Vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium

The relationship between vascular endothelial growth factor (VEGF) expression and the pattern of vascular proliferation in the rhesus macaque endometrium has not been studied. In this report, we used in situ hybridization to evaluate VEGF, VEGF receptor type 1 and VEGF receptor type 2 mRNA expression during hormonally regulated menstrual cycles in ovariectomized macaques. Proliferating endothelial cells were identified by a double immunocytochemistry procedure that detected Ki-67 antigen and von Willebrand factor in the same endothelial cells. One and 2 d after progesterone withdrawal (premenstrual), VEGF mRNA was up-regulated in the glands and stroma of the superficial endometrial zones, a finding that supports our previous suggestion that VEGF may play a role in the menstrual induction cascade. During the postmenstrual repair phase, the healing surface epithelium showed a further, dramatic increase in expression of VEGF mRNA, accompanied by strong increases in signals for VEGF receptor types 1 and 2 in multiple profiles of small blood vessels immediately below the surface epithelium. This finding implicates VEGF in the early angiogenic processes associated with endometrial healing and regeneration. Vascular endothelial proliferation persisted throughout the cycle in the upper endometrial zones and showed a dramatic estrogen- dependent peak during the midproliferative phase. This proliferative peak coincided with a peak in VEGF expression in the endometrial stroma. Endothelial proliferation was also significantly correlated with the degree of stromal VEGF expression during the proliferative and secretory stages of the cycle. These results implicate VEGF of stromal origin in endometrial vascular proliferation.     

Expression of vascular endothelial growth/permeability factor by endometrial glandular epithelial and stromal cells in baboons during the menstrual cycle and after ovariectomy

Vascular endothelial growth/permeability factor (VEG/PF) has a crucial role in angiogenesis, and neovascularization is essential in preparing the uterine endometrium for implantation. However, the regulation of VEG/PF synthesis by particular cell types of the endometrium during the human menstrual cycle is not well understood. Therefore, in the present study the baboon was used as a nonhuman primate to determine the role of the ovary in vivo in endometrial VEG/PF expression. VEG/PF mRNA levels were quantified by competitive RT-PCR in whole uterine endometrium and in glandular epithelial and stromal cells isolated from the endometrium by laser capture microdissection of baboons during the normal menstrual cycle and after ovariectomy, which decreased serum estradiol and progesterone to undetectable levels. Mean (+/-SE) levels (attomoles per micrograms of total RNA) of the 323-bp VEG/PF mRNA product, which reflected collective expression of all VEG/PF isoforms, in whole endometrium were 785 and 727 +/- 158 during the mid and late follicular phases, respectively, and 1108 +/- 320 during the midcycle surge in serum estradiol. VEG/PF mRNA levels then declined briefly before increasing to 1029 +/- 365 attomoles/ micro g RNA during the late luteal phase of the menstrual cycle. VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) were similar in glandular epithelial (2.27 +/- 1.11) and stromal (2.54 +/- 0.70) cells at the midcycle estradiol peak and the midluteal phase of the menstrual cycle (2.34 +/- 1.30 and 1.49 +/- 0.53, respectively). Immunocytochemical expression of VEG/PF protein was abundant in glandular and luminal epithelium, stroma, and vascular endothelium. Endometrial vessel density and percent vascularized area, determined by morphometric image analysis, were similar during the various stages of the baboon menstrual cycle. After ovariectomy, VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) in the endometrial glands (0.52 +/- 0.21) and stroma (0.22 +/- 0.11) were decreased to values that were approximately 20% and 10% (P < 0.05), respectively, of those in intact baboons during the midcycle estrogen surge. Moreover, there was relatively little VEG/PF protein immunostaining in the endometrial glands, stroma, and vascular endothelium after ovariectomy. In summary, VEG/PF mRNA and protein expression in glandular epithelial and stromal cells were markedly suppressed after ovariectomy, indicating that synthesis of this angiogenic factor in these endometrial cells is dependent upon a product(s) secreted by the ovary. Moreover, endometrial VEG/PF expression remained relatively constant and thus was available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.     

The role of vascular endothelial growth factor and estradiol in the regulation of endometrial angiogenesis and cell proliferation in the marmoset

The present studies explore the roles of vascular endothelial growth factor (VEGF) and estradiol on angiogenesis and stromal and epithelial cell proliferation in the marmoset endometrium during the proliferative phase of the ovulatory cycle. At the start of the proliferative phase, marmosets were 1) treated with vehicle, 2) treated with a VEGF inhibitor (VEGF Trap, aflibercept), 3) ovariectomized, 4) ovariectomized and given replacement estradiol, or 5) treated with VEGF Trap and given replacement estradiol. The uterus was examined 10 d later in the late proliferative phase. Changes in endothelial and epithelial cell proliferation were quantified using a volumetric density method after immunohistochemistry for bromodeoxyuridine to localize proliferating cells, CD31 to visualize endothelial cells, and dual staining to distinguish endothelial cell proliferation. Endothelial proliferation was elevated in late proliferative controls but virtually absent after VEGF Trap. Ovariectomy had a similar inhibitory effect, whereas angiogenesis was restored by estrogen replacement. Estradiol replacement in VEGF Trap-treated marmosets resulted in only a small increase in endothelial cell proliferation that remained significantly below control values. VEGF Trap treatment and ovariectomy also markedly reduced stromal cell proliferation but resulted in increased stromal cell density associated with a reduction in overall endometrial volume. Estrogen replacement in both ovariectomized and VEGF Trap-treated animals restored stromal proliferation rates and cell density. These results show that endometrial angiogenesis and stromal proliferation during the proliferative phase are driven by estradiol and that the effect of estrogen on angiogenesis is mediated largely by VEGF.     

Progesterone withdrawal up-regulates vascular endothelial growth factor receptor type 2 in the superficial zone stroma of the human and macaque endometrium: potential relevance to menstruation

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The upregulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.     

Angiogenesis in the endometrium

Human endometrial vasculature has the unique property of undergoing benign angiogenesis during each menstrual cycle under the influence of the ovarian steroids estradiol and progesterone. However, neither has intrinsic angiogenic activity and endometrial angiogenesis involves stimulation by ovarian steroids of angiogenic factor release by the epithelium and stroma which then act on the endothelium. In vitro models using cultures of isolated endometrial epithelium, stroma and endothelium now allow mechanistic questions to be addressed. Vascular endothelial growth factor and platelet-derived endothelial cell growth factor/thymidine phosphorylase at present appear to be key players in endometrial angiogenesis.     

Vascular endothelial growth factor mediates the estrogen-induced breakdown of tight junctions between and increase in proliferation of microvessel endothelial cells in the baboon endometrium

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4-6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 +/- 0.99-fold (mean +/- se) and 5.70 +/- 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 +/- 0.22 nm at 0 h to 7.27 +/- 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67- immunolabeled endothelial cells, increased (P < 0.05) from 2.9 +/- 0.3% at 0 h to 21.4 +/- 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.     

Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.

The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.     

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.     

血清雌激素与肝细胞癌转移的关系

目的了解血清雌激素升高与原发性肝细胞癌（ＨＣＣ）转移的关系．方法应用放射免疫分析法（ＲＩＡ）对ＨＣＣ患者血清雌二醇（Ｅ２）含量进行了检测，并应用原位杂交研究外源性Ｅ２对肝癌细胞系ＣＢＲＨ７９１９血管内皮生长因子（ＶＥＧＦ）和层粘蛋白受体（ＬＮＲ）表达的影响．结果ＨＣＣ患者血清Ｅ２水平较正常者显著升高，且ＨＣＣ有转移者血清Ｅ２水平较无转移者亦显著升高；原位杂交结果显示，外源性Ｅ２能够增加ＣＢＲＨ７９１９细胞表达ＶＥＧＦ和ＬＮＲｍＲＮＡ．结论血清Ｅ２水平增高与ＨＣＣ转移密切相关，它有可能通过促进ＶＥＧＦ和ＬＮＲ的合成而影响肿瘤的转移过程．     

Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia

Endometrial growth and repair after menstruation are associated with profound angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns and are common in many women. It is therefore important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and pathological angiogenesis. In this study we show that expression of VEGF is regulated by hypoxia in human endometrium. Culture in vitro for 24 h under hypoxic conditions resulted in a 2- to 6-fold increase in VEGF secretion by both stromal and epithelial cells isolated from human endometrium. Quantitative RT-PCR was used to measure VEGF messenger ribonucleic acid (mRNA) levels in these cells. After hypoxia, VEGF mRNA levels increased 1.8-fold in stromal cells and 3.4-fold in glandular epithelial cells. The mRNA for each VEGF splice variant increased to an equal extent. The increase in VEGF secretion by stromal and epithelial cells in response to hypoxia was not altered by treatment at the same time with estradiol or progesterone. In situ hybridization of human endometrium during menstruation, when steroid levels are low but the tissue is subject to ischemia, showed strong hybridization to VEGF mRNA in both stromal and glandular cells. These results show that local factors, such as hypoxia, can regulate VEGF expression in the endometrium. This may play an important part in normal endometrial repair after menstruation. The secretion of VEGF by endometrial cells under hypoxic conditions may also be important in the pathogenesis of endometriosis, because it would be predicted to assist revascularization of desquamated endometrial explants when they attach at ectopic sites.     

Vascular endothelial cadherin regulates vascular permeability: Implications for ovarian hyperstimulation syndrome

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. It is characterized by increased vascular permeability and simultaneous overexpression of vascular endothelial growth factor (VEGF) in ovarian cells. OBJECTIVE: We tested the hypothesis that the endothelium and endothelial cell-to-cell junctions are downstream targets of VEGF during OHSS pathogenesis. We investigated the potential involvement of vascular endothelial (VE)-cadherin, an interendothelial adhesion molecule, in the capillary hyperpermeability in OHSS. DESIGN: Human endothelial cells from umbilical veins (HUVEC) were used as an in vitro model of OHSS. INTERVENTION: Cell cultures were treated with varying doses of estradiol (E2), human chorionic gonadotropin (hCG), VEGF, and antihuman VEGF antibodies, either alone or in combination, and the effect on VE-cadherin release was evaluated at different time points. Permeability assays were performed using fluoresceinisothiocyanate-labeled albumin, and actin filaments rearrangement was evaluated by fluorescent microscopy. RESULTS: Culturing of HUVEC with high doses of E2 produced no significant changes in VE-cadherin concentration, but hCG and VEGF produced a significant increase in VE-cadherin release. Time-course experiments showed that VE-cadherin was secreted 12 h after VEGF addition. Antihuman VEGF antibodies prevented these changes. Permeability assays demonstrated that, although E2 did not alter the arrangement of HUVEC in vitro, hCG and VEGF caused changes in the actin fibers indicative of increased capillary permeability. VEGF also induced an increase in paracellular permeability of HUVEC at the same doses used in the previous experiments. CONCLUSIONS: Adhesion molecules like VE-cadherin may play a role in the development and progression of increased capillary permeability in severe OHSS.     

Acute temporal regulation of vascular endothelial growth/permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration. Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 +/- 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 +/- 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 +/- 0.30, glands and 1.20 +/- 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (+/-SE) of 71.6 +/- 4.6 nm at 0 h to 101.1 +/- 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.     

Cellular expression and hormonal regulation of neuropilin-1 and -2 messenger ribonucleic Acid in the human and rhesus macaque endometrium

Although much is known about the biology of vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs), VEGFR1 and VEGFR2, little is known about the roles of the VEGFRs neuropilin (NP)-1 and NP-2 in the primate endometrium. In this study, we investigated the cellular localization and hormonal regulation of NP-1 and NP-2 mRNA by in situ hybridization in the endometrium of ovariectomized, hormonally cycled rhesus macaques and women during the natural menstrual cycle. NP-1 mRNA was highly expressed in vascular endothelium and in stromal cells, but in these cells, NP-1 expression did not change during the menstrual cycle. However, NP-1 mRNA was also expressed in the luminal epithelium (not the glands), and its expression in these cells was elevated during the mid- to late proliferative phase and completely suppressed during the secretory phase. The increase in NP-1 level in the luminal epithelium was estradiol dependent because such expression was not detectable in the absence of estradiol in ovariectomized, hormone-deprived animals. Moreover, NP-1 expression in the luminal epithelium was highly correlated with the degree of proliferation in these cells. A recent study showed that blockade of VEGF action can inhibit luminal epithelial cell proliferation, but there is no evidence of VEGFR1 and VEGFR2 expression in these cells. Therefore, NP-1 may be the relevant VEGFR that mediates proliferation in this epithelium. NP-2 mRNA, unlike NP-1, was expressed only by the endothelium of veins, and in these cells, its expression was hormonally regulated in the converse manner: it was very low during the proliferative phase and high during the secretory phase. The increased permeability and edema observed during the secretory phase in the primate endometrium may be mediated in part by VEGF-NP-2 interaction. In the human endometrium, the pattern of expression and cellular localization of both NP-1 and NP-2 during the menstrual cycle were essentially identical with that seen in the rhesus macaque endometrium. These are the first data to specify the hormonal regulation and cell-specific expression of NP-1 and NP-2 mRNA in the endometrium of both women and nonhuman primates. The findings extend our understanding of VEGF action in the primate endometrium.     

Cell-mediated delivery of fibroblast growth factor-2 and vascular endothelial growth factor onto the chick chorioallantoic membrane: endothelial fenestration and angiogenesis.

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.     

Lysophosphatidic acid mediates interleukin-8 expression in human endometrial stromal cells through its receptor and nuclear factor-kappaB-dependent pathway: a possible role in angiogenesis of endometrium and placenta

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid molecule involved in inflammation, angiogenesis, would healing, and cancer invasion. Whereas serum lysophospholipase D activity increases in women with pregnancy, the role of LPA in pregnancy remains unclear. We investigated the expression of LPA receptors and function of LPA in endometrial stromal cells. Histologically normal endometrium was obtained from surgical specimens of women undergoing hysterectomy for leiomyoma. First-trimester decidua was obtained from women receiving elective termination of pregnancy. We examined the expressions of LPA1, LPA2, and LPA3 receptors in endometrial stromal cells. The effects of LPA on the expression of vascular endothelial growth factor, IL-6, and IL-8 were examined. Signal pathways of LPA were delineated. Functions of secretory angiogenic factors were tested using human endometrial microvascular endothelial cells. Immunoreactivity and mRNA of LPA1 receptors were identified in endometrial stromal cells. LPA enhanced IL-8 expression in a dose- and time-dependent manner, whereas vascular endothelial growth factor or IL-6 expression was not affected by LPA treatment. Mechanistic dissection disclosed that LPA functioned via the Gi protein, MAPK/p38 and nuclear factor-kappaB pathway. LPA-induced IL-8 enhanced migration, permeability, capillary tube formation, and proliferation of human endometrial microvascular endothelial cells. Endometrial stromal cells express LPA1 receptors. Through the LPA1 receptor, LPA induces IL-8 expression via a nuclear factor-kappaB-dependent signal pathway. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.     

Differential effects of thrombin and hypoxia on endometrial stromal and glandular epithelial cell vascular endothelial growth factor expression

Ovarian steroids and/or premenstrual endometrial hypoxia are thought to restore the endometrial vasculature shed during menstruation by elevating endometrial vascular endothelial growth factor (VEGF) levels. During the luteal phase, VEGF levels peak, progesterone induces estradiol (E(2))-primed human endometrial stromal cells (HESCs) to decidualize and express tissue factor (TF), and endometrial vascular permeability is enhanced. The latter would present circulating clotting factors to decidual cell-expressed TF to form local thrombin. HESCs were incubated in serum-supplemented medium containing vehicle (control) or 10(-8) M E(2) or 10(-7) M medroxyprogesterone acetate (MPA) or E(2) + MPA for 7 d to induce decidualization, while monolayers of human endometrial glandular epithelial cells (HEGECs) formed during 4-d incubation of glands. The medium was exchanged for a defined medium containing corresponding vehicle or steroids +/- thrombin under normoxia or hypoxia (0-1% O(2)). Hypoxia enhanced secreted immunoreactive VEGF levels by severalfold in HESCs and HEGECs, but the steroids did not affect VEGF output in either cell type under normoxia or hypoxia. In E(2) + MPA-decidualized HESCs, VEGF levels were elevated by 0.1 U/ml of thrombin, and 0.5-2.5 U/ml of thrombin elicited maximum effects. The addition of 0.5 U/ml of thrombin evoked a time-dependent enhancement of VEGF levels and about an 8-fold increase at 48 h (P < 0.02; n = 6). Northern blotting indicated that E(2) + MPA-decidualized HESCs expressed VEGF(121), VEGF(165), and VEGF(189) mRNA, which were enhanced severalfold during 5- to 20-h incubation with thrombin. Moreover, TRAP, a synthetic peptide activator of the constitutively expressed protease activated receptor-1 thrombin receptor in decidualized HESCs, also elevated secreted VEGF levels. By contrast, HEGECs were unresponsive to thrombin added alone or with ovarian steroids. These results suggest that thrombin formed by progestin-augmented TF levels acts as an autocrine enhancer of VEGF expression in decidualized HESCs. Because angiogenesis occurs in a matrix of decidualized HESCs, these in vitro results provide a novel mechanism to account for both the peak in VEGF and angiogenesis in luteal phase human endometrium.     

Effects of estradiol-17beta on expression of mRNA for seven angiogenic factors and their receptors in the endometrium of ovariectomized (OVX) ewes

We have previously established an ovariectomized (OVX) ewe model to study how steroid removal and replacement affects uterine blood vessel and tissue growth. Using this model, endometrial expression of mRNA for 14 angiogenic factors (7 genes and their respective receptors) in caruncular (CAR) and intercaruncular (ICAR) endometrium were evaluated by quantitative real time RT-PCR at 0 (control), 2, 4, 8, 16, or 24 h after treating OVX ewes with an estradiol-17β (E2) implant. In CAR and ICAR, compared to 0 h, the mRNA expression of vascular endothelial growth factor (VEGF), VEGF receptor (R)1, soluble guanylate cyclase (GUCY1B3; the R for nitric oxide [NO]), hypoxia inducible factor (HIF) 1α, and placental growth factor (PIGF) increased by 4 h after E2-treatment, but basic fibroblast growth factor (FGF2), endothelial NO synthase (NOS3), angiopoietin (ANGPT)1, ANGPT2, ANGPT receptor Tie2 by 2 h after E2. Expression of mRNA for FGFR2IIIc was increased at 2 h by E2-treatment in ICAR, but not in CAR. By contrast, expression of neuropilin (NP) 1 mRNA was increased at 2 h in CAR, but not ICAR. The mRNA expression of VEGF, FGF2, HIF1α, and PIGF was positively correlated with mRNA expression of NOS3, VEGFR1, and Tie2 suggesting some E2-stimulated interactions between these factors in promoting blood vessel growth. Thus, several major angiogenic factors and their receptors are increased within hours after E2-treatment, which indicates that E2 plays a role in regulation of angiogenesis in the uterus. By using the OVX ewe model, we may begin to understand the molecular basis of E2 effects on angiogenesis in the endometrium and, eventually, how angiogenesis is regulated in normal versus pathological conditions.