularization data are shown in Fig. 5. Values represent the means (± SE) of 3 to 6 placentas per group

Fig. 7

Size distribution of blood vessels determined in the villous placenta from baboons untreated or treated with CGS 20267 or CGS 20267 plus estradiol (E₂). Values are the means (± SE) of 3 to 6 placentas per group