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Centre for Women’s Health Research, Monash Institute of Medical Research and Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Rd, Clayton, Victoria 3168, Australia
Centre for Women’s Health Research, Monash Institute of Medical Research and Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Rd, Clayton, Victoria 3168, Australia
Centre for Women’s Health Research, Monash Institute of Medical Research and Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Rd, Clayton, Victoria 3168, Australia
While vascular endothelial growth factor (VEGF)-A mediates endometrial vascular remodelling during early pregnancy in mice, individual VEGF-A isoforms have not been investigated, despite their different biological properties. Using mice as a model, the expression of VEGF-A isoforms and receptors in the mouse uterus during early pregnancy was quantified. It was postulated that selected isoform expression would increase concurrent with increased endometrial endothelial cell proliferation at this time. Uteri were collected on days 1–5 of pregnancy and mRNA expression was quantified by quantitative reverse-transcription polymerase chain reaction, VEGF-A protein by Western blot and VEGF receptor (VEGFR)-2 by immunohistochemistry. The lowest expression of isomers Vegf120 and Vegf164 was observed on day 2 of pregnancy, increasing thereafter. Vegfr-2 mRNA expression was significantly higher on days 3–5 of pregnancy relative to days 1–2 (P < 0.05). No significant changes were noted in Vegf188, Nrp1 or Nrp2 mRNA. VEGF188 protein expression was consistently higher than other isoforms. These data demonstrate differential regulation of VEGF-A isoforms in mouse uterus during early pregnancy.
During early pregnancy in all mammals, the blood vessels in the endometrium (uterine lining) undergo a process of growth and remodelling known as angiogenesis. Without appropriate angiogenesis, neither implantation nor placental development will be successful. The blood vessels in the endometrium respond to various growth factors released from the surrounding tissue. The best known of these factors is vascular endothelial growth factor (VEGF)-A, which interacts with a receptor called VEGFR-2. There are several different forms of VEGF-A (isoforms), which display different biological properties. The aim of this study was to quantify the expression of each of the isoforms (VEGF120, VEGF164 and VEGF188), as well as the receptor (VEGFR-2), in the mouse uterus during early pregnancy. We have shown that the expression of two of the VEGF-A isoforms (VEGF120 and VEGF164) and VEGFR-2 increases in the uterus during early pregnancy. This increase occurs at the same time as the blood vessels are remodelling in the endometrium. The remaining isoform (VEGF188) did not show any change in expression level during early pregnancy. These results indicate that the VEGF-A isoforms are controlled in different ways and also suggests they may have different roles in the vascular remodelling that occurs. By understanding the mechanisms that regulate the different VEGF-A isoforms, we may be better able to understand the reasons for implantation failure.
During the early stages of pregnancy in humans and other mammals, considerable vascular, remodelling takes place in the endometrium both prior to and following implantation and placentation (
). Mouse models are used to investigate the mechanisms regulating this remodelling. It has been previously shown that endometrial endothelial and mural cell proliferation increases concurrently with increasing plasma progesterone concentrations prior to implantation in the mouse (
). Following implantation in the rat, endometrial endothelial cell proliferation continues to increase at implantation sites, but decreases at intersites (
). A key regulator involved in this remodelling is the potent endothelial cell mitogen vascular endothelial growth factor-A (VEGF-A), which acts via the tyrosine kinase receptors VEGF receptor (VEGFR)-1 and -2 and the semaphorin family receptors neuropilin (Nrp)-1 and -2 (
). Inhibition studies in rats have shown that VEGF-A is essential for this pre-implantation endometrial angiogenesis (growth of new microvessels from pre-existing vasculature) (
). In a model designed to mimic early pregnancy, progesterone treatment stimulated endometrial endothelial cell proliferation in ovariectomized mice; this proliferation was significantly reduced when an antibody against VEGF-A was administered in conjunction with the progesterone (
To fully understand the mechanisms responsible for endometrial vascular remodelling during early pregnancy, it is important to understand the differential regulation and function of the various VEGF-A isoforms present within the uterus. VEGF-A has multiple isoforms (115, 120, 144, 164, 188 and 205 amino acids) that arise from alternative splicing of the eight murine VEGF-A exons (
Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines.
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and posttranscriptional regulatory sequences.
). These isoforms have different biological and solubility properties and consequently the angiogenic effects on the blood vasculature may vary depending on which VEGF-A isoform predominates (
). The properties of the different isoforms depend in part upon the presence or absence of two distinct heparin-binding domains; the presence of these domains confers on the isoform the ability to bind to the extracellular matrix (ECM) via cell surface heparin-containing proteoglycans. For example, VEGF188 possess both heparin-binding domains and binds to the ECM, but VEGF120, which lacks both domains, is highly diffusible; VEGF165 exhibits intermediate properties (
reported Vegf-A mRNA accumulated in the luminal epithelium on days 1 and 2 of pregnancy and in the luminal epithelia and subepithelial stromal bed on days 3 and 4. Following embryo attachment on day 5, Vegf-A mRNA accumulated in the luminal epithelial and stromal cells immediately surrounding the blastocyst. A similar pattern was observed by
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
, who examined Vegf164/188 transcript mRNA expression. While the above studies indicate that Vegf-A expression varies in different endometrial tissues, it does not illustrate whether these changes reflect variation in any particular Vegf-A isoform.
performed a ribonuclease protection assay analysis of Vegf-A mRNA isoform concentrations in adult mouse organs. They found Vegf164 and Vegf188 mRNA constituted 45%, 47% and 7%, respectively, of the total Vegf-A mRNA expression in the uterus of normal cycling mice (cycle stage not given). While these studies illustrate that the different isoforms are present in the mouse uterus, they do not quantify changes in the specific forms during different cycle or pregnancy stages. The goal of the current study was to quantify changes in different Vegf-A isoforms in the mouse uterus during early pregnancy and to determine whether the changing patterns were isoform specific and/or corresponded with the increased endothelial cell proliferation that occurs from day 3 of pregnancy (
There has only been limited work examining VEGF-A receptors in mouse uterus during early pregnancy. In-situ hybridization studies showed an accumulation of Vegfr2 mRNA in the uterine stromal cells of mice on day 4 of pregnancy but not in any uterine cell types on day 1 (
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
found Vegfr2 and Nrp-1 mRNA expression at low levels on days 1 and 2 of pregnancy, increasing thereafter. This study also aimed to quantify Vegf-A isoform and receptor expression in the mouse uterus during early pregnancy. As VEGF164 is believed to be the major isoform responsible for the biological actions of VEGF-A (
), it was postulated that Vegf-A164 in particular would increase during early pregnancy concurrent with the endometrial endothelial cell proliferation that is known to occur at this time (
Adult female mice (3–7 weeks, 18–28 g, C57BL/6J × CBA) were housed under controlled environmental conditions (20°C, 12 h light per day) and provided with food and water ad libitum. This study was approved by the Monash Medical Centre Animal Ethics Committee A. Female mice were housed overnight with stud males and the presence of a vaginal plug indicated successful mating. The day of a successful mating was considered day 1 of pregnancy (
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
). The mice were anaesthetized with an injection of Avertin (i.p. 25 mg/100 g body weight, 25 mg/ml of 2,2,2-tribromoethanol (Aldrich Chemical Company, Milwaukee, WI, USA), in butan-2-ol (BDH Laboratory Supplies, Poole, England)). One uterine horn from each mouse was snap frozen in dry ice immediately after collection and stored at −80°C until RNA extraction. The other uterine horn was immersion fixed in 10% buffered formalin for 2 h before processing for paraffin sections, which were used for immunohistochemistry.
Total RNA was extracted from whole uteri using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Genomic DNA contamination was removed by DNase treatment (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The RNA was quantified and an estimation of purity was measured by spectrophotometry (Eppendorf BioPhotometer, Hamburg, Germany). RNA (2 μg) from each sample was mixed with 1 μl random primers (Invitrogen), 2 μl 10 mM dNTP (Roche, Mannheim, Germany), 4 μl 5× RT Buffer (Roche), 0.5 μl RNAsin (Promega), 2 μl dithiothreitol (Promega), 0.2 μl avian myeloblastosis virus reverse transcriptase (Roche) and sterile water to make up a total volume of 20 μl. The mixture was incubated for 1 h at 42°C. Quantitative reverse-transcription polymerase chain reaction was performed using a Roche Light Cycler and the Light Cycler Fast Start Dna Master SYBR Green kit (Roche), according to the manufacturer’s instructions.
The primer sequences used are shown in Table 1. Total Vegf-A refers to a forward and reverse primer set that is common to all of the Vegf-A isoforms (as all isoforms share exons one–five). The reverse primers designed for the individual isoforms amplify each splicing variant using its specific sequence. Primer concentrations were 0.5 μmol/l. Each set of primers was optimized for annealing temperature and extension times (Table 2). The housekeeping gene 18S rRNA was used to normalize all results. To confirm that the appropriate Vegf-A isoform was being amplified, the products were sequenced by The Gandel Charitable Trust Sequencing Centre (Monash Health Research Precinct, Monash Medical Centre).
Table 1Primer sequences for PCR amplification of murine VEGF-A isoforms and receptors.
). Therefore, both the raw and the normalized data were analysed. Similar patterns of Vegf-A isoform and receptor mRNA expression were observed in both instances (data not shown). Thus, it was decided to use the normalized data for analysis as is common practice.
Western blot analysis
Following the removal of the aqueous-phase RNA fraction during the RNA extraction procedure using Trizol reagent, total protein was extracted from the protein/DNA fraction of each sample (whole uterine samples only), according to the manufacturer’s instructions. The protein pellet was washed and dissolved in 1% sodium dodecyl sulphate (SDS). Protein quantification was performed using the Bicinchoninic Acid (BCA) Protein Assay kit (Pierce Biotech, Rockford, USA), following the manufacturer’s instructions. Protein (15 μg) was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) under reducing conditions. The protein samples were mixed with 2× SDS loading buffer with 2-mercaptoethanol, heated at 95°C for 5 min and then run on a SDS–PAGE, 15% agarose gel. Electrophoresis was carried out at 100 V for 3 h. Proteins were transferred to a nitrocellulose membrane (BioRad, Hercules, CA, USA) at 35 V for 90 min. The blots were treated with blocking solution (SuperBlock; Pierce Biotech), followed by incubation with a 1:500 dilution of VEGF-A monoclonal antibody (c-7269; Santa Cruz Biotechnology, CA, USA) for 1 h. This was followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibody and finally developed using Supersignal West Dura Extended Duration Substrate (Pierce Biotech), according to manufacturer’s instructions. Pre-stained SDS–PAGE standard protein markers (BioRad) were used to calibrate the molecular mass. A mouse monoclonal β-actin antibody (1:4000 dilution; Sigma–Aldrich) was used as the loading control, using the same method as for VEGF. The developed films were scanned and assessed by densitometry (Quantity One Software, Biorad).
Immunohistochemistry
After dewaxing and rehydration, sections (3 μm) were microwaved for 15 min in EDTA buffer (pH 8) for antigen retrieval. Endogenous peroxidase was quenched using 3% H2O2 in methanol (10 min) and a protein block (Serum-free; DakoCytomation, Carpentaria, CA) was used to prevent nonspecific binding (10 min). Sections were incubated with rabbit monoclonal anti-VEGFR-2 (0.047 μg/ml, over two nights at 4°C; Cell Signaling Technology, Danvers, MA) and DakoCytomation rabbit Envision HRP (30 min). Staining was visualized by DAB (3,3′-diaminobenzidine; Sigma–Aldrich). Slides were then counterstained with Harris haematoxylin and mounted. A negative isotype matched control was prepared by replacing the VEGFR-2 primary antibody with rabbit IgG (0.047 μg/ml) at the same concentrations as that of the primary antibodies. From each section, the intensity of VEGFR-2 immunostaining in the endometrial luminaland glandular epithelium, stroma and myometrium was semi-quantified using a graded scale: 0 = no staining; 1 = weakly positive; 2 = moderately positive; 3 = strongly positive; and 4 = very strongly positive. These gradings were used for a semi-quantitative and qualitative description of the VEGFR-2 immunostaining in uterine samples collected from day 1 to 5 of pregnancy.
Statistics
Statistical analysis was performed using the Statistical Package for Social Sciences for Windows version 14.0 (SPSS, Chicago, IL, USA). VEGF-A isoform and (co)receptor mRNA data were expressed as the fold change compared with the median value of day 1 of pregnancy. mRNA and VEGFR-2 immunohistochemistry intensity data were analysed using the non-parametric Kruskal–Wallis test (H) and Mann–Whitney U-test (U). The Spearman rank correlation test was used to determine whether a correlation was present between VEGF-A isoform and VEGFR-2 mRNA expression. A P-value of <0.05 was considered significant.
Results
Total Vegf-A, Vegf120 and Vegf164, but not Vegf188, mRNA expression change significantly in mouse uterus during early pregnancy
Very similar patterns of change were exhibited by total Vegf-A mRNA, Vegf120 and Vegf164. For Vegf-A mRNA, H = 15.8, P = 0.003; day 1, median fold change = 1.0 (range: 0.4–3.5); day 2 = 0.4 (0.2–1.1); day 3 = 0.9 (0.3–3.2); day 4 = 3.1 (0.51–3.81); day 5 = 3.81 (1.51–4.31; Figure 1A). For Vegf120 mRNA, H = 12.5, P = 0.01; day 1 = 1.01 (0.1–4.7); day 2 = 0.1 (0.04–0.4); day 3 = 1.0 (0.2–6.5); day 4 = 4.5 (0.7–10.1); day 5 = 1.5 (0.6–7.2; Figure 1B). For Vegf164 mRNA, H = 17.6, P = 0.001; day 1 = 1.0 (0.2–3.9); day 2 = 0.2 (0.05–1.3); day 3 = 1.04 (0.2–5.8); day 4 = 4.7 (0.57–6.0); day 5 = 7.1 (2.0–12.5; Figure 1C). For each of these isoforms, the lowest levels of mRNA expression were observed on day 2 of pregnancy. In the case of Vegf120, expression increased significantly between days 2 and 3 of pregnancy (P < 0.05). For total Vegf-A and Vegf164, a significant increase was not observed until day 4 (groups that are significantly different are shown in Figure 1; P < 0.05). Expression remained high on day 5 of pregnancy (day of implantation). There was no significant change in the expression of Vegf188: H = 5.46, P = 0.2; day 1 = 1.0 (0.2–1.3); day 2 = 0.26 (0.1–0.7); day 3 = 0.8 (0.2–3.689); day 4 = 1.6 (0.2–2.3); day 5 = 0.9 (0.4–2.0; Figure 1D).
Figure 1Uterine vascular endothelial growth factor (VEGF)-A isoform mRNA expression in CBA/C57 mice from day 1 to 5 of pregnancy. The mRNA expression of (A) total Vegf-A, (B) Vegf120, (C) Vegf164 and (D) Vegf188 in pregnant mice was normalized against 18S rRNA. Data is expressed as the fold change compared with the median value on day 1 of pregnancy. The lines in the boxes represent the medians and the boxes extend from the 25th to the 75th centiles. The error bars indicate the minimum and maximum values (n = 6). Groups that do not share a letter in common are significantly different (P< 0.05).
Vegfr2, but not Nrp-1 and Nrp-2, mRNA expression increases significantly in early pregnancy in the mouse uterus
There was a significant increase in relative mRNA expression of the VEGF-A receptor Vegfr2 across the first 5 days of pregnancy: H = 18.3, P = 0.001; day 1, median = 1.0 (range: 0.2–2.3); day 2 = 0.9 (0.2–1.7); day 3 = 7.4 (1.8–16.2); day 4 = 5.7 (2.6–15.0); day 5 = 4.0 (1.1–11.8) (Figure 2A). Vegfr2 mRNA expression was significantly increased on days 3, 4 and 5 relative to days 1 and 2 (P < 0.05). Vegfr2 mRNA expression also had a moderate, but significant positive correlation with Vegf120, Vegf164 and Vegf188 mRNA expression levels (r = 0.51, P = 0.004; r = 0.42, P = 0.02; and r = 0.46, P = 0.02, respectively).
Figure 2Uterine vascular endothelial growth factor (VEGF)-A receptor mRNA expression in CBA/C57 mice from day 1 to 5 of pregnancy. The mRNA expression of (A) Vegfr-2, (B) Nrp-1 and (C) Nrp-2 in pregnant mice were normalized against 18S rRNA. Data is expressed as the fold change compared with the median value on day 1 of pregnancy. The lines in the boxes represent the medians and the boxes extend from the 25th to the 75th centiles. The error bars indicate the minimum and maximum values (n = 6). Groups that do not share a letter in common are significantly different (P< 0.05).
However, the increase in relative mRNA expression of the co-receptor Nrp-1 across the first 4 days of pregnancy did not reach significance: H = 9.35; day 1 = 1.0 (0.2–45.9); day 2 = 0.5 (0.2–37.1); day 3 = 67.7 (0.3–191.0); day 4 = 29.2 (1.1–215.1); day 5 = 82.4 (6.8–149.3; Figure 2B). Although the highest median levels were seen on days 3, 4 and 5 (as for Vegfr2), there was a large range in relative expression among samples. There was no significant change in Nrp-2 expression: H = 3.26; day 1 = 1.0 (0.1–1.4); day 2 = 0.9 (0.1–2.3); day 3 = 1.4 (0.1–3.2); day 4 = 1.2 (0.4–7.2); day 5 = 1.2 (0.7–6.0; Figure 2C).
VEGF-A protein isoform expression in pregnant mouse uterus
Western blot detection of VEGF-A protein showed several distinct bands (Figure 3). A strong band was noted at approximately 27 kDa consistent with the VEGF188 isoform. Other bands greater than 27 kDa were also noted, including a distinctive band at approximately 51 kDa. These may be various cleaved products and dimers of VEGF-A isoforms. VEGF164 was represented by two distinct bands at approximately 20–23 kDa; these may reflect differentially glycosylated forms. A very faint band, consistent with the VEGF120 isoform, was detected at approximately 18 kDa in some samples. No changes were noted in the intensity of the VEGF188 band (27 kDa) or VEGF164 bands (20–23 kDa) among uterine samples collected from female mice on days 1–5 of pregnancy. However, VEGF188 isoform expression was consistently higher than both VEGF164 bands.
Figure 3Uterine vascular endothelial growth factor (VEGF)-A protein expression in CBA/C57 mice from day 1 (D1) to 5 (D5) of pregnancy. Representative Western blot analysis of VEGF-A isoforms under reducing conditions. Bands at 20–23 kDa and 27 kDa represent VEGF164 and VEGF188, respectively. A very pale band was observed at ∼18 kDa in some samples (thought to be VEGF120, white arrows). Molecular weight of bands determined by comparison with pre-stained, broad range standards (left-hand side). BC = Breast cancer sample used as positive control.
The strongest VEGFR-2 immunostaining was observed in endothelial cells (Figure 4); however, there was no significant difference in expression across the days of pregnancy in either the endometrium (H = 3.2; day 1 median = 3 (range 2–3); day 2 = 2.5 (2–3); day 3 = 3 (all three samples); day 4 = 2 (1.5–3); day 5 = 2.5 (2–3)) or myometrium (H = 3.2; day 1 = 2 (2–3); day 2 = 2.5 (2–3); day 3 = 3 (all three samples); day 4 = 2 (2–3); day 5 = 2.5 (2–3)). Immunostaining was also observed in pericytes and vascular smooth muscle cells. VEGFR-2 immunostaining was present in the glandular epithelial cells (day 1 = 2 (1–2.5); day 2 = 2.5 (2–3); day 3 = 2 (1–2.5); day 4 = 2 (2–3); day 5 = 2.5 (2–3)), and to a lesser extent, the luminal epithelium (day 1 = 1.5 (1–2); day 2 = 1.5 (1.5–2.5); day 3 = 1 (1–2); day 4 = 2.5 (2–2.5); day 5 = 1.5 (1.5–3)). Epithelial staining was variable both within and among samples and consistently higher expression was observed in the nuclei relative to the cytoplasm. There was no significant difference across the days of pregnancy for either glandular or luminal epithelial cells (H = 3.8 and 7.7, respectively). In some sections examined (not specific to any particular day of pregnancy), immunostaining was also observed in subepithelial stromal fibroblasts.
Figure 4Vascular endothelial growth factor receptor (VEGFR)-2 immunostaining in CBA/C57 mouse uterus during early pregnancy. Representative photomicrograph of endometrium from pregnant CBA/C57 mice immunostained with VEGFR-2 antibody. Inset; negative control (rabbit IgG). Black arrows indicate blood vessels; e = luminal epithelium; g = gland; l = uterine lumen; s = stroma. Bar = 100 μm.
Immunostaining was generally low within the myometrium and no significant changes in intensity were observed across the days of pregnancy (myometrial longitudinal muscle: H = 3.1; day 1 = 1.5 (1.5–2); day 2 = 1.5 (1.5 all samples); day 3 = 1.5 (1.5–2.5); day 4 = 2 (1.5–2); day 5 = 1.5 (1–2.5); myometrial circular muscle: H = 1.8; day 1 = 1.5 (1.5–2); day 2 = 1.5 (1–1.5); day 3 = 1.5 (1.5 all samples); day 4 = 1.5 (1–2); day 5 = 1.5 (1.5–2.5)).
Discussion
It has been shown that VEGF-A isoform mRNA is differentially expressed in the mouse uterus during early pregnancy. While Vegf120 and Vegf164 showed a significant increase in expression after day 2 of pregnancy, there was no significant change in Vegf188 expression. These results suggest differential regulation of VEGF-A isoform expression and have implications for the type of vascular remodelling that takes place within the endometrium. The most common VEGF-A protein present in the mouse uterus was VEGF188, consistent with the idea that a store of VEGF-A is maintained within the extracellular matrix of the uterus until required. Such a source could be expected to play a role in the localized vascular remodelling that occurs during embryo implantation. It is postulated that the increase in Vegf120 and Vegf164 expression, as well as the increase in Vegfr2 that was also observed, plays a role in driving the increased endometrial endothelial cell proliferation that occurs during early pregnancy in the mouse (
). In previous studies, Vegf-A mRNA expression was observed in the luminal epithelium on days 1 and 2 of mouse pregnancy and in both the luminal epithelium and subepithelial stromal cells on days 2 and 4 of pregnancy, prior to implantation (
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
). A significant increase in total Vegf-A and Vegf120/Vegf164 mRNA in mouse uterus was observed on days 3 and 4 of pregnancy, respectively, concurrent with previously reported increases in circulating progesterone concentrations and endothelial cell proliferation (
). In contrast, no change in Vegf188 mRNA was observed. It is possible that the increased expression of Vegf120 and Vegf164 mRNA reflects increased expression by stromal cells in particular, as could be postulated from the earlier studies (
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
also described how VEGF-A immunostaining was most intense in the uterine epithelium.
Therefore, as VEGF120 and VEGF164 are soluble and partially soluble isoforms, respectively, it is also possible that the increased mRNA expression observed in the whole uterine tissue in the current study, reflects changes occurring in the epithelium. While it has been shown that most epithelial VEGF-A is secreted into the uterine lumen (
). Whether specific VEGF-A isoforms produced by the epithelium act in a paracrine manner to regulate endometrial vascular remodelling is yet to be determined.
Although increases in total Vegf-A, Vegf120 and Vegf164 mRNA expression were observed in the current study, corresponding increases in VEGF-A protein from Western blot analysis were not observed. This later observation is consistent with previously published data examining VEGF-A immunoexpression in mouse uterus (
). No changes in the intensity of VEGF-A immunostaining were observed across early pregnancy in either the stroma or the epithelium. Unfortunately, there are a number of problems associated with precise quantification of the VEGF-A isoform proteins. Neither Western blotting nor immunohistochemistry have the specificity as a single technique to detect both the expression levels of the different isoforms and their localization. Nor do they have the sensitivity to detect subtle changes in expression. Western blotting is unable to localize protein expression within the tissue, thereby the level of expression in one region may be masked by the level of expression in another region. In contrast, there are not the different VEGF-A isoform antibodies available for immunohistochemistry, which would enable their localization. Furthermore, soluble VEGF-A (VEGF120 and VEGF164) is most likely either rapidly used, or secreted into the lumen of the uterus, and is no longer detectable at the time of immunohistochemistry. Until specific antibodies to the VEGF-A isoforms are available, precise protein quantification and localization is not possible.
In the current study, the protein isoform with the highest expression overall during early pregnancy was VEGF188, although there were no changes in either mRNA or protein expression of this isoform across the days of pregnancy. This isoform contains two heparin-binding domains and binds closely with the ECM, thereby providing a pre-formed source of VEGF-A for localized requirements. It is postulated that VEGF188 is the dominant VEGF-A isoform responsible for the vascular remodelling that takes place immediately around the implanting blastocyst, in contrast to the uterine-wide changes in VEGF-A expression and endometrial endothelial cell proliferation occurring prior to implantation. The mechanisms by which VEGF-A, and in particular VEGF188, is regulated at the sites of implantation remain to be elucidated. Molecules such as heparanase are known to increase in expression around implantation sites in early pregnancy in the mouse and its inhibition negatively impacts on implantation (
). Heparanase is involved in releasing various growth factors and cytokines from their interactions with heparin sulphate proteoglycans and is a likely candidate for investigating the mechanism of VEGF188 release from the cytoplasm.
Although this study has shown that VEGF-A isoforms are differentially regulated in the pregnant mouse uterus, the functional consequences of this differential regulation are still to be addressed. However, the functional significance of VEGF-A splice variants have been considered in other physiological systems. Using a mouse tumour model, it has been shown that the properties of the blood vasculature may vary depending on which VEGF-A isoform is prominent. Only VEGF164 was able to fully rescue tumour growth when transfected cells expressing only one VEGF-A isoform were injected into immunocompromised mice. The vascular density of VEGF188-expressing tumours was significantly greater than wild-type tumours, but most vessels were of small caliber and failed to connect the tumour vessels to the systemic vasculature. VEGF120 induced fewer and less-branched vessels that did not pervade the entire tumour mass (
). In the mouse mammary gland, specific VEGF-A isoforms were localized to either the stroma or the epithelium of the mammary gland, depending upon whether the mice were nulliparous or lactating (
). These isoforms functioned in an autocrine or paracrine manner under hormonal regulation. Furthermore, using data from mice that expressed only one VEGF-A isoform,
was able to show that VEGF-A isoforms have distinct roles in vascular patterning of the retina. Only VEGF(164/164) mice had normal retinal angiogenesis, whereas VEGF(120/120) mice had defects in vascular outgrowth and patterning. Although VEGF(188/188) mice had normal venular outgrowth, they had impaired arterial development.
Two previous studies have used northern blots and in-situ hybridization to investigate the temporal and spatial expression of Vegfr2 mRNA during the peri-implantation period in mouse uterus (days 1–8 of pregnancy).
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
study, the current study only observed positive VEGFR-2 immunostaining in the stromal fibroblasts of a few samples, with no correlation to any particular day of pregnancy. However, similar to the
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
study, the current study also observed VEGFR-2 immunostaining in stromal endothelial cells throughout early pregnancy. It remains to be determined whether the different patterns reported for mRNA (
Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.
) versus protein (this study) reflects a redistribution of VEGFR-2 following translation.
The current study also observed positive immunostaining for VEGFR-2 within the endometrial epithelium, in particular the glandular epithelium. It is curious that high levels of both VEGF-A and VEGFR-2 expression are observed in the endometrial epithelium. These expression patterns argue for a specific function for VEGF-A within the epithelium, distinct from the important roles of this growth factor in vascular remodelling. In some studies examining the effect of VEGF-A inhibition on endothelial cell proliferation, a decrease in endometrial epithelial cell proliferation was also observed (
). Future studies specifically examining the role of VEGF-A/VEGFR-2 interactions in the endometrial epithelium are required.
In conclusion, it has been shown that VEGF-A isforms are differentially expressed and regulated in the mouse uterus during early pregnancy. The timing of the increase in VEGF-A isoforms VEGF120 and VEGF164 corresponded with the increase in endometrial endothelial cell proliferation that takes place during early pregnancy in mice (
). The increase in these specific isoforms may indicate a requirement for a rapidly available and soluble source of VEGF-A throughout the endometrium during a time of increased endometrial angiogenesis prior to implantation. However, VEGF188 was shown to be the most highly expressed VEGF-A protein during early pregnancy in the mouse. It is postulated that this provides a source of VEGF-A available for locally regulated vascular remodelling, such as that occurring around the implanting blastocyst. Further research is required to fully elucidate the biological significance of the different VEGF-A isoforms during early pregnancy both in humans and other animal models.
Acknowledgements
The authors wish to thank the staff of the Monash Medical Centre Animal House for their technical help and assistance. Thanks are also due to Dr Anna Ponnampalam for her advice on molecular biology protocols.
References
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Effect of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells.
Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines.
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Declaration: The authors report no financial or commercial conflicts of interest.
Footnotes
Dr Lisa Walter completed her Biomedical Science (Honours) degree in 2003 and her PhD in 2008 in the Centre for Women’s Health Research, Monash Institute of Medical Research, Monash University. Her studies investigated the molecular regulation of endometrial angiogenesis. Dr Walter joined the Ritchie Centre as a Research Fellow in February 2008 and her post-doctoral research involves the cardiovascular implications of sleep-disordered breathing in pre-school children.
Dr Lisa Walter completed her Biomedical Science (Honours) degree in 2003 and her PhD in 2008 in the Centre for Women’s Health Research, Monash Institute of Medical Research, Monash University. Her studies investigated the molecular regulation of endometrial angiogenesis. Dr Walter joined the Ritchie Centre as a Research Fellow in February 2008 and her post-doctoral research involves the cardiovascular implications of sleep-disordered breathing in pre-school children.
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