Lack of correlation between vascular endothelial growth factor production and endothelial cell proliferation in the human endometrium.

The role of vascular endothelial growth factor (VEGF) in endometrial angiogenesis was examined by measuring its production in human endometrial tissues from different stages of the menstrual cycle and relating these data to endothelial cell proliferation in the same tissues. Conditioned medium was collected from explant, and separated glandular epithelial and stromal cells cultured from 24 normal human endometrial biopsies and VEGF measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was also used to assess VEGF and the percentage of proliferating microvessels in the samples. Wide variation in results between individual endometrial samples at each stage of the menstrual cycle was observed for all parameters measured. There was no significant difference in VEGF secretion by explant, glandular epithelial or stromal cell cultures across the menstrual cycle, or in the percentage of proliferating vessels. VEGF immunostaining in the stroma was elevated during the early proliferative stage (P = 0.03). Epithelial cells secreted more VEGF than stromal cells (1.76 +/- 0.46 versus 0.46 +/- 0.06 ng per 10(5) cells; P = 0.002). There was no correlation between VEGF secreted by cultured explants, epithelial or stromal cells, VEGF immunostaining and the proportion of proliferating microvessels. These results show that the majority of endometrial VEGF is produced by glands, but neither total glandular nor stromal VEGF is correlated with endometrial endothelial cell proliferation. There is still no clear understanding on the regulation of human endometrial angiogenesis.     

Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils

Vascular endothelial growth factor (VEGF) is expressed in human endometrium, but the cellular source of VEGF for endometrial angiogenesis has not been determined. In the present study the relationship between focal VEGF associated with microvessels and endothelial cell proliferation was examined in three layers of human endometrium at various stages of the menstrual cycle (menstrual, proliferative and secretory). Immunohistochemical analysis of full thickness endometrium from 18 hysterectomy samples without endometrial pathology were examined. The percentage of proliferating vessels was higher in proliferative compared to secretory endometrium, but this was only statistically significant in the basalis layer. A significantly greater percentage of VEGF-expressing microvessels was observed in proliferative than secretory endometrium (P < 0.05). The most VEGF-expressing microvessels were observed in the subepithelial capillary plexus, followed by the functionalis and least were present in the basalis. There was a significant correlation between focal VEGF-expressing microvessels and proliferating vessels for the subepithelial capillary plexus (R(s) = 0.70, P = 0.008), the functionalis (R(s) = 0.70, P = 0.001) and the basalis (R(s) = 0.76, P < 0.001). Focal VEGF associated with microvessels was found in marginating and adherent neutrophils. These data suggest that neutrophils in intimate contact with endometrial endothelium may be a source of intravascular VEGF for vessels undergoing angiogenesis by elongation or intussusception, particularly during the proliferative phase of rapid endometrial growth.     

Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system. Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1 (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show no variation. Epiregulin (EP) is detectable in some samples. EGF is undetectable. HER1, HER2, HER3 and HER4 were localized to the epithelium and glands HER3 and HER4 solely in the secretory phase. Amphiregulin was seen in leucocytes and stromal cells, TGFalpha and betacellulin in the epithelial lining, epiregulin in stromal cells whereas HB-EGF and EGF are undetectable. In conclusions, we observed cyclical expression of the four EGF receptors and two ligands and localized all four receptors and four ligands in endometrial biopsies. This suggests a role for the EGF system in growth of the endometrium.     

Angiogenesis, vascular endothelial growth factor and the endometrium

Angiogenesis is an essential component of endometrial renewal. The formation of new vessels depends on interactions between various hormones and growth factors, and this review focuses on the expression of angiogenic growth factors in the human endometrium. Peptide and non-peptide angiogenic factors interact during endometrial renewal, including epidermal growth factor (EGF), transforming growth factors (e.g. TGF-beta), platelet-derived endothelial growth factor/thymidine phosphorylase (PD-ECGF/TP), tumour necrosis growth factors and vascular endothelial growth factor (VEGF). Their role in the proliferation and migration of endothelial cells from pre-existing vessels is described, concentrating on VGEF and its receptors (VEG-R1 and -R2), and the fibroblast growth factor (FGF) family. The actions of the products of the VEGF gene are outlined, and the hormonal and non-hormonal control of their localization in the human endometrium and biological actions on vasculature and coagulation are described. Finally, the role of VEGF in menorrhagia is assessed.     

Expression of vascular endothelial growth factors and their receptors in human endometrium from women experiencing abnormal bleeding patterns after prolonged use of a levonorgestrel-releasing intrauterine system

BACKGROUND: Menstrual bleeding disturbances are a common initial complaint among users of the levonorgestrel-releasing intrauterine system (LNG-IUS). In this study, women who experienced bleeding disturbances recurring after a previous period of problem-free use and who therefore wanted removal of their LNG-IUD were investigated. Vascular endothelial growth factors (VEGFs) and their receptors are thought to be involved in normal endometrial angiogenesis. The aim of the study was to elucidate the possible association of these VEGF and receptors with bleeding disturbances among users of LNG-IUS. METHODS: Endometrial biopsies were obtained from users of the LNG-IUS who complained of bleeding disturbances (n = 17) and from women without such problems (n = 14). The endometrial expression of these VEGFs and their receptors was analysed using immunohistochemistry. RESULTS: Endometrial endothelial cells from LNG-IUS users with menstrual bleeding disturbances exhibited significantly higher immunoreactivity for VEGFR-1 and VEGFR-3 than those from women without bleeding disturbances. Stromal cells showed significantly lower immunoreactivity for VEGF-A in samples from LNG-IUS users with bleeding disturbances than in those without. CONCLUSION: Changes in the expression of these angiogenic growth factors and their receptors in LNG-IUS-exposed endometrium might be involved in the formation of fragile and dysfunctional blood vessels that subsequently give rise to bleeding disturbances.     

Expression of cystic fibrosis transmembrane conductance regulator in human endometrium

BACKGROUND: As a cAMP-regulated Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile, generally assumed to be due to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mRNA levels in human endometrium were analysed using immunohistochemical and in situ hybridization methods, respectively. Significant expression of CFTR protein was only seen in the glandular cells from late proliferative to all secretory phases, consistent with western blot analysis. High levels of CFTR mRNA were present only around the ovulatory period. In cultured glandular cells, the production of CFTR protein and mRNA was stimulated by estradiol and inhibited by progesterone. A forskolin-activated Cl- current in endometrial epithelial cells with a linear I-V relationship was detected by the whole-cell patch-clamp technique. CONCLUSIONS: (i) CFTR mRNA and protein were localized in human endometrial epithelial cells and the amounts varied in a cyclic manner; (ii) CFTR expression in cultured glandular cells was up- and downregulated by estradiol and progesterone, respectively; and (iii) CFTR in human endometrium functions as a cAMP-activated Cl- channel.     

Expression of gap junction connexins in the human endometrium throughout the menstrual cycle

Expression of connexins, the proteins which comprise gap junctionchannels, is regulated by ovarian hormones in the female reproductivetract of rodents. In order to determine if these hormones alsoaffect connexin expression in the human uterus, the distributionpatterns of different connexins (cx26, cx32, cx43) were investigatedby immunohistochemistry in human endometrial tissue collectedthroughout the menstrual cycle. During the early proliferativephase of the cycle extremely low staining for connexin 43 wasobserved and connexin 26 antigens could not detected. An increasein the amount of connexin 43 stromal cells and of connexin 26in glandular and luminal epithelial cells was seen from days11–15 of the cycle. Following ovulation, the expressionof both connexinswas suppressed and was completely abolishedin the late secretory phase. Weak staining for connexin 32 wasfoundmainly in the late proliferative and the early secretoryphase and was restricted to the basal membrane region the glandularcells. These results suggest that the different onnexins couldrepresent cell biological markers for the proliferation anddifferentiation of the human endometrium throughout the menstrualcycle.     

Aberrant expression of angiopoietins-1 and -2 and vascular endothelial growth factor-A in peri-implantation endometrium after gonadotrophin stimulation

BACKGROUND: Ovarian stimulation affects normal endometrial development.The expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2)and vascular endothelial growth factor-A (VEGF-A) and the vascularstate in the peri-implantation endometrium in women with naturaland gonadotrophin-stimulated cycles were compared. METHODS: The expression of these angiogenesis-associated molecules inendometrial biopsies, collected on Day 7 after human chorionicgonadotrophin injection or luteinizing hormone surge in stimulatedor natural cycles respectively, or at mid-luteal phase of womenundergoing diagnositic laparoscopy, were analysed. RESULTS: Women with gonadotrophin-stimulation had lower Ang-1, but higherAng-2, mRNA and protein expression (P < 0.05), and increasedconcentrations of von Willebrand factor (vWF) and blood vesseldensity than those with natural cycles (P < 0.05). Althoughstimulated cycles had higher VEGF-A mRNA expression (P = 0.023),VEGF-A protein expression was similar between the groups. LowerAng-1/Ang-2 but higher Ang-2/VEGF-A mRNA ratios (P = 0.025)were found after gonadotrophin-stimulation. The ratios werenegatively (P < 0.001) and positively correlated (P <0.001) with estradiol levels, respectively. Cyclical changesin Ang-1 and Ang-2, but not in VEGF-A expression were noted. CONCLUSIONS: The decreased Ang-1 concentration and Ang-1/Ang-2 ratio andthe increased Ang-2 concentration, with the increased vWF concentrationand blood vessel density, in stimulated cycles suggests advancedendometrial angiogenesis after gonadotrophin-stimulation.     

Endocrinology and paracrinology: The presence of platelet-derived endothelial cell growth factor in human endometrium and its characteristic expression during the menstrual cycle and early gestational period

Decidualized tissues are characterized by the intensive outgrowthof the microvasculature. Several angiogenic factors are assumedto be involved during the drastic change in the vasculatureoccurring in the process of decidualization. We examined thepossible role of platelet-derived endothelial cell growth factor(PD-ECGF), a known angiogenic factor, during the process ofearly decidualization in humans. The expression of PD-ECGF inhuman endometrium was demonstrated by Western blot analysis,a marked increase being found in decidualized endometrium. Immunohistochemicalstaining showed that PD-ECGF immunoreactivity was present mainlyin decidualized endometrial stromal cells. We established aprimary cell culture of human endometrial stromal cells whichwere differentiated into decidualized cells in vitro by theaddition of progesterone. In this cell culture system, progesteroneaugmented the expression of PD-ECGF in a dose-dependent fashion.The addition of progesterone also resulted in an increased releaseof prolactin, a well-known marker for decidualization. Thesefindings suggest that PD-ECGF may play a physiological roleas a possible angiogenic factor in the process of decidualizationof human endometrium.     

Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle

Endometrial biopsies were obtained from 73 normal women throughoutthe menstrual cycle. Using a polyclonal antibody and a streptavidin–biotin–peroxidasemethod, formalinfixed paraffin sections of the tissue were stainedfor von Willebrand factor (vWF). Both subjective scoring andobjective quantitative colour image analysis were used to assessthe staining intensity, and the results obtained by the twomethods were in concordance with each other. Positive stainingwas observed at all stages of the menstrual cycle. Specificstaining was confined to the vascular endothelium and showedcyclical changes. The staining intensity was the weakest duringthe menstrual phase and was significantly (P < 0.02) reducedfrom all other stages of the cycle, except late secretory phase.This was followed by a rapid increase in the early proliferativephase to reach a peak in the mid cycle before gradually fallingoff towards the end of the cycle. The staining intensity inthe late secretory phase was significantly reduced (P < 0.05)from other stages except menstrual, early proliferative andmid secretory phase. Vascular staining for vWF was heterogeneouswith some vessels devoid of any positive staining.     

Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women

Although the role of the reproductive hormone, relaxin, in rodents is well documented, its potential contribution to human reproduction is less well defined. In this study, we examine the effects of relaxin on human endometrial cells in vitro and describe the clinical effects of relaxin on menstrual flow in women. In cultured endometrial cells, relaxin specifically induces the expression of an angiogenic agent, vascular endothelial growth factor (VEGF). cAMP is implicated as a second messenger involved in VEGF stimulation. VEGF expression is temporally regulated in the endometrium, and our results suggest that relaxin, which is secreted by the corpus luteum and is present in the endometrium during the menstrual cycle and pregnancy, may be involved in regulating endometrial VEGF expression. Relaxin was recently tested in a clinical trial for efficacy in the treatment of progressive systemic sclerosis, and was administered at levels up to 10 times higher than that measured during pregnancy. The most frequent relaxin-related adverse event reported during the course of the study was the onset of menometrorrhagia, defined in this study as heavier-than-usual or irregular menstrual bleeding. The intensification of menstrual flow observed in these patients is consistent with the hypothesis that relaxin mediates neovascularization of the endometrial lining.     

Variant progesterone receptor mRNAs are co-expressed with the wild-type progesterone receptor mRNA in human endometrium during all phases of the menstrual cycle

Progesterone receptor (PR) variant mRNAs in human endometrium could encode proteins with the potential to alter progesterone action in states of normal and abnormal endometrial development. We have assessed the expression levels of mRNA for the wild-type PR and splice variants of PR mRNA lacking exon 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4&6 PR), and part of exon 4 (del-p4 PR) or part of exon 6 (del-p6 PR) in the human endometrium throughout menstrual cycle development. Eighty-eight endometrial specimens (47 proliferative, 41 secretory) were collected from patients undergoing hysterectomy for benign gynaecologic causes. Measurements by RT-PCR indicated that mRNAs for wild-type PR, and splice variants del-4 PR, del-6 PR, del-4&6 PR, del-p6 PR, and a novel del-p4 PR were detected in all endometrial specimens throughout the menstrual cycle. Higher levels of wild-type PR and all PR variant mRNAs were found in the early and mid-proliferative endometrial phases than in secretory endometrium. The relative expression of mRNA for all PR variants compared to wild-type PR mRNA, however, did not change through all stages of endometrial development. We, therefore, found no evidence of differential co-expression of the PR variants compared with wild-type PR during normal menstrual development. Future studies will determine if the expression profile of PR variant mRNAs will be different in the endometrium of patients with infertility, recurrent pregnancy loss, or endometrial adenocarcinoma.     

Effect of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells

BACKGROUND: We recently showed that vascular endothelial growth factor (VEGF) expression by endometrial glandular epithelial and stromal cells, and endometrial microvascular endothelial cell permeability, an early step in angiogenesis, were rapidly increased by estradiol (E(2)) administration to ovariectomized baboons. We proposed that estrogen promotes endometrial angiogenesis by regulating VEGF expression by glandular epithelial and stromal cells. In the present study, we developed a co-culture of human endometrial cells and microvascular endothelial cells to determine whether the regulatory role shown for estrogen on endometrial angiogenesis in vivo in the non-human primate would be demonstrable in vitro in the human. METHODS AND RESULTS: Human endometrial glandular epithelial and stromal cells were co-cultured with human myometrial microvascular endothelial cells (HMMECs) and E(2). HMMEC tube formation (means +/- SEM, % endothelial tube area/total endothelial cell area), an index of angiogenesis, was 65% (P < 0.05) and 2-fold (P < 0.01) greater in cells co-cultured with human glandular epithelial cells (54 +/- 7%) and glandular epithelial cells plus E(2) (66 +/- 11%), respectively, compared with medium (33 +/- 4%). In contrast, endothelial tube formation was not altered in HMMECs incubated with endometrial stromal cells (32 +/- 4%), stromal cells plus E(2) (36 +/- 2%) or E(2) (39 +/- 3%). CONCLUSIONS: We propose that estrogen, by regulating expression and secretion of angiogenic factors such as VEGF by glandular epithelial cells of the endometrium, regulates endometrial angiogenesis.     

Modulation of placental vascular endothelial growth factor by leptin and hCG

Vascular endothelial growth factor (VEGF) has been identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival. Leptin and hCG have also been suggested as possible regulators of angiogenesis in various models. In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that the vascular endothelium is a target for leptin. Thus, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis and we therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion. We incubated cytotrophoblastic cells (CTB) with recombinant human leptin (rhLept) (0-4 pg/ml) or hCG (0-30000 IU/ml) for 4 h. rhLept significantly stimulated hCG (P = 0.0045) and decreased VEGF release (P = 0.0008) by CTB in a concentration-dependent manner. On the other hand, increasing concentrations of hCG (0-30000 IU/ml), induced a significant inhibition of leptin secretion (P = 0.0028) and a marked dose-dependent stimulation of VEGF(165) secretion (P < 0.0001). We observed an increase of >1000-fold in basal trophoblastic VEGF secretion with physiological concentrations of hCG in vitro. An inhibitory effect of hCG on trophoblastic leptin secretion was also observed, suggesting that hCG might exert a possible negative feedback on trophoblastic release of leptin. We hypothesize that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal-fetal interface.     

Coexpression of heparanase, basic fibroblast growth factor and vascular endothelial growth factor in human esophageal carcinomas

Heparan sulfate (HS), which is degraded by heparanase, plays an important role in cell adhesion, insolubility of the extracellular matrix (ECM) and as a reservoir for various growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). In the present study, we examined the immunohistochemical expression of heparanase, bFGF and VEGF, and evaluated the correlation between their expression and microvessel density (MVD) in human esophageal carcinomas. Heparanase, bFGF and VEGF were immunolocalized predominantly to the carcinoma cells, but they were also localized to the endothelial cells of microvessels near the carcinoma cell nests. In carcinomas with invasion of the muscular layer or adventitia, heparanase staining was stronger at the invasive areas of carcinomas than the intraepithelial spread. Expression of heparanase and bFGF and the degree of MVD were associated with tumor invasion, lymph node metastasis and pathological stages. Cases with positive staining for heparanase, bFGF or VEGF tended to have a higher MVD than those without staining, and carcinomas with concomitant expression of heparanase, bFGF and VEGF showed the highest MVD. The level of heparanase mRNA expression was directly correlated with the MVD. In addition, heparanase-positive cases had a higher positive ratio of bFGF and VEGF compared with the heparanase-negative cases. These data suggest the possibility that heparanase may contribute to not only cancer cell invasion but also angiogenesis probably through degradation of HS in the ECM and release of bFGF and VEGF from the HS-containing ECM.     

血管内皮生长因子及其受体在肝癌细胞中的表达及意义

目的 探讨人肝癌细胞株血管内皮生长因子（VEGF）及其受体的表达,进一步认识VEGF在肝癌血管形成中的作用机制,方法 以人脐静脉血管内皮细胞系ECV304和小鼠成纤维细胞系L929作为对照,采用免疫组化染色及RT-PCR,检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF及其受体的表达。结果 SMMC7721、HHCC和HepG2细胞均有VEGF的表达。同时VEGF受体1（Flt-1)在SMMC7721细胞中也有表达;而HHCC和HepG2细胞则表达VEGF的受体2（KDR）。结论 在肝癌的血管形成中可能存在VEGF的自分泌机制。     

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy

We have demonstrated the presence of epidermal growth factor(EGF) and its receptors in human non-gestational corpora lutea.To determine further the characteristics of EGF receptor binding,we examined 30 human corpora lutea throughout the luteal phaseand during pregnancy. Scatchard plots of EGF binding in 29 ofthe 30 corpora lutea were curvilinear, suggesting negative co-operativity.The mean ± SE of the association constant K_a was (0.9± 0.2) x 10⁹ 1/mol, the dissociation constant K_d was(2.2 ± 0.3) x10^–9 mol/1 and the number of bindingsites (Rt) was (15.8 ± 2.1) x10^–19 mol/µgprotein for non-gestational corpora lutea. The K_d increasedsignificantly in late pregnancies compared to early pregnancies(P = < 0.005), while Rt was significantly higher in termpregnancies than in either early pregnancy (P < 0.01) orthe menstrual cycle (P < 0.001). Corpora lutea atretica (n= 2) and ovarian stroma (n = 6) did not show any EGF bindingactivity. Our findings demonstrate the presence of specificEGF receptors in human corpora lutea of both the menstrual cycleand pregnancy. The changes in EGF binding parameters in earlypregnancy suggest that there may be a relationship between therole of EGF and ovarian steroidogenesis.     

Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation

BACKGROUND: Classical studies in monkeys suggested that menstruation results from vasoconstriction, hypoxia and necrosis. The heterodimeric hypoxia-inducible factor (HIF) complex is critical in oxygen homeostasis via increased stability of HIF-1alpha/2alpha monomers, and these act as markers of hypoxia. We hypothesized that focal hypoxia in perimenstrual endometrium results in locally increased matrix metalloproteinases (MMP), leading to tissue destruction. METHODS: HIF-1alpha, HIF-2alpha and HIF-1beta were immunolocalized in cycling endometrium. Endometrial stromal cells were cultured under hypoxic and normoxic conditions and MMP measured by zymography and Western blots. RESULTS: HIF-1alpha and HIF-2alpha were detected in only some endometrial samples, and not confined to the perimenstrual tissue. Where present, they were primarily cytoplasmic, not nuclear. HIF-1beta was localized in epithelium, leukocytes and some decidual cells. Cultured endometrial stromal cells responded to hypoxia with increased cellular HIF-1alpha and secreted vascular endothelial growth factor. ProMMP-1 and proMMP-3 production was reduced in response to hypoxia regardless of the steroidal milieu (no added steroids, estrogen or estrogen plus progesterone). Active MMP-2 and membrane type 1 MMP but not proMMP-2 or proMMP-9 production were also inhibited by hypoxia. CONCLUSIONS: These results do not support a role for hypoxia in the focally increased production and activation of MMP observed prior to and during menstruation.