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Department of Obstetrics and Gynecology, New York University School of Medicine, New York, New York 10016
Abstract
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 (E2)-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 E2 or 10-7 M medroxyprogesterone acetate (MPA) or E2 + 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% O2). 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 E2 + 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 E2 + MPA-decidualized HESCs expressed VEGF121, VEGF165, and VEGF189 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.
WITHDRAWAL OF CIRCULATING ovarian steroids elicits sloughing of the functional endometrial layer in the late luteal phase of the nonfertile human menstrual cycle. The endometrium is replenished in the next cycle under the sequential control of estradiol (E2) and progesterone (P4). Abundant studies indicate that E2 induces mitosis of follicular phase epithelial and stromal cells and primes these cells for the differentiating effects of luteal phase P4. However, ovarian steroids do not appear to control the angiogenic restoration of the vasculature of the functional endometrial layer (1, 2). Although several angiogenic factors are present in human endometrium (3, 4, 5), most studies have focused on vascular endothelial growth factor (VEGF), which is expressed cyclically by endometrial stromal and glandular epithelial cells (6, 7, 8).
Alternative splicing of a single VEGF gene produces four molecular species (VEGF121, VEGF165, VEGF189, and VEGF206; Ref. 9). The major isoform, VEGF165, corresponds to a basic, heparin-binding glycoprotein of 45,000 kDa. Binding of VEGF to its tyrosine kinase endothelial cell-specific surface receptors, flt-1 and kinase domain region (KDR), promotes angiogenesis by enhancing endothelial cell proliferation, migration, vascular permeability, and protease activity (9). In several cell types, hypoxia enhances VEGF synthesis by augmenting VEGF mRNA transcription and stability. These effects are mediated by the cis-acting hypoxia inducible factor-1
site on the VEGF promoter (10, 11, 12).
Endothelin and prostanoid expression by the premenstrual human endometrium is thought to evoke vasoconstriction, ischemia, and local hypoxia (13, 14). These events occur in a milieu of progestin withdrawal-enhanced plasminogen activator (PA) and matrix metalloproteinase expression, and inhibited perivascular stromal cell expression of the PA inhibitor (PAI-1) and tissue factor (TF) (15, 16, 17, 18, 19, 20, 21). These orchestrated changes provoke extracellular matrix (ECM) degradation, endothelial disruption, and menstrual hemorrhage. Hypoxia enhances VEGF expression in human endometrial stromal cells (HESCs; Refs. 22, 23, 24) and human endometrial glandular epithelial cells (HEGECs; Ref. 23). These effects are consistent with the marked angiogenesis that follows the onset of menstruation in follicular phase endometrium. However, they do not account for either the angiogenic activity that persists throughout the luteal phase (7, 24, 25) or the peak in VEGF expression that has been observed in the luteal phase endometrium (26, 27).
Decidualization of HESCs is initiated in the midluteal phase and spreads throughout the late luteal phase endometrium (28). Decidualized HESCs express high levels of TF (29, 30, 31), which binds to plasma factors VII/VIIa to generate factor Xa. The resulting formation of thrombin from prothrombin initiates hemostasis by converting fibrinogen to fibrin (32). Moreover, thrombin mediates various cellular effects by binding to a family of ubiquitous cell receptors (33). The luteal phase is accompanied by increased vascular permeability and stromal edema (34), which would generate thrombin via enhanced access of circulating plasma clotting factors to perivascular decidual cell (DC)-expressed TF. Because thrombin induces VEGF expression in several cell types (35, 36, 37), we hypothesized that thrombin could play a key role in regulating luteal phase endometrial VEGF expression. Thus, we evaluated thrombin effects in the presence and absence of ovarian steroids on VEGF expression in the predominant endometrial cell types, HESCs and HEGECs, and determined whether such effects are independent of hypoxia.
Materials and Methods
Tissues
Written informed consent and approval was received from the Institutional Research Board of New York University Medical Center and Bellevue Hospital. Fourteen specimens of predecidualized endometrium from the follicular and luteal phases were obtained from hysterectomies for benign conditions (e.g. myomas without abnormal uterine bleeding) from women of reproductive age and transported to a sterile laminar flow hood. A small portion of each specimen was formalin-fixed and histologically dated by the criteria of Noyes et al. (28). The remainder was used to isolate and culture glandular epithelial and stromal cells.
Isolation of HESCs and HEGECs
Endometrial fragments were digested with 0.25% type I collagenase (267 U/mg; lot no. 47P1428; Worthington, Lakewood, NJ) for 30 min in a shaking water bath at 37 C. The digestate was filtered through a 38-µm stainless steel sieve. The glands were retained by the sieve and collected after backwashing. The stromal cells pass through the sieve along with some epithelial cells and blood elements. Purification of the stromal cell-enriched fraction to virtual homogeneity, as determined by immunostaining for cytokeratin and vimentin (29), used a Percoll gradient together with incubation for 30 min at 37 C in a standard humidified 95% air/5% CO2 incubator. During this period, the stromal cells, but not the other cell types, attach to polystyrene tissue culture plastic. Conversely, incubation of the glandular epithelial cell fraction for 30 min at 37 C enables contaminating stromal cells to adhere to the tissue culture plastic surface (Falcon, BD Biosciences, Bedford, MA) while purified glands and glandular epithelial cells remain floating in the supernatant (see Refs. 29, 38 , and 39 for details of cell isolation).
Cell culture
HESCs. HESCs were grown to confluence in BMS, which consists of basal medium (BM), a phenol red-free 1:1 vol/vol mix of DMEM (Life Technologies, Inc., Grand Island, NY) and Ham’s F-12 (Flow Laboratories, Rockville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone that was supplemented with 10% charcoal-stripped calf serum(s).
Confluent HESCs were incubated in parallel for 7 d in BMS plus 0.1% ethanol (vehicle control) or 10-8 mol/liter E2 or 10-7 mol/liter medroxyprogesterone acetate (MPA; Sigma, St. Louis, MO) or E2 + MPA to decidualize the cells, with one change of medium. The cultures were washed twice with Hanks’ balanced salt solution to remove residual serum components and switched to a serum-free defined medium (DM) containing corresponding vehicle or steroid(s) added with or without thrombin (American Diagnostica, Greenwich, CT) or thrombin receptor activating peptide (TRAP; Bachem Bioscience Inc. , King of Prussia, PA). DM consists of BM plus ITS+ (BD Biosciences, Bedford, MA), 5 µM FeSO4, 50 µM ZnSO4, 1 nM CuSO4, 20 nM Na2SeO3, trace elements (Life Technologies, Inc.), and 50 µg/ml ascorbic acid (Sigma). Conditioned DM was centrifuged, and the cells were washed with Hanks’ balanced salt solution, lysed with 0.4% SDS, and used to determine DNA and protein content. Medium supernatants and cell lysates were stored at -70 C. RNA was extracted from parallel cultures for Northern blot analysis.
HEGECs. The purified glandular epithelial cell fraction was suspended in BMS and distributed equally among polystyrene tissue culture dishes coated with 2% gelatin (Sigma). After 24 h in the standard incubator to enable the glands to attach and begin to form monolayers, the medium was exchanged for fresh BMS containing vehicle (control) or 10-8 mol/liter E2 or 10-7 mol/liter MPA or E2 + MPA. After 3 additional days in the incubator, the medium was exchanged for corresponding DM. The experimental treatment, collection of conditioned DM, and lysing of the cells were performed as described above for cultured HESCs.
Hypoxia
Confluent HESCs or HEGECs were placed in a humidified sealed chamber containing a portable gas oxygen analyzer. The chambers were purged with 5% CO2/95% N2 for 15 min after the oxygen analyzers read 0–1% O2 (12–14 mm Hg). The sealed chambers were placed in a standard 37 C incubator for 48 h. Only cultures in chambers reading 2% O2 or less after 48 h were used for this study. Control cultures were maintained in a standard incubator (pO2, 120–130 mm Hg). Parallel incubations under normoxia vs. hypoxia were performed in DM.
Assays
A commercial ELISA that detects VEGF165 and VEGF121 was used to measure immunoreactive levels of VEGF in the cell-conditioned medium according to instructions provided by the manufacturer (R&D Systems, Minneapolis, MN). Total cell DNA and protein levels were measured by the method of Hinegardner (40) and the Bio-Rad Assay (Bio-Rad Laboratories, Inc. Hercules, CA), respectively.
Northern blotting was performed on 15 µg of total cell RNA that was separated on a 1% agarose gel containing 2.2 mol/liter formaldehyde and then transferred to a Zeta-Probe nylon membrane (Bio-Rad Laboratories, Inc.). VEGF mRNA was detected with a probe generously supplied by Dr. Judith Abrams (Scios, Inc. Sunnyvale, CA). The probes were labeled with [32P]deoxy-CTP to high specific activity by random priming (Roche Molecular Biochemicals, Indianapolis, IN) as previously described (29). Hybridization was performed by standard methods, and the washed filters were exposed to Kodak XAR film (Eastman Kodak Co., Rochester, NY). Total RNA loads were standardized by reprobing the stripped membranes with a 32P-labeled probe for actin. Densitometric analysis was carried out with the Kodak 1D scientific imaging system program (Kodak, New Haven, CT).
Statistical analysis
Comparisons of control vs. treatment groups were performed with the Kruskall-Wallis signed rank test with P value less than 0.05 representing statistical significance.
Results
Effects of steroids and hypoxia on VEGF output by HESCs and HEGECs
Figure 1
displays the separate and interactive effects of hypoxia and E2 + MPA on VEGF output by cultured HESCs (Fig. 1A) and HEGECs (Fig. 1B). A similar pattern of response is evident for both cell types. Thus, hypoxia elicited a severalfold increase in secreted VEGF levels compared with parallel normoxic incubations. However, both cell types were unresponsive to E2 + MPA under normoxia as well as hypoxia. Both HESCs and HEGECs were also refractory to either 10-8 M E2 or 10-7 M MPA (results not shown).
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Endometrial VEGF expression peaks at a time when TF expression in decidualized HESCs is expected to generate steady-state levels of thrombin (see introduction). Therefore, thrombin effects were evaluated on VEGF output in HESCs that had been decidualized by incubation with E2 + MPA. Figure 2A indicates that immunoreactive VEGF levels were about 3-fold greater in response to 0.1 U/ml thrombin at 48 h compared with 24 h. The dose-response results displayed by Fig. 2B reveal that after 48 h, 0.1 U/ml of thrombin elevated VEGF levels by severalfold in the decidualized HESCs, with peak effects evident between 0.5 and 2.5 U/ml. By contrast with the marked responsiveness of the HESCs, Fig. 2B indicates that cultured HEGECs from the same endometrial specimens were refractory to thrombin. The absence of a thrombin response was also evident in HEGECs incubated in vehicle or in E2-containing medium and in an additional experiment in which HEGECs were incubated with E2 + MPA and 5 U/ml of thrombin (results not shown). As revealed by Fig. 2C, secreted levels of VEGF were elevated by about 8-fold (P < 0.02) after incubation of HESCs from six specimens for 48 h with 0.5 U/ml of thrombin. Figure 2D compares the effects of thrombin with TRAP, which activates the protease activating receptor-1 (PAR1) directly without proteolytic modification (33), on VEGF levels in HESCs. At 50 ng/ml, TRAP was an effective agonist, eliciting about half the maximum effects seen in response to thrombin.
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shows that HESCs express three previously described VEGF mRNA forms in human endometrium (VEGF121, VEGF165, and VEGF189; Ref. 27). Steady-state levels of all three forms were higher in thrombin-treated than in control cultures at 5, 7, and 20 h. For example, after 7 h, densitometric comparison with mRNA for the actin housekeeping gene indicated a 5-fold elevation in the VEGF165 peak in thrombin-treated cells vs. cells treated in parallel with E2 + MPA alone. Further determinations are required to distinguish between enhancement of VEGF mRNA transcription and inhibition of VEGF mRNA turnover in response to thrombin.
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Consistent with results observed in Figs. 1A
and 2C, respectively, Table 1 confirms that hypoxia and thrombin each elevated secreted VEGF levels by severalfold in in vitro decidualized HESCs. The effects of adding various concentrations of thrombin under hypoxia appeared additive, but the differences among the groups were not significant. This situation contrasts with the reported synergy between TGF- or IL-1ß (24) and hypoxia in elevating VEGF output in HESCs.
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In nonfertile menstrual cycles, progesterone withdrawal appears to elicit ischemia, hypoxia, and endometrial sloughing. A model has been proposed in which ruptured endometrial spiral arterioles and venules serve as an angiogenic nidus that restores the vasculature in the next cycle. According to this model, endothelial cells sprout from the disrupted vessels and recruit or induce pericytes or smooth muscle cells to form capillaries, venules, or arterioles [for review, see Smith (7)]. Of the angiogenic factors identified in human endometrium, which also includes epidermal growth factor, basic fibroblast growth factor-2, platelet-derived growth factor, and TGF-ß (3, 6, 7), VEGF has emerged as the most likely mediator of endometrial angiogenesis. Thus, a series of transgenic and gene knockout experiments identified crucial roles for VEGF and its flt-1 and KDR receptors in angiogenesis (41). Moreover, among candidate angiogenic agents described in human endometrium, only the VEGF gene promoter contains a hypoxia inducible factor-1 response element (9, 11, 12).
The current study confirms reports that hypoxia markedly up-regulates VEGF expression in cultured HESCs and HEGECs (22, 23, 24). It extends previous reports that the hypoxic effects are unaffected by E2 or progestin (23, 24) to now include similar observations with E2 plus progestin. The latter mimics ovarian steroid stimulation during the luteal phase. This nonresponsiveness is consistent with the absence of the ovarian steroid response elements from the VEGF gene promoter (9), as well as the premenstrual withdrawal of circulating ovarian steroids. By contrast, both TGF-, which stimulates cells via the EGF receptor (EGFR), and IL-1ß are reported to synergize with hypoxia to augment VEGF expression in cultured HESCs (24). The luteal phase endometrium expresses the EGFR as well as EGFR agonists (42, 45, 46), and IL-1ß is produced by leukocytes, which represent an estimated 40% of the resident cells of the perimenstrual endometrium (20). Under the chemoattractant influence of IL-8 expression, neutrophils constitute a significant portion of this leukocyte population (20, 47, 48). Recently, neutrophils were found to express VEGF (49), suggesting an additional source of angiogenic stimulation.
Diverse angiogenic stimuli arising from the perimenstrual endometrium are thought to control the initial phase of angiogenesis, which involves repair of the endometrial vasculature during the early follicular phase (50). However, this stimulation is unlikely to account for the persistence of angiogenesis in the luteal phase, which includes endothelial cell proliferating activity, increased microvascular density, and coiling of the spiral arteries (24, 25, 51). The latter is integral to implantation-related trophoblast-endometrial interactions. Moreover, endometrial VEGF levels peak in the luteal phase (26, 27).
Enhanced endometrial VEGF expression in the luteal phase has been attributed to P4-mediated up-regulation (reviewed in Ref. 23). However, evidence that ovarian steroids enhance VEGF expression in either HEGECs or HESCs is conflicting. Thus, Classen-Linke et al. (52) recently reported that E2 and MPA each elevated VEGF output by HEGECs. By contrast, the current study confirms a report that neither E2 nor P4 altered secreted VEGF levels in cultured HEGECs (23) and extends it to show a lack of response during incubation with E2 plus a progestin (MPA). In cultured HESCs, E2, P4, and E2 + P4 were each shown to increase VEGF mRNA levels (8). Interestingly, the effects on VEGF protein expression were modest (8, 24), particularly in response to decidualization-inducing stimulation. Thus, in primary HESCs, E2 + P4 elicited a mean 4.7 ± 3.8-fold increase in VEGF mRNA levels, but only an 0.2-fold increase in secreted immunoreactive VEGF levels (8).
These in vitro results suggest that VEGF expression by decidualized HESCs is inconsistent with the prolonged VEGF output required to mediate angiogenesis in the luteal phase. Alternatively, secreted VEGF levels were reportedly either inhibited during in vitro decidualization of HESCs (7, 52) or unaltered by E2 or P4 (23). The current study confirmed the latter results and extended them to show that VEGF levels were also unaffected in incubations of HESCs with E2 + MPA.
Unlike the apparent absence of a notable effect of E2 + MPA on VEGF protein expression, numerous reports indicate that incubation of HESC monolayers with E2 plus progestin alters the expression of an array of decidualization markers. These include prolactin, IGF binding protein-1, fibronectin, laminin, stromelysin-1, interstitial collagenase, the tissue-type and urokinase-type PA, PA inhibitor-1, and TF (reviewed in Ref. 53). These in vitro responses are characteristically prolonged. In the case of TF, levels are up-regulated for at least 3 wk in HESCs incubated with E2 + MPA (54), thereby mimicking the chronic enhancement of TF levels in DCs during the luteal phase and pregnancy (29, 30, 31). The mid- to late luteal phase endometrium also experiences increased vascular permeability, accompanied by stromal edema, increased blood flow, and transudation of plasma proteins (34, 55), which would enhance access of clotting factors to perivascular DC TF, and thus promote thrombin generation. Because thrombin has been reported to stimulate VEGF expression in various cell types (35, 36, 37), we theorized that it could act as a paracrine stimulator of glandular VEGF production and/or an autocrine stimulator of HESC-expressed VEGF. The current study revealed that although thrombin did not affect glandular epithelial VEGF expression, it was a potent enhancer of VEGF output in decidualized HESCs. Moreover, our in vitro results suggest that this luteal phase effect may not extend into the perimenstrual endometrium because thrombin and hypoxia did not synergize to enhance VEGF output.
Although immunohistochemical staining for VEGF is greater in glandular epithelial cells than in stromal cells of luteal phase endometria (8, 26, 27), several criteria suggest that stromal cell-derived VEGF is more likely to mediate angiogenesis than is that originating from the glands. Thus, polarized glandular epithelial cell monolayers secrete VEGF primarily in an apical direction (56), i.e. toward the uterine lumen. Moreover, signaling from any residual epithelial-derived VEGF secreted basally is expected to be weak because contact with the distal endothelial cells would require diffusion, and the freely diffusible VEGF isoform (VEGF121) is far less angiogenic than ECM-bound isoforms (VEGF165 and VEGF189). Finally, angiogenesis takes place in a matrix of stromal cells that exhibit decidualization-dependent TF expression in the luteal phase (29, 30, 31), and thus thrombin-generating potential.
Thrombin acts via PAR1 to augment an array of cellular responses including DNA synthesis (57) and the expression of proinflammatory cytokines (58, 59, 60). Previously, we demonstrated the constitutive expression of PAR1 in HESCs (61). To confirm the involvement of PAR1 in thrombin-enhanced VEGF expression in cultured HESCs, we evaluated the effects of TRAP, which activates PAR1 directly without enzymatic cleavage of the receptor (33). The observed dose-response effects indicate that TRAP was less effective than thrombin in eliciting VEGF secretion. This observation is consistent with reports in several cell types in which thrombin and TRAP enhance proliferation and cytokine production (33, 57, 58, 59). In other cell systems, similar results have been interpreted to indicate that some of the effects of thrombin are mediated by the PAR3 and/or PAR4 receptors (33). Thrombin-induced platelet activation involves an initial phase of PAR1-mediated Ca2+ signaling and a later phase of PAR4-mediated Ca2+ signaling (62). Recently, PAR3 was also shown to mediate thrombin-induced Ca2+ signaling in rat astrocytes (63).
The current study proposes that thrombin mediates angiogenesis indirectly by enhancing VEGF expression in HESCs. However, thrombin has also been shown to stimulate angiogenesis directly by enhancing endothelial proliferation and by elevating levels of the KDR receptor in endothelial cells (57). In the model depicted in Fig. 4, DC-derived thrombin and VEGF act via their respective receptors to synergistically enhance luteal phase angiogenesis. Moreover, thrombin and VEGF each enhance endothelial permeability by different pathways (64, 65). This would increase access of clotting factors to DC-expressed TF to promote a feed-forward cycle of enhanced thrombin and VEGF production. These changes are consistent with the persistence of angiogenesis, as well as the peak in VEGF expression in the luteal phase endometrium.
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, the latter would elicit a prolonged increase in vascular permeability, lead to chronic TF-derived thrombin generation, and result in sustained, aberrant angiogenesis. Moreover, we found that thrombin is a potent inducer of ECM-degrading PA and matrix metalloproteinase activity in HESCs (61, 68). Aberrant angiogenesis in a milieu of compromised vascular support structure would produce the enlarged, distended, fragile vessels observed in these patients (31, 66, 69, 70). Ultimately, vessel fragility overcomes TF-thrombin mediated hemostasis, and bleeding ensues. Similar pathological processes may underlie the abnormal uterine bleeding associated with polyps and submucous myomas, which have also been linked to aberrant angiogenesis (7).
Acknowledgments
Footnotes
Address all correspondence and requests for reprints to: Frederick Schatz, Ph.D., Department of Obstetrics and Gynecology, New York University School of Medicine, 550 First Avenue, New York, New York 10016.
This work was supported in part by National Institutes of Health (NIH) Grant 5RO1 HL33937-06 (to C.J.L.) and NIH General Clinical Research Center Grant M01 RR00096.
Abbreviations: BM, Basal medium; DC, decidual cell; DM, defined medium; E2, estradiol; ECM, extracellular matrix; EGFR, EGF receptor; HEGEC, human endometrial glandular epithelial cell; HESC, human endometrial stromal cell; KDR, kinase domain region; MPA, medroxyprogesterone acetate; P4, progesterone; PA, plasminogen activator; PAR, protease-activating receptor; TF, tissue factor; TRAP, thrombin receptor activating peptide; VEGF, vascular endothelial growth factor.
Received December 10, 2001.
Accepted June 9, 2002.
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