help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0825
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/12/5568    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hewitt, D. P.
Right arrow Articles by Waddell, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hewitt, D. P.
Right arrow Articles by Waddell, B. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*DEXAMETHASONE
Medline Plus Health Information
*Steroids
Endocrinology Vol. 147, No. 12 5568-5574
Copyright © 2006 by The Endocrine Society

Glucocorticoids Prevent the Normal Increase in Placental Vascular Endothelial Growth Factor Expression and Placental Vascularity during Late Pregnancy in the Rat

Damien P. Hewitt, Peter J. Mark and Brendan J. Waddell

School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia 6009, Australia

Address all correspondence and requests for reprints to: Brendan J. Waddell, Ph.D., School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. E-mail: bwaddell{at}anhb.uwa.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased glucocorticoid exposure reduces fetal growth and predisposes to an increased risk of disease in later life. In addition to direct effects on fetal growth, glucocorticoids also compromise fetal growth indirectly via detrimental effects on placental growth and function. The current study investigated the effects of dexamethasone-induced intrauterine growth restriction on placental vascular development and expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF). Separate analyses were conducted for the three main VEGF isoforms (VEGF120, VEGF164, and VEGF188) in the two functionally and morphologically distinct regions of the rat placenta, the basal and labyrinth zones. Quantitative PCR and immunohistochemical analysis demonstrated that expression of VEGF was markedly up-regulated specifically in the rapidly growing labyrinth zone over the final third of normal pregnancy. Unbiased stereological analyses showed an associated increase in the volume and surface area of maternal and fetal blood spaces, including vascular remodeling of the fetal capillary network near term. In contrast, dexamethasone-induced fetal and placental growth restriction reduced expression of the Vegf120 and Vegf188 isoforms and prevented normal labyrinthine vascular development near term. Most notably, dexamethasone impaired the normal increase in fetal vessel density over the final third of pregnancy, with no effect on the density of maternal blood spaces. Overall, this study quantifies the labyrinth zone-specific increases in placental VEGF expression and vascular development during normal pregnancy, and shows that these increases are prevented by maternal dexamethasone treatment. Our data suggest that glucocorticoid-induced restriction of fetal and placental growth is mediated, in part, via inhibition of placental VEGF expression and an associated reduction in placental vascularization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS ARE CRUCIAL for maturation of the fetus late in pregnancy, and clinical glucocorticoid administration is used to accelerate fetal lung development in mothers at risk of preterm delivery (1, 2). However, increased fetal glucocorticoid exposure reduces birth weight, and is associated with an increased risk of cardiovascular and metabolic disease in later life (3, 4). Placental development is a critical determinant of fetal growth, and it appears that the effects of glucocorticoids on fetal growth may be mediated in part via effects on placental growth and function. Specifically, rodent models suggest that placental growth is more sensitive to altered glucocorticoid exposure than fetal growth, the former being compromised by increased levels of trophoblast apoptosis (5). Moreover, maternal glucocorticoid treatment appears to reduce the transplacental passage of glucose (6) and leptin (7) without a reduction in the expression of associated placental transporter proteins. This suggests that increased glucocorticoid exposure is likely to impact on the vascular exchange network of the placenta. Indeed, glucocorticoids down-regulate expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF) in various tissues (8, 9, 10, 11, 12), but the impact of glucocorticoids on placental VEGF expression remains unknown.

VEGF (also referred to as VEGF-A) is a potent angiogenic factor belonging to a gene family that includes VEGF-B, -C, -D, and placental growth factor (13). VEGF promotes angiogenensis through induction of endothelial cell proliferation and increased vascular permeability (13), and gene deletion studies show that VEGF is essential for normal embryonic development. Indeed, even heterozygotes show an embryonic lethal phenotype associated with a failure of placental vascular development (13, 14). In normal pregnancy, placental VEGF expression increases near term in the rodent (15), baboon (16, 17, 18), and sheep (19) in association with increased placental vascularization (16, 17, 18, 20, 21, 22). In contrast, reduced placental expression of VEGF has been observed in cases of intrauterine growth restriction (23), and pharmacological inhibition of VEGF is associated with impaired placental vascularity (24).

The rodent Vegf gene is encoded from eight exons with alternate splicing of the sixth and seventh exons, resulting in the generation of five alternate splice variants (VEGF120, VEGF144, VEGF164, VEGF188, VEGF205) with 120, 144, 164, 188, and 205 amino acids, respectively. VEGF120 is a freely secreted isoform that does not bind heparin and is thus a highly potent endothelial cell mitogen. The VEGF188 and VEGF205 isoforms display reduced bioactivity due to their sequestration within the extracellular matrix. VEGF164 has intermediate activity between the small and large isoforms because it is secreted and, therefore, is functionally active, but significant quantities are retained bound to the cell surface and extracellular matrix (13). Four of the VEGF isoforms (120, 164, 188, and 205) have been identified in the rat placenta with the 120 and 164 isoforms increasing near term (15). Homologs of rodent VEGF144 have also been identified in human (25) and sheep (26) placenta.

In the current study, we investigated the impact of increased glucocorticoid exposure on the expression of the major VEGF isoforms (VEGF120, VEGF164, and VEGF188) and vascularization of the rat placenta over the final third of pregnancy, the period of maximal fetal and placental growth. Increased glucocorticoid exposure was achieved via maternal administration of dexamethasone in a model known to restrict fetal and placental growth (27, 28). Separate analyses were conducted for the two functionally and morphologically distinct regions of the placenta, the basal and labyrinth zones, because only the latter is involved in fetal-maternal transport and undergoes dramatic growth and vascular development over the final third of pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Nulliparous albino Wistar rats aged between 8–12 wk were obtained from the Animal Resources Center (Murdoch, Australia) and maintained under controlled conditions as described previously (29). Rats were mated overnight, and the day on which spermatozoa were present in a vaginal smear was designated d 1 of pregnancy. All procedures involving animals were conducted only after approval by the Animal Ethics Committee of The University of Western Australia.

Glucocorticoid manipulations
Increased fetal and placental glucocorticoid exposure was achieved by maternal administration of dexamethasone acetate (1 µg/ml maternal drinking water; Sigma, St. Louis, MO) from d 13–22 of pregnancy (term = 23 d). We have previously shown that this treatment reduces fetal weight by 25–30% at d 22 (5, 27).

Tissue collection
Rats were anesthetized with halothane/nitrous oxide at either d 16 or 22 of gestation, and three fetuses and placentas were removed from each mother. Placental zones were separated by blunt dissection and snap frozen in liquid nitrogen for real-time quantitative RT-PCR, or alternatively, whole placentas were fixed in Histochoice MB (Amresco, Solon, OH) and processed for routine paraffin histology for subsequent immunohistochemical or stereological analyses.

RNA isolation
Total RNA was isolated from placental samples using Tri-Reagent (Molecular Resources Center, Cincinnati, OH) as per the manufacturer’s instructions. RNA integrity was assessed by ethidium bromide staining of the nucleic acids after agarose gel electrophoresis (data not shown). Total RNA was treated to remove contaminating genomic DNA using DNA-free reagent (Ambion, Austin, TX). DNase-treated total RNA (1 µg) was used to synthesize cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison, WI) as per the manufacturer’s instructions. The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA).

Real-time RT-PCR
Analyses of expression levels for Vegf transcripts were performed by real-time RT-PCR on the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and the amplification conditions outlined in Table 1Go. Primers for Vegf isoform amplification were designed using Primer 3 software (MIT/Whitehead Institute; http://www-genome.wi.mit.edu) (30) and the reverse primers for VEGF120, VEGF164, and VEGF188 were positioned at distinct exon boundaries for each isoform. Each of the selected primer pairs were positioned to span introns to ensure no product was amplified from genomic DNA, and the resulting amplicons were sequenced to confirm specificity (data not shown). All samples were standardized against Rpl19 as previously described (31). Standard curves for each assay were generated from gel extracted (QIAEX II; Qiagen, Melbourne, Australia) PCR products using 10-fold serial dilutions and the Rotorgene 3000 software.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers and conditions used for Vegfisoforms and Rpl19 real-time RT-PCR

 
Immunohistochemistry
Sections were cut at 4 µm, deparaffinized, and rehydrated, then incubated for 10 min in 3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary antibody to CD31 (BD Biosciences, New South Wales, Australia) and VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) were each used at a dilution of 1:50 overnight at 4 C followed by a 30-min room-temperature incubation in biotinylated antimouse secondary at 1:200 (Vector Laboratories, Burlingame, CA) and streptavidin-peroxidase complex at 1:50 (Vectastain ABC Kit; Vector Laboratories). Sections were visualized by application of diaminobenzidine substrate (Sigma) and counterstained with hematoxylin before dehydration in graded alcohols and mounting in DPX.

Stereological measures
Detailed analysis of the labyrinth zone vasculature was achieved by random sampling of 12 fields of view (objective lens = x40 magnification) of placentas collected from untreated mothers at either d 16 or 22, or from dexamethasone-treated mothers at d 22. Relative volumes of labyrinth zone components were calculated by a 35-point grid overlaid on each field of view, counting points that fell in each placental structure according to the Cavalieri principle (32). Surface area measurements were estimated by using the same fields obtained for volume measurements and overlaying a grid of cycloid arcs as previously reported in Coan et al. (20). Points of intersection of cycloid arcs with the lumen of maternal blood spaces and fetal capillaries were used to calculate surface area densities in vertically oriented sections (20), with CD31 immunostaining used to aid in the distinction between maternal and fetal blood spaces. Combined length density, mean diameter, and overall density of fetal capillaries were all estimated by counting the number of capillary profiles in a counting square containing two contiguous forbidden lines (20). Volume, surface area, and length densities were adjusted for shrinkage of the tissue by measurement of average maternal erythrocyte diameter before and after tissue processing (33, 34). Measures of density were converted to absolute measures by multiplying by the total volume of the labyrinth zone assuming a specific gravity of 1 g/cm3.

Statistical analysis
All group values were expressed as means ± SEM, standardized by Rpl19 in the case of real-time RT-PCR data. Two-way ANOVAs (GenStat7, Hemel Hempstead, UK) were used to assess variation in expression levels of Vegf mRNA, volume and surface area estimates for gestational age and placental zone. Where the F test for the ANOVA reached statistical significance (P < 0.05), differences were assessed by least significant difference (LSD) test. Changes in mean capillary diameter and length of fetal capillaries over gestation and after dexamethasone treatment were assessed by unpaired t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gestational changes in VEGF expression
Vegf mRNA was readily detectable in both zones of the rat placenta over the final third of pregnancy (Fig. 1Go). Increased labyrinthine expression of Vegf120 (3.2-fold increase; P < 0.01), Vegf164 (2.4-fold increase; P < 0.01) and Vegf188 (4.7-fold increase; P < 0.001) was observed from d 16–22 of normal rat pregnancy, but no changes were observed in basal zone expression. Expression of the VEGF protein was localized predominantly to the cytoplasm of the labyrinth zone trophoblast and some basal trophoblast cells, with no expression observed in fetal endothelial cells (Fig. 2Go). Localization of the VEGF protein was consistent at d 16 and 22 (data not shown).


Figure 1
View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. Expression of Vegf120 (A), Vegf164 (B), and Vegf188 (C) mRNA in the basal and labyrinth zones of the rat placenta on d 16 and 22 of pregnancy. Values are the mean ± SEM (n = 5–6 per group). *, P < 0.01; **, P < 0.001, compared with corresponding d-16 value (two-way ANOVA and LSD test).

 

Figure 2
View larger version (81K):
[in this window]
[in a new window]
  		FIG. 2. Spatial distribution of VEGF by immunohistochemistry at d 22 of rat pregnancy. A, No primary antibody (negative control). B, VEGF expression primarily localized to the cytoplasm of labyrinthine trophoblast cells with some basal zone trophoblast cells exhibiting weak expression. C, Higher power view of labyrinthine VEGF distribution showing positive trophoblast cells (black arrowheads) and negative fetal endothelium (white arrowheads). LZ, Labyrinth zone; BZ, basal zone; scale bar, 10 µm.

 
Vascular changes in the rat placenta over the final third of pregnancy
Stereological analyses of the labyrinth zone during normal pregnancy (Figs. 3Go and 4Go) revealed that this region of the placenta underwent a marked increase (P < 0.001) in the absolute volumes and surface areas of both maternal blood spaces (5-fold increase in volume and surface area; Fig. 3Go, A and B) and fetal capillaries (volume, 4-fold increase; surface area, 5-fold increase; Fig. 3Go, A and B) over the final third of pregnancy. The increases in volume and surface area were accompanied by a marked rise in the total combined length of the fetal capillary network (7-fold increase, P < 0.001; Fig. 4AGo). These changes were due in part to a higher density of maternal blood spaces (volume, 46% increase, P < 0.001; and surface area, 49% increase, P < 0.01; Fig. 3Go, C and D) and fetal capillaries (volume, 48% increase; and surface area, 54% increase, P < 0.01; Fig. 3Go, C and D) at d 22, including an increase in the relative density of fetal capillaries (2.2-fold increase, P < 0.001; Fig. 4CGo) and a reduction in the mean diameter of fetal vessels (19% reduction, P < 0.05; Fig. 4BGo). Tissue volume (i.e. labyrinthine volume excluding vascular lumen) also increased (2.2-fold, P < 0.01; Fig. 3AGo) from d 16–22, although its proportional volume fell by 30% (P < 0.01; Fig. 3CGo) over this period.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. Stereological analysis of labyrinth zone components on d 16 and 22 showing changes in absolute volume (A) and absolute surface area (B) of maternal blood spaces (MBS) and fetal capillaries (FC) and changes in relative vascular volumes (C) and relative surface areas (D) of the vasculature. Values are the mean ± SEM (n = 4 per group). *, P < 0.01; **, P < 0.001, compared with corresponding d-16 value (two-way ANOVA and LSD test).

 

Figure 4
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 4. Stereological analysis of fetal capillary network on d 16 and 22 of pregnancy showing changes in total combined capillary length (A), mean capillary diameter (B), and capillary density (C). Values are the mean ± SEM (n = 4 per group). *, P < 0.05; **, P < 0.001, compared with corresponding d-16 values (unpaired t test).

 
Changes in VEGF expression after dexamethasone treatment
Administration of dexamethasone over the final third of pregnancy reduced fetal weight (22% reduction; P < 0.01) and the weight of the placental labyrinth zone (35%; P < 0.01) (Table 2Go). Maternal dexamethasone treatment also reduced labyrinthine expression of Vegf120 (40% reduction; P < 0.05) and Vegf188 (64% reduction; P < 0.01), but had no effect on the expression of Vegf164 (Fig. 5Go). No change was observed in basal zone expression of any isoform. Immunolocalization of the VEGF protein did not change after treatment with dexamethasone (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Fetal and placental zone weights following maternal dexamethasone treatment

 

Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5. Effects of dexamethasone treatment from d 13 of pregnancy on zonal expression of Vegf120 (A), Vegf164 (B), and Vegf188 (C) mRNA at d 22. Values are the mean ± SEM (n = 5–6 per group) for placentas obtained from dexamethasone-treated (Dex) and untreated control (Con) mothers. *, P < 0.05; **, P < 0.01, compared with corresponding control value (two-way ANOVA and LSD test).

 
Vascular changes in the rat placenta after dexamethasone treatment
Stereological analyses of the placental labyrinth zone showed that dexamethasone treatment reduced (P < 0.01) absolute volumes and surface areas associated with maternal (volume, 48% reduction; surface area, 46% reduction; Fig. 6Go, A and B) and fetal blood spaces (volume, 73% reduction; surface area, 61% reduction; Fig. 6Go, A and B). Absolute placental tissue volume was also reduced (36%, P < 0.05; Fig. 6AGo) by dexamethasone, despite a relatively higher density (18% increase, P < 0.05; Fig. 6CGo). Dexamethasone also impaired the normal increase in fetal vessel density (volume, 51% reduction; surface area, 30% reduction, P < 0.01; Fig. 6Go, C and D) and total combined fetal capillary length (66% reduction, P < 0.01; Fig. 7AGo) over the final third of pregnancy, but had no effect on the density of maternal blood space (i.e. relative volume) or its surface area (Fig. 6Go, C and D). In addition, treatment with dexamethasone slightly reduced the size of fetal capillaries (12% reduction, P < 0.05; Fig. 7BGo) and the relative density of fetal capillary profiles (37% reduction, P < 0.01; Fig. 7CGo).


Figure 6
View larger version (44K):
[in this window]
[in a new window]
  		FIG. 6. Effects of dexamethasone treatment from d 13 of pregnancy on the absolute volume (A), absolute surface area (B), relative volume (C), and relative surface area (D) of the labyrinthine maternal blood spaces (MBS) and fetal capillaries (FC) at d 22. Values are the mean ± SEM (n = 4 per group) for placentas obtained from dexamethasone-treated (Dex) and untreated control (Con) mothers. *, P < 0.05; **, P < 0.01, compared with corresponding control value (two-way ANOVA and LSD test).

 

Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7. Effects of dexamethasone treatment from d 13 of pregnancy on total combined capillary length (A), mean capillary diameter (B), and relative capillary density (C) of the labyrinthine vasculature at d 22. Values are the mean ± SEM (n = 4 per group) for placentas obtained from dexamethasone-treated (Dex) and untreated control (Con) mothers. *, P < 0.05; **, P < 0.001, compared with corresponding control values (unpaired t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the impact of increased glucocorticoid exposure on the spatial and temporal expression of the endothelial cell-specific mitogen, VEGF, and associated changes in placental vascularization over the final third of rat pregnancy. The expression of Vegf mRNA increased specifically in the rapidly growing labyrinth zone between d 16 and 22 of normal pregnancy, and this change coincided with a marked increase in vascularization of this placental zone. In contrast, treatment with dexamethasone, which reduced fetal and placental growth, prevented the normal rise in Vegf expression and the associated increase in labyrinthine vascularity over the final third of pregnancy. Most notably, dexamethasone-induced reductions in absolute vascular volume and surface area were accompanied by a reduction in the density of the fetal vasculature, but there was no effect on the density of maternal blood spaces. Thus, dexamethasone appears to reduce placental growth by preventing the normal development of the fetal vasculature within the labyrinthine placenta.

Unbiased stereological analyses of the rat placenta in this study clearly demonstrated rapid vascular development within the labyrinth zone, the site of fetal-maternal exchange, over the final third of pregnancy. This increase in vascularization would be expected to enhance the efficiency of fetal-maternal transport and is thus consistent with the greater demands of the fetus late in pregnancy. In absolute terms, fetal vessels and maternal blood spaces exhibited a 4- to 5-fold increase over the final third of pregnancy, whereas relative densities (i.e. luminal volume expressed as a proportion of total labyrinth volume) increased by approximately 50% over the same period. Previous studies in the rodent have reported comparable increases in placental vascularization (20, 21, 22); however, the results of this study include a wider range of pregnancy, and demonstrate a similar rate of vascular growth in the rat to that observed in the mouse (20). In addition, the increase in the total combined length of fetal capillaries as well as the reduction in mean capillary diameter observed near term suggest that fetal vascular remodeling is involved in the development of the fetal capillary network of the rat placenta in a similar manner to that observed in the mouse (20).

While these vascular changes are potentially due to the effect of various angiogenic factors, the parallel increase in the expression of the endothelial cell-specific mitogen, VEGF, in labyrinth zone trophoblast cells, suggests that this is likely to be a major stimulus. Thus, a 2.4- to 4.7-fold increase in the expression of Vegf isoforms was associated with a 4-fold increase in absolute volume, a 5-fold increase in absolute surface area and a 7-fold increase in total combined length of fetal capillaries. In addition to the current data, prior studies have demonstrated the importance of VEGF in the development of placental vasculature and subsequent fetal growth via promotion of endothelial cell proliferation and differentiation in the mouse and human (13, 14). In particular, gene deletion studies have shown that the loss of even one VEGF allele results in impaired placental vasculature and embryonic lethality by d 13 of pregnancy (13, 14). Our observation that the major Vegf mRNA isoforms and fetal vascularity both increase in the labyrinth zone toward term, together with similar observations in other species (16, 17, 18), points to a key role for VEGF in stimulating the major vascular remodeling that occurs in the placenta to ensure continued fetal growth in late pregnancy.

In contrast to the increase in placental vascularity and Vegf mRNA expression during normal pregnancy, maternal dexamethasone treatment between d 13–22 reduced Vegf120 and Vegf188 mRNA expression and absolute volumes and surface areas of both maternal blood spaces and fetal capillaries. Previous reports show that increased glucocorticoid exposure reduces transplacental passage of glucose (6) and leptin (7) with no reduction in the expression of placental transporter proteins. It seems likely that these effects on transport are nonspecific, reflecting a glucocorticoid-induced reduction in the effective vascular exchange area within the labyrinth zone. A key finding of this study was that the effects of dexamethasone were specific to the fetal vasculature, with no apparent effect on the density of maternal blood spaces. In effect, it appears that dexamethasone treatment suppressed the normal increase in the density of fetal capillary volume and surface area over the final third of pregnancy, because the vascularity of the dexamethasone-treated placentas at d 22 was comparable to that at d 16. Therefore, these data suggest that dexamethasone treatment suppresses fetal vessel growth, consistent with the prevention of the normal increase in Vegf mRNA observed over the final third of pregnancy. With respect to total placental volume, a recent study suggests that the reduction in nonvascular tissue volume observed in cases of intrauterine growth restriction may reflect the reliance on adequate fetal vascularity (24). Thus, the dexamethasone-mediated effects on labyrinth zone growth observed in the present study may be a consequence of the primary effects on VEGF-mediated fetal capillary growth.

Although the present study is the first to report an inhibitory effect of glucocorticoids on placental VEGF expression, similar effects have been observed in other tissues including airway smooth muscle cells and chondrocytes (8, 9, 10, 11, 12). Exactly how glucocorticoids suppress Vegf expression remains uncertain, but potentially it could involve reduced transcription of the Vegf gene due to interaction of the activated glucocorticoid receptor with the Vegf promoter, and/or indirect effects via other placental genes that regulate VEGF expression. For example, we recently demonstrated that labyrinth zone expression of peroxisome proliferator-activated receptor-{gamma}, activation of which up-regulates VEGF expression in various cell types (35, 36), is suppressed by dexamethasone (27). In addition, dexamethasone up-regulates the expression of secreted frizzled related protein-4 in rat trophoblast (37), which could reduce Vegf gene transcription via inhibition of WNT signaling (38). Interestingly, dexamethasone did not affect labyrinthine expression of the Vegf164 isoform, raising the possibility that glucocorticoids regulate VEGF by means of an isoform-specific degradation of Vegf mRNA. Indeed, a previous report indicates that dexamethasone decreases Vegf mRNA stability in cultured keratinocytes (39). Further studies are required to elucidate the specific mechanisms by which dexamethasone suppresses placental VEGF expression in our model of fetal and placental growth restriction.

In conclusion, we have shown that VEGF expression in the labyrinth zone of the rat placenta is up-regulated near term, and that maternal dexamethasone treatment prevents this increase. These changes are consistent with the marked increase in labyrinthine vascular development observed over the final third of normal pregnancy, and impaired vascularization observed during dexamethasone-induced fetal and placental growth restriction. Although the control of VEGF expression and vascular growth is likely to involve a complex balance of diverse molecular signals, the results of the current study suggest that glucocorticoids restrict fetal and placental growth via inhibition of placental VEGF expression and subsequent placental vascularization.


   Footnotes
 
This work was supported by National Health and Medical Research Council of Australia (Project Grant 254576).

Disclosure statement: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: LSD, Least significant difference; VEGF, vascular endothelial growth factor.

Received June 19, 2006.

Accepted for publication August 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sloboda DM, Challis JR, Moss TJ, Newnham JP 2005 Synthetic glucocorticoids: antenatal administration and long-term implications. Curr Pharm Des 11:1459–1472[CrossRef][Medline]
  2. Ward RM 1994 Pharmacologic enhancement of fetal lung maturation. Clin Perinatol 21:523–542[Medline]
  3. Seckl JR 2004 Prenatal glucocorticoids and long-term programming. Eur J Endocrinol 151(Suppl 3):U49–U62
  4. Seckl JR, Meaney MJ 2004 Glucocorticoid programming. Ann NY Acad Sci 1032:63–84[CrossRef][Medline]
  5. Waddell BJ, Hisheh S, Dharmarajan AM, Burton PJ 2000 Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids. Biol Reprod 63:1913–1917[Abstract/Free Full Text]
  6. Langdown ML, Sugden MC 2001 Enhanced placental GLUT1 and GLUT3 expression in dexamethasone-induced fetal growth retardation. Mol Cell Endocrinol 185:109–117[CrossRef][Medline]
  7. Smith JT, Waddell BJ 2003 Leptin distribution and metabolism in the pregnant rat: transplacental leptin passage increases in late gestation but is reduced by excess glucocorticoids. Endocrinology 144:3024–3030[Abstract/Free Full Text]
  8. Alagappan VK, McKay S, Widyastuti A, Garrelds IM, Bogers AJ, Hoogsteden HC, Hirst SJ, Sharma HS 2005 Proinflammatory cytokines up-regulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells. Cell Biochem Biophys 43:119–129[CrossRef][Medline]
  9. Alonso G, Galibert E, Duvoid-Guillou A, Vincent A 2005 Hyperosmotic stimulus induces reversible angiogenesis within the hypothalamic magnocellular nuclei of the adult rat: a potential role for neuronal vascular endothelial growth factor. BMC Neurosci 6:20[CrossRef][Medline]
  10. Heiss JD, Papavassiliou E, Merrill MJ, Nieman L, Knightly JJ, Walbridge S, Edwards NA, Oldfield EH 1996 Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor. J Clin Invest 98:1400–1408[Medline]
  11. Koedam JA, Smink JJ, van Buul-Offers SC 2002 Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes. Mol Cell Endocrinol 197:35–44[CrossRef][Medline]
  12. Smink JJ, Buchholz IM, Hamers N, van Tilburg CM, Christis C, Sakkers RJ, de Meer K, van Buul-Offers SC, Koedam JA 2003 Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis. Osteoarthritis Cartilage 11:864–871[CrossRef][Medline]
  13. Ferrara N 2004 Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25:581–611[Abstract/Free Full Text]
  14. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A 1996 Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439[CrossRef][Medline]
  15. Ni Y, May V, Braas K, Osol G 1997 Pregnancy augments uteroplacental vascular endothelial growth factor gene expression and vasodilator effects. Am J Physiol 273:H938–H944
  16. Albrecht ED, Robb VA, Pepe GJ 2004 Regulation of placental vascular endothelial growth/permeability factor expression and angiogenesis by estrogen during early baboon pregnancy. J Clin Endocrinol Metab 89:5803–5809[Abstract/Free Full Text]
  17. Hildebrandt VA, Babischkin JS, Koos RD, Pepe GJ, Albrecht ED 2001 Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy. Endocrinology 142:2050–2057[Abstract/Free Full Text]
  18. Robb VA, Pepe GJ, Albrecht ED 2004 Acute temporal regulation of placental vascular endothelial growth/permeability factor expression in baboons by estrogen. Biol Reprod 71:1694–1698[Abstract/Free Full Text]
  19. Reynolds LP, Borowicz PP, Vonnahme KA, Johnson ML, Grazul-Bilska AT, Wallace JM, Caton JS, Redmer DA 2005 Animal models of placental angiogenesis. Placenta 26:689–708[CrossRef][Medline]
  20. Coan PM, Ferguson-Smith AC, Burton GJ 2004 Developmental dynamics of the definitive mouse placenta assessed by stereology. Biol Reprod 70:1806–1813[Abstract/Free Full Text]
  21. Doherty CB, Lewis RM, Sharkey A, Burton GJ 2003 Placental composition and surface area but not vascularization are altered by maternal protein restriction in the rat. Placenta 24:34–38[CrossRef][Medline]
  22. Lewis RM, Doherty CB, James LA, Burton GJ, Hales CN 2001 Effects of maternal iron restriction on placental vascularization in the rat. Placenta 22:534–539[CrossRef][Medline]
  23. Lyall F, Young A, Boswell F, Kingdom JC, Greer IA 1997 Placental expression of vascular endothelial growth factor in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth restriction does not support placental hypoxia at delivery. Placenta 18:269–276[CrossRef][Medline]
  24. Rutland CS, Mukhopadhyay M, Underwood S, Clyde N, Mayhew TM, Mitchell CA 2005 Induction of intrauterine growth restriction by reducing placental vascular growth with the angioinhibin TNP-470. Biol Reprod 73:1164–1173[Abstract/Free Full Text]
  25. Anthony FW, Wheeler T, Elcock CL, Pickett M, Thomas EJ 1994 Short report: identification of a specific pattern of vascular endothelial growth factor mRNA expression in human placenta and cultured placental fibroblasts. Placenta 15:557–561[CrossRef][Medline]
  26. Matsumoto LC, Bogic L, Brace RA, Cheung CY 2002 Prolonged hypoxia up-regulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta. Am J Obstet Gynecol 186:303–310[CrossRef][Medline]
  27. Hewitt DP, Mark PJ, Waddell BJ 2006 Placental expression of peroxisome proliferator-activated receptors in rat pregnancy and the effect of increased glucocorticoid exposure. Biol Reprod 74:23–28[Abstract/Free Full Text]
  28. Waddell BJ, Benediktsson R, Brown RW, Seckl JR 1998 Tissue-specific messenger ribonucleic acid expression of 11ß-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action. Endocrinology 139:1517–1523[Abstract/Free Full Text]
  29. Benbow AL, Waddell BJ 1995 Distribution and metabolism of maternal progesterone in the uterus, placenta, and fetus during rat pregnancy. Biol Reprod 52:1327–1333[Abstract]
  30. Rozen S, Skaletsky H 2000 Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386[Medline]
  31. Orly J, Rei Z, Greenberg NM, Richards JS 1994 Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. Endocrinology 134:2336–2346[Abstract]
  32. Gundersen HJ, Osterby R 1981 Optimizing sampling efficiency of stereological studies in biology: or "do more less well!" J Microsc 121:65–73[Medline]
  33. Ali KZ, Burton GJ, Morad N, Ali ME 1996 Does hypercapillarization influence the branching pattern of terminal villi in the human placenta at high altitude? Placenta 17:677–682[CrossRef][Medline]
  34. Burton GJ, Palmer ME 1988 Eradicating fetomaternal fluid shift during perfusion fixation of the human placenta. Placenta 9:327–332[CrossRef][Medline]
  35. Yamakawa K, Hosoi M, Koyama H, Tanaka S, Fukumoto S, Morii H, Nishizawa Y 2000 Peroxisome proliferator-activated receptor-{gamma} agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells. Biochem Biophys Res Commun 271:571–574[CrossRef][Medline]
  36. Yasuda E, Tokuda H, Ishisaki A, Hirade K, Kanno Y, Hanai Y, Nakamura N, Noda T, Katagiri Y, Kozawa O 2005 PPAR-{gamma} ligands up-regulate basic fibroblast growth factor-induced VEGF release through amplifying SAPK/JNK activation in osteoblasts. Biochem Biophys Res Commun 328:137–143[CrossRef][Medline]
  37. Hewitt DP, Mark PJ, Dharmarajan AM, Waddell BJ 2006 Placental expression of secreted frizzled related protein-4 in the rat and the impact of glucocorticoid-induced fetal and placental growth restriction. Biol Reprod 75:75–81[Abstract/Free Full Text]
  38. Zhang X, Gaspard JP, Chung DC 2001 Regulation of vascular endothelial growth factor by the Wnt and K-ras pathways in colonic neoplasia. Cancer Res 61:6050–6054[Abstract/Free Full Text]
  39. Gille J, Reisinger K, Westphal-Varghese B, Kaufmann R 2001 Decreased mRNA stability as a mechanism of glucocorticoid-mediated inhibition of vascular endothelial growth factor gene expression by cultured keratinocytes. J Invest Dermatol 117:1581–1587[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. J. De Blasio, M. Dodic, A. J. Jefferies, K. M. Moritz, E. M. Wintour, and J. A. Owens
Maternal exposure to dexamethasone or cortisol in early pregnancy differentially alters insulin secretion and glucose homeostasis in adult male sheep offspring
Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E75 - E82.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/12/5568    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hewitt, D. P.
Right arrow Articles by Waddell, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hewitt, D. P.
Right arrow Articles by Waddell, B. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*DEXAMETHASONE
Medline Plus Health Information
*Steroids

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals