BIOLOGY OF REPRODUCTION 64, 1821–1825 (2001)
Relationship Between Placental Vascular Endothelial Growth Factor Expression and Placental/Endometrial Vascularity in the Pig1 Kimberly A. Vonnahme, Matthew E. Wilson,3 and Stephen P. Ford2 Department of Animal Science, Iowa State University, Ames, Iowa 50011 ABSTRACT We investigated the temporal association between placental vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis and vascular permeability, and changes in placental/endometrial vascularity on selected days throughout gestation in the pig. Placental and endometrial tissues were collected from sows on Days 25 (n 5 4), 36 (n 56), 44 (n 56), 70 (n 55), 90 (n 55), and 112 (n 57) of gestation. Cross sections of the placental/endometrial interface of each conceptus were used to estimate the number of blood vessels per unit area via image analysis and the intensity of VEGF staining via immunohistochemistry. Placental tissues were also collected on these days to evaluate VEGF mRNA expression. Placental VEGF mRNA expression and the numbers of blood vessels per unit area of placental and adjacent endometrial tissue were low and decreasing from Day 25 to Day 44, before increasing (P , 0.05) markedly and progressively through Day 112. These data are consistent with the marked increase in VEGF immunostaining in the chorionic and uterine luminal epithelium from early to late gestation. Further, these increases in placental VEGF mRNA were positively correlated with fetal weight (r 5 0.73; P , 0.0001) and placental efficiency (fetal weight/placental weight ratio; r 5 0.66, P , 0.0001). These data are consistent with a role for VEGF in increasing the number of blood vessels at the placental endometrial interface, resulting in an increased capacity for nutrient transfer from the maternal to the fetal compartment.
conceptus, developmental biology, female reproductive tract, placenta, pregnancy
MATERIALS AND METHODS
INTRODUCTION
Animals
After microvillar attachment of the porcine conceptus to the uterine wall on about Day 18 [1], there is a progressive increase in conceptus estrogen production from Day 20 to Day 30 of gestation [2]. There is an increased permeability of placental cells to water [3], resulting in a rapid increase in chorioallantoic (CA) fluid volume during this period. This expansion of conceptus volume allows the CA membranes to establish an intimate contact between the placenta and the uterine wall [2, 4]. From Day 30 to Day 50, CA fluid volume declines as a pronounced interdigitation occurs between the chorionic and uterine luminal epithelium Supported in part by Hatch Act and State of Iowa funds. This article is journal paper no. J-19055 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Projects 0239 and 3505. 2 Correspondence: Stephen P. Ford, Department of Animal Sceince, Iowa State University, 2356F Kildee Hall, Ames, IA 50011. FAX: 515 294 4471; e-mail:
[email protected] 3 Current address: Division of Animal & Vet Science, West Virginia University, Morgantown, WV 26505. 1
Received: 24 October 2000. First decision: 12 December 2000. Accepted: 7 February 2001. Q 2001 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
[4, 5]. The increasing metabolic demands of the rapidly growing fetus from Day 50 to term result in additional increases in placental size and surface area during this period [3, 4, 6]. Vascular endothelial growth factor (VEGF), a secreted 45kDa disulfide-linked dimeric heparin-binding glycoprotein, is produced from a large eight-exon pre-mRNA [7]. This premRNA can be alternatively spliced into four different isoforms: VEGF121, VEGF165, VEGF189, and VEGF206. All isoforms are present in the placenta throughout gestation in the sheep [8] and the rat [9]. Other workers have proposed that the VEGF121 and VEGF165 isoforms are secreted and act to increase permeability of vessels [10, 11], whereas VEGF189 and VEGF206 remain membrane bound and may serve to facilitate maintenance of existing vessels. Hypoxia is a potent stimulus for VEGF production [12–14]. It is logical to assume that as nutrient and particularly oxygen demands by the rapidly growing fetus increase after Day 50, both the placental and the adjacent endometrial tissues would become hypoxic, potentially upregulating the synthesis and secretion of VEGF. The production of VEGF is stimulated by estrogens in the rat [15], sheep [16], and human [17]. Placental estrogen secretion also increases markedly and progressively from Day 50 to term [6]. This study was conducted to determine the temporal associations among the levels of placental VEGF mRNA, placental/endometrial vascular development, and conceptus growth throughout gestation in the pig.
All procedures and protocols involving the use of animals were approved by the Iowa State University Committee on Animal Care. Thirty-three females of white composite breeding (Landrace, Large White, and/or Yorkshire) were slaughtered at the Iowa State University Meat Laboratory on Days 25 (n 5 4), 36 (n 5 6), 44 (n 5 6), 70 (n 5 5), 90 (n 5 5), or 112 (n 5 7) of gestation (Day 0 5 first day of estrus). Gravid uteri were collected and immediately placed on ice for transport back to the laboratory, where the mesometrium was trimmed from the tract. Small incisions were made on the antimesometrial side of each uterine horn to expose individual placentae. Each uterine horn was opened by connecting the incisions over each conceptus on the antimesometrial side. Each fetus had its umbilical cord double ligated and transected, and location, weight, and crown-rump length (CRL) were recorded. A 33 4-cm section of the uteroplacental interface located 2–3 cm from the umbilicus of each conceptus was excised, placed into a tissue cassette (Fisher Brand; Fisher Scientific, Pittsburgh, PA), and fixed in 10% neutral buffered formalin for histologic evaluation. An additional quantity (4 g) of placental tissue adjacent to the excised uteroplacental section was then collected and immediately snap frozen in liquid nitrogen for later RNA extraction. Individual placentae
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TABLE 1. Conceptus measurements on selected days of gestation in the pig. Day of gestation 25 36 44 70 90 112 a,b,c,d,e,f
No. litters
No. conceptuses
4 6 6 5 5 7
79 86 81 53 42 61
Fetal weight (g) 0.50 4.86 19.93 248.22 540.29 1363.45
6 6 6 6 6 6
CRL (cm)
0.01a 0.08b 0.32c 5.48d 18.40e 58.23f
2.76 4.72 8.25 18.01 22.90 30.14
6 6 6 6 6 6
0.03a 0.03b 0.06c 0.13d 0.31e 0.37f
Placental weight (g) 4.73 50.77 67.14 214.06 202.74 335.16
6 6 6 6 6 6
0.26a 1.86b 3.01c 10.88d 18.06d 22.76e
Mean 6 SEM within a column with different superscripts differ (P , 0.05).
were manually separated from the endometrium and weighed. To obtain a measure of placental efficiency (grams of placenta required to support 1 g of fetus) for each conceptus, the weight of each fetus was divided by the weight of its placenta. Histology and Vascular Density Determination
Uteroplacental tissue sections were fixed and embedded using procedures previously established in our laboratory [18] except that each sample was trimmed before a final rinse in 100% ethanol. Vascular density was determined for uteroplacental sections from seven conceptuses (randomly selected from four to seven litters) from each of the assigned slaughter days (i.e., Days 25–112 of gestation). Paraffin-embedded placental/endometrial tissues were sectioned at 5 mm, stained with periodic acid-Schiff reagent, counterstained with hematoxylin, and traced for quantitation of vascular measurements as previously described [19] with the following modifications. Images of two fields from each of four slides with stained tissues were projected onto a sheet of paper using a projecting microscope (XM150; Kramer Scientific, New York, NY) and the area occupied by the placental and endometrial tissues was traced. In these areas, the placental and endometrial vessels were traced so that the area and number of all vessels could be determined. Areas were then quantified via image analysis [19] and evaluated for the relative number of placental and endometrial vessels per unit tissue area. Placental VEGF mRNA Expression Probe. To examine the levels of VEGF mRNA in the pig placenta, a pig-specific probe was needed for use in the RNase protection assay. Primers for VEGF were designed using a known porcine VEGF nucleotide sequence (sense 59-CGGAATTCTGCTGTCTTGGGTGC-39 and antisense 59-CTGGATCCGCATAATCTGCATGG-39) [20]. Porcine placental RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Conditions were optimized, and the polymerase chain reaction (PCR) was performed. A 5-min extension time was added to the cycling program to insure ample addition of dATP to the 39 end of the PCR amplicon for ligation into the pCR2.1 plasmid (Invitrogen, Carlsbad, CA). The VEGF insert (containing sequence representing exons 1–5 and 7) was then subcloned into Bluescript SK 1/2 plasmid (Stratagene, La Jolla, CA). RNase protection assay. The in vitro transcription assay and RNase protection assay were performed as previously described [21] in our laboratory with the following exceptions. Plasmids containing either VEGF or pyruvate dehydrogenase (PD) were linearized with EcoRV or XbaI, respectively. Radiolabeled RNAs were synthesized from these linearized DNA templates with 10 U of T3 or T7
RNA polymerase according to the manufacturer’s protocol (Ambion, Austin, TX). Protected fragments resulted in two bands using the VEGF probe representing messages that coded for the VEGF165 fragment (exons 1–5 and 7) or the VEGF121, VEGF189, and VEGF206 fragments (exons 1–5). To quantitate VEGF mRNA levels for each placental sample (n 5 7/day), counts from both VEGF bands were summed and divided by the counts in the PD band to correct for slight differences in sample loading. To correct for between-gel variation, the ratio of VEGF to PD was further divided by the average ratio of VEGF to PD in a placental pool sample included in each assay and therefore as a percentage of control. Although the pattern of VEGF165 was positively correlated (r 5 0.87; P , 0.0001) with the pattern of total VEGF mRNA expression, we cannot determine the exact patterns of VEGF isoforms 121, 189, and 206, and therefore we will only report the total amount of VEGF mRNA expression. Immunohistochemistry
Uteroplacental sections from each conceptus in the study (n 5 42) were analyzed via immunohistochemistry as previously described [18] for the localization and overall density of VEGF with the following modifications. VEGF immunostaining was accomplished using a polyclonal antirabbit antibody recognizing variants VEGF121, VEGF165, and VEGF189 (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted at 1:200 and incubated with tissue sections overnight at 48C. The presence of the primary antibody was detected by means of a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. In preliminary characterization experiments, we employed primary antibody preabsorbed with a limited amount of antigen. We confirmed in subsequent experiments that omission of the primary antibody served as a sufficient control. Therefore, a negative control consisted of omission of the primary antibody. Statistical Analysis
Data were analyzed using the general linear model procedure of SAS [22]. day of gestation was included in the class statement. Effects of day of gestation were tested for the variables fetal weight, CRL, placental weight, placental efficiency, and vessel number. Separation of means was accomplished using the least squared means procedure. P values of ,0.05 were considered significant. RESULTS
Data from a total of 402 conceptuses were collected over the course of gestation (Table 1). There were progressive increases in fetal weight, CRL, and placental weight from Day 25 through Day 112 of gestation. Increase in fetal
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PLACENTAL VEGF EXPRESSION DURING GESTATION IN THE PIG
sured components of placental/endometrial vascularity and their impacts on fetal and placental weight and placental efficiency. Placental VEGF mRNA was positively correlated (P , 0.0001) with fetal and placental weight and placental efficiency. Further, fetal and placental weights and placental efficiency were positively associated (P , 0.05) with the number of blood vessels per unit area at the placental/endometrial interface. Staining for VEGF in endometrial/placental sections was localized to the chorionic and uterine lumenal epithelium at all stages of gestation examined. Further, a marked increase in immunostaining was observed from early (Fig. 2B) to late (Fig. 2C) gestation, which is consistent with levels of VEGF mRNA expression in the pig placenta during the second half of gestation. DISCUSSION
FIG. 1. Changes in VEGF mRNA (A) and the relative number of blood vessels per unit area of placental tissue (B) throughout gestation in the pig. Mean and SEM with different superscripts within a measurement differ (P , 0.05).
weight accelerated as pregnancy advanced, with increases of 4.36 g from Days 25 to 36, 15.07 g from Days 36 to 44, 228.29 g from Days 44 to 70, 292.07 g from Days 70 to 90, and 823.16 g from Days 90 to 112. Levels of placental VEGF mRNA declined slightly from Day 25 to Day 36, before exhibiting a marked and progressive increase (P , 0.01) from Day 44 through Day 112 (Fig. 1A). Similarly, the number of vessels per unit area of placental tissue also declined from Day 25 through Day 44, declining slightly by Day 44 before increasing progressively from Day 44 to Day 112 of gestation (P , 0.05; Fig. 1B). Further, the blood vessel number per unit area of placenta was correlated with the increasing amounts of VEGF mRNA in the same placenta (r 5 0.34; P , 0.05) and number of blood vessels per unit area of endometrium (r 5 0.34; P , 0.05) from Day 25 to Day 112 (Table 2). Table 2 also depicts the associations between the mea-
Placental VEGF mRNA expression increases progressively from Day 44 through Day 112 of gestation in the pig in association with marked increases in the number of blood vessels (placental and endometrial) at the fetal/maternal interface. This increase in the number of blood vessels per unit area would allow for a greater surface area of nutrient and waste product exchange between the sow and her rapidly growing offspring [23]. Although not significantly different, a visual decline in both placental VEGF mRNA and the number of placental blood vessels occurs from Day 25 to Day 44 of gestation. Although this decline may be an artifact, it may be associated with the continued growth of the placenta during this period, resulting in decreased vascular density in the face of a decline in placental estrogen secretion [6]. The decline in placental blood vessel numbers may reflect either a decreased angiogenic stimulus or a reduction in the maintenance of existing vessels. Further, the similarity in the proliferation of blood vessels in the placenta and the adjacent endometrium of each conceptus throughout gestation suggests a coordinated and conceptus-specific pattern of vascular development. Besides its role in promoting angiogenesis, the VEGF receptor system also has the ability to increase permeability of blood vessels, which is believed to result in access to serum factors essential for new blood vessel growth [24]. The ability of VEGF to increase permeability of the microvasculature is a unique property of this molecule; other angiogenic factors (e.g., placental growth factor, basic and acidic fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-b) do not possess a similar ability [25]. The VEGF165 isoform, which increased progressively in association with fetal growth in the present study, possesses a potent ability to increase vascular permeability [26, 27]. Thus there is a potential for increased
TABLE 2. The correlations (r ) between the measured components of placental endometrial vascularity and their impacts on fetal and placental weights and placental efficiency. Vascular measurements
Placental VEGF mRNA No. placental vessels No. endometrial vessels
No. placental vessels
No. endometrial vessels
0.34 (P , 0.05)
0.34 (P , 0.05) 0.64 (P , 0.0001)
Conceptus measurements Fetal weight
Placental weight
Placental efficiency
0.73 (P , 0.0001) 0.52 (P , 0.0001) 0.50 (P , 0.005)
0.60 (P , 0.0001) 0.52 (P , 0.001) 0.56 (P , 0.0001)
0.66 (P , 0.0001) 0.48 (P , 0.002) 0.40 (P , 0.05)
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FIG. 2. Immunolocalization of VEGF to the chorionic and uterine epithelium. Omission of the primary antibody (A), and immunolocalization of VEGF in uterine epithelial cells (*) and chorionic epithelial cells (**) on Day 36 (B) and Day 70 (C) of gestation.
vascular permeability and density of placental and endometrial blood vessels with the advancement of gestation. VEGF proteins not only act to change the permeability of the endothelium but also act as specific mitogen- and migration-stimulating factors for endothelial cells [28–30]. The consistent localization of VEGF to the chorionic and uterine luminal epithelium of the pig throughout gestation ([31, 32], this study) may function to induce a closer association between placental and endometrial capillary endothelium at the fetal/maternal interface. In agreement with
Winther et al. [31], we also observed an increase in VEGF immunostaining in the chorionic epithelium and uterine lumenal epithelium during the later part of gestation, when the distance between fetal and maternal capillaries decreases. During the first trimester of gestation in the pig, the chorionic epithelium and uterine luminal epithelium form a continuous barrier of at least 40 mm between the fetal and maternal capillaries [33]. By the end of gestation, and largely as a consequence of progressive indentation of capillaries on both sides of the placenta, the distance is reduced to ,2 mm at the summit and lateral sides of the chorionic ridges. Oxygen and carbon dioxide pass across the placental membranes by simple diffusion, which is dependent on the surface area available for transfer and the physical thickness separating fetal and maternal capillaries [34]. Thus, by increasing the density of capillaries at the fetal/maternal interface and decreasing the distances between them, gaseous exchange should be markedly increased. Friess et al. [33] presented morphologic and histologic evidence that the transport of less diffusable material takes place in the chorionic troughs, where the chorionic and uterine luminal epithelium remain high columnar and no indentation or protruding capillaries could be seen on either side. However, even in these areas, an increased density of capillaries (placental and endometrial) would facilitate the transfer mechanisms, such as facilitated diffusion, active transport, and pinocytosis [35]. Data from this study support the concept that the increasing vascularity of the placenta with the advancement of gestation is a response of the conceptus to insufficient delivery of oxygen and/or nutrients to the fetus, as has been shown in sheep [14]. It would logically follow that as nutrient and particularly oxygen delivery becomes insufficient at the endometrial/placental interface in association with conceptus growth, both placental and adjacent endometrial tissues would upregulate the synthesis and secretion of VEGF to compensate. Additionally, there is evidence that estrogens upregulate VEGF expression in uterine tissues from rats [15], sheep [16], and humans [17]. This upregulation is likely a primary estrogen receptor-mediated effect, because VEGF induction is very rapid, is blocked by pure antiestrogens [36], and is inhibited by actinomycin D but not by pure pyromycin or cycloheximide [15, 36]. This regulation by estrogens is consistent with finding of most studies on the expression of VEGF in the uterus throughout the estrous cycle in rodents [37, 38] and the menstrual cycle in humans [39]. Both fetal demand for oxygen and placental secretion of estrogen increase dramatically throughout late gestation in the pig. An increase in placental VEGF secretion could potentially affect not only the number of blood vessels at the fetal/maternal interface but also intercapillary distance and/ or permeability, allowing a more efficient delivery of nutrients to the fetus. We have recently confirmed an increase in VEGF protein in CA fluid and fetal blood during the latter part of gestation in the pig [40]. Further, VEGF concentrations in fetal blood were positively correlated with fetal weight (r 5 0.45; P , 0.01) and placental efficiency (r 5 0.40; P , 0.01). Based on these data, we hypothesize that VEGF is playing an active role in the coordinated and progressive vascularization of the placenta and adjacent uterine wall throughout gestation in the pig. The coordinated patterns of vascular development of the placenta and adjacent endometrium suggest a conceptus-specific effect. Our data are consistent with a role for VEGF in increasing the numbers
PLACENTAL VEGF EXPRESSION DURING GESTATION IN THE PIG
(and possibly permeability) of blood vessels at the placental/endometrial interface, resulting in an increased capacity for nutrient transfer from the maternal to the fetal compartment.
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