Connetics Corporation, 3400 W. Bayshore Rd., Palo Alto, California 94303, USA
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Abstract |
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Key words: endometrium/menometrorrhagia/menstrual cycle/relaxin/vascular endothelial growth factor
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Introduction |
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In primates, however, there is evidence that relaxin, like other secretory products of the ovary, affects the morphology and secretory profile of the uterine lining. Relaxin binds specifically and with high affinity to human endometrial cells (Osheroff and King, 1995). In addition, levels of expression of glycodelin (Stewart et al., 1997
), insulin-like growth factor binding protein-1, and prolactin (Tseng et al., 1992
) in human endometrial cells increase in response to relaxin treatment in vitro. In-vivo studies testing relaxin in primates are limited in number; however, one such study examining the effect of a relatively crude preparation of relaxin on the uterus in immature or castrated macaque monkeys found that relaxin, in conjunction with oestrogen treatment, stimulated new blood vessel growth in the endometrium (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967
).
Angiogenesis in the endometrium, as in other sites, is stimulated by exposure of pre-existing endothelial cells to angiogenic agents. Studies have correlated an increase in vascular endothelial growth factor (VEGF) expression (Ferrara and Davis-Smyth, 1997) at both the protein and mRNA levels with new vessel growth in the endometrium in multiple species (Charnock-Jones et al., 1993
; Cullinan-Bove and Koos, 1993
; Schweik et al., 1993; Das et al., 1997
; Torry and Torry, 1997
). There is also evidence that inhibiting VEGF activity contributes to lack of endometrial maturation (Ferrara et al., 1998
). For these reasons, we tested the effect of relaxin on expression of VEGF in human endometrial cells in vitro. We have found that relaxin treatment specifically increases the expression of VEGF but not other angiogenic agents, such as placental growth factor (PlGF), angiogenin, transforming growth factor-ß (TGF-ß), or basic fibroblast growth factor (bFGF). In a recently concluded clinical trial (Seibold et al., 1997
), women receiving relaxin treatment reported menometrorrhagia significantly more often than women receiving a placebo. Menometrorrhagia was not accompanied by systemic elevations in VEGF. The increased frequency of menometrorrhagia in women receiving relaxin is consistent with the hypothesis that relaxin stimulates neovascularization in the endometrial lining of the uterus, and may reflect relaxin's normal physiological role in women.
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Materials and methods |
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Tissue culture reagents
Recombinant human relaxin-2 (Hudson et al., 1984) (Lot 63601, 1.5 mg/ml in 25 mmol/l acetate, pH 5.5) was manufactured by Connetics Corporation (Palo Alto, CA, USA). Forskolin, insulin, 17ß-oestradiol, and medroxyprogesterone were purchased from Sigma Chemical Company (St Louis, MO, USA). Insulin-like growth factor (IGF-1) was purchased from R&D Systems (Minneapolis, MN, USA). Dideoxyadenosine was purchased from Calbiochem (San Diego, CA, USA).
[32P]-relaxin binding assays
Relaxin was phosphorylated as previously described (Parsell et al., 1996). NHE cells were incubated at room temperature with [32P]-relaxin (20400 pmol/l) in the presence or absence of 1.4 µmol/l unlabelled relaxin. The number of relaxin binding sites and their affinity for relaxin were determined using the LIGAND program (Munson and Rodbard, 1980
).
Enzyme-linked immunosorbent assay (ELISA)
A quantitative sandwich ELISA specific for human VEGF was purchased from R&D Systems and performed according to manufacturer's specifications. The lower limit of detection of VEGF was 15.6 pg/ml. Immunoassays for TGF-ß, angiogenin, bFGF, and PlGF were also purchased from R&D Systems. Lower limits of detection in the respective assays were 31.2 pg/ml for TGF-ß, 78.1 pg/ml for angiogenin, 5 pg/ml for bFGF, and 15.6 pg/ml for PlGF. Serum relaxin levels were assayed in a quantitative sandwich ELISA immunoassay utilizing a goat affinity-purified anti-human relaxin polyclonal antibody and an affinity-purified, peroxidase conjugated anti-human relaxin polyclonal antibody, as previously described (Unemori et al., 1996). The assay has a lower limit of detection of 20 pg/ml.
Relaxin clinical trial
A randomized, placebo-controlled, double-blind clinical trial was conducted to test relaxin's safety and efficacy in patients with progressive systemic sclerosis (Seibold et al., 1997). Relaxin was formulated in a buffer of 20 mmol/l acetate, pH 5.5. The placebo was identical except for the active ingredient. Relaxin was delivered by continuous subcutaneous infusion at two doses, 25 µg/kg/day and 100 µg/kg/day, for 24 weeks. Patients were screened 48 weeks prior to initiation of treatment and came for a follow-up assessment 2 weeks after cessation of therapy. Patients ranged from 1566 years in age and gave written informed consent. There were 22 women who received relaxin at 25 µg/kg/day, 20 women who received 100 µg/kg/day, and 16 women who received the placebo. Adverse events were defined as: (i) any sign or symptom observed by the investigator or reported by the subject which was not being assessed as an efficacy parameter; (ii) any efficacy parameter which required medical intervention; (iii) any clinically significant laboratory abnormality or any clinically significant abnormality of any other clinical test; or (iv) any abnormality detected on physical examination.
Patients were instructed prior to administration of relaxin to report any physical changes or new symptoms they noticed during the course of the study. They were not prospectively instructed to report incidents of menstrual irregularities using the specific terms menorrhagia or metrorrhagia. The information was recorded on case report forms regardless of whether they were believed to be associated with the study medication and entered verbatim into a database. As part of standard clinical trial safety data analysis, the verbatim terms were coded to preferred terms, which are standard terms assigned to adverse events and link an event to a body system for summarization of adverse events. The terms menorrhagia, metrorrhagia, and menstrual disorder were the preferred terms assigned to an event based on the verbatim adverse event description and the patient's menstrual history. Events describing heavy menses were coded to menorrhagia, and events describing prolonged or irregular menses were coded to metrorrhagia. Postmenopausal patients reporting any type of menstruation were coded to menstrual disorder. Date of first reported onset of events was recorded. All reports of reproductive system-related adverse events were retrieved from the database following completion of the study.
Serum samples that were collected from patients at screening, first day of dosing and at 1, 2, 4, 8, 12, 16, 20, and 24 weeks after initiation of relaxin treatment were assayed for relaxin concentrations as part of the clinical trial study design. Serum samples drawn at screening and at visits immediately prior to and at the first report of adverse event onset were assayed for serum VEGF concentrations.
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Results |
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Relaxin stimulation of VEGF expression in endometrial cells in vitro
NHE cells were treated with relaxin (0.001100 ng/ml) for 24 h, then the conditioned media were collected and assayed for VEGF content by ELISA. The cells secreted a baseline level of VEGF of 875.0 ± 93.6 pg/ml, and the addition of relaxin resulted in an increase in secretion in a dose-dependent manner with an almost twofold increase detectable following addition of relaxin at 1 ng/ml (Figure 1a). Relaxin did not cause an alteration in cellular morphology or proliferation rate, measured by [3H]-thymidine incorporation (data not shown). Following the addition of 100 ng/ml of relaxin to NHE cells, elevated levels of VEGF protein expression were detectable by 24 h and secretion persisted at a constant rate over 96 h (Figure 1b
). Pretreatment of NHE cells with concomitant addition of oestrogen and/or progesterone did not alter their production of VEGF in response to relaxin, nor did it alter [32P]-relaxin binding from control levels (data not shown). Other members of the relaxin gene family, insulin and IGF-1, were tested for their ability to induce VEGF expression in NHE cells (Figure 2
). IGF-1 was a weak inducer of VEGF protein expression in comparison to relaxin. Insulin was unable to induce expression at the doses tested (0.1100 ng/ml).
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Nine of the 41 women receiving relaxin and three of the women on the placebo had previously had a hysterectomy and were excluded from the analysis on menometrorrhagia. Of the remaining 32 women in the relaxin treatment groups, 23 (72%) reported experiencing menometrorrhagia, compared with two of the 13 women (15%) on the placebo (P = 0.0003 by Fisher's exact test). Patients reporting menometrorrhagia were about equally represented in both relaxin dose groups, 13 in the 25 µg/kg/day group and 10 in the 100 µg/kg/day group. Their circulating relaxin concentrations at the time of adverse event reporting ranged from 2.0083.31 ng/ml (Table III). The ages of the women experiencing menometrorrhagia ranged from 1560 years. Although complete reproductive histories were not always taken for the purposes of this trial, it is likely that 17 of the 23 relaxin-treated women who reported menometrorrhagia were cycling. These included all of those who were recorded in their charts as being of childbearing potential (ages 27 to 44 years), and, judging by patient age, also those that were of non-childbearing potential due to tubal ligation (ages 30 to 42 years). Four other women were recorded as postmenopausal and were on hormone replacement therapy. One patient (no.7) was postmenopausal and was not reported to be taking hormone supplements. Another woman (patient no. 14) was listed as of non-childbearing potential, but was only 31 years old at the start of the trial; she reportedly had experienced pelvic inflammatory disease in her history but no further information was detailed regarding her reproductive status. The nine relaxin-treated women who did not report menometrorrhagia ranged in age from 2762 years old. Five of them were of childbearing age. The other four were postmenopausal and two of them were on hormone replacement therapy.
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Because of the finding that relaxin stimulates VEGF production by endometrial cells in vitro, and because we postulate that this is the mechanism by which menometrorrhagia is stimulated in relaxin-treated women, we looked for changes in serum VEGF concentrations in response to relaxin administration. VEGF content of serum drawn at screening was compared with VEGF concentrations demonstrable in serum drawn at the visit preceding first report of menometrorrhagia onset, as well as at the visit at which the report was made. Baseline VEGF concentrations proved to be highly variable, ranging from 71 to 1337 pg/ml prior to initiation of drug treatment (Figure 3). There were no consistent patterns in VEGF concentrations observed when comparing the three sampling dates that would indicate a positive, or negative, response to relaxin. Generally, VEGF concentrations remained within the range of individual baseline readings. The two placebo patients who reported menometrorrhagia had VEGF concentrations of 710 and 469 ng/ml at screening and demonstrated VEGF patterns that were not inconsistent with those seen in relaxin-treated patients (data not shown).
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Discussion |
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In the present study, we examine relaxin's potential effects on the human endometrium and demonstrate that one of relaxin's specific effects on isolated human endometrial cells is the stimulation of VEGF expression. In vitro, VEGF is stimulated by relaxin in a dose-dependent manner, with the threshold of response occurring between 0.01 and 0.1 ng/ml relaxin, roughly equivalent to circulating levels present in women during the menstrual cycle and early pregnancy (Stewart et al., 1990). VEGF is present in the endometrium in a variety of species (Shweiki et al., 1993
; Das et al., 1997
), including the human (Charnock-Jones et al., 1993
; Shifren et al., 1996
) during periods of active blood vessel proliferation, leading to the inference that VEGF may be an important mediator regulating cycle-specific vessel growth in the endometrial lining of the uterus. Baseline levels of VEGF secretion by the NHE cells are significant, perhaps consistent with its expression observed in situ in the endometrium during periods of relative quiescence (Torry and Torry, 1997
). It has been postulated that VEGF may be necessary for maintenance of the differentiated phenotype of endothelial cells, as well as for stimulating proliferation.
The endometrial cells used in this study are most likely stromal, since IGF-BP1 is induced in these cells by relaxin, consistent with a stromal cell phenotype (data not shown). Neither oestrogen nor progesterone had any discernible effect on these cells in vitro. Others have reported that endometrial stromal cells are stimulated to produce VEGF in vitro by treatment with oestradiol (Shifren et al., 1996). However, it has also been reported that these cells have the potential to synthesize oestrogen in culture (Huang et al., 1989
), suggesting the possibility of endogenous stimulation of a VEGF response, producing the baseline levels that we observe. Our data suggest that induction of VEGF over baseline is mediated, at least in part, by cAMP; the involvement of more than one second messenger system is consistent with VEGF regulation in other systems (Claffey et al., 1992
). It is possible that relaxin stimulates transcriptional activation of the VEGF gene via cAMP, as the promoter contains four AP-1 and two AP-2 binding sites, which are believed to be able to transduce cAMP-mediated transcriptional activation (Tischer et al., 1991
).
The results of our findings in vitro allow that, in women, relaxin expressed during the luteal phase of the menstrual cycle and early in pregnancy may be directly involved in inducing VEGF expression, and consequently neovascularization, in the endometrium. This is consistent with an analysis of factors contributing to uteroplacental circulation, which found that circulating relaxin levels were positively correlated with uterine flow in early pregnancy (Jauniaux et al., 1994). Our observation that exogenous relaxin administration was associated with menometrorrhagia in women supports this hypothesis, as does the previous finding that a crude preparation of relaxin administered to monkeys stimulated blood vessel proliferation in the endometrium (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967
). Because relaxin administration in the clinical trial resulted in supraphysiological circulating levels (~3 ng/ml and ~10 ng/ml in the lower and higher dose groups respectively), it is perhaps not surprising that we observed not only excess menstrual bleeding, but also bleeding at irregular times during the menstrual cycle. All of the relaxin-treated women, who reported menometrorrhagia, except one, were likely either cycling or postmenopausal and on hormone replacement therapy. This suggests that oestrogen may be required for endometrial stimulation by relaxin, which is consistent with results observed in a study of the endometrium following steroid and relaxin supplementation in castrated or juvenile primates (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967
).
Although we have shown here that exogenous, systemically administered relaxin can cause heavier than usual menstrual bleeding, the endometrium itself is also normally a source of relaxin (Yki-Jarvinen et al., 1985; Bryant-Greenwood et al., 1993
). Endometrial relaxin is first immunocytochemically detectable 4 days following ovulation in the early secretory phase of the cycle and is inducible with exogenous progesterone administration in anovulatory and postmenopausal women. The timing of appearance of relaxin in the endometrium is also consistent with its potential role in neovascularization.
Circulating VEGF levels were not altered by relaxin treatment, which is consistent with the hypothesis that relaxin stimulates the expression and distribution of VEGF only in certain sites, such as in the endometrium. The lack of a measurable elevation in VEGF in the serum during early pregnancy has been previously reported and is contrasted with an increase in circulating VEGF that occurs during follicular luteinization in women undergoing in-vitro fertilization (Lee et al., 1997).
In conclusion, one of relaxin's physiological roles in women may be to promote the VEGF-mediated stimulation of endometrial neovascularization that occurs during the luteal phase of the menstrual cycle and in early pregnancy.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 19, 1998; accepted on November 23, 1998.