Hum. Reprod. Advance Access originally published online on April 3, 2006
Human Reproduction 2006 21(8):2158-2166; doi:10.1093/humrep/del089
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reduced levels of VEGF-A and MMP-2 and MMP-9 activity and increased TNF-
in menstrual endometrium and effluent in women with menorrhagia
Department of Obstetrics and Gynaecology, The Rosie Hospital, Robinson Way, Cambridge, UK
1 To whom correspondence should be addressed at: Academic Department of Obstetrics & Gynaecology, Mint Wing, St. Mary’s Hospital, Praed Street, London W2 1NY, UK. E-mail: shazymalik{at}yahoo.co.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: Heavy regular menstrual periods (menorrhagia) are an important cause of ill health in women and remain the leading indication for hysterectomy. Abnormalities of the endometrial blood vessels are among the possible causes of this condition. Many different factors affect endothelial cell growth, function and vessel remodelling. We sought to determine whether the levels of vascular endothelial growth factor-A (VEGF-A), tumour necrosis factor-
(TNF-), matrix metalloproteinase (MMP)-2 and MMP-9 and soluble VEGF receptor-1 (VEGF-R1) were altered in the menstrual effluent of women with objective menorrhagia. We have also quantitated the VEGF-A mRNA in the menstruated endometrium. METHODS AND RESULTS: We recruited 37 women and determined their menstrual blood loss (MBL) over two cycles and collected menstrual effluent during the 2nd day of bleeding for 4 h. There was no difference in the total level of VEGF-A, and neither latent MMP. However, the concentration of VEGF-A was significantly reduced in the women with menorrhagia, as was the VEGF-A mRNA level. In addition, the active forms of both MMPs were markedly reduced and the total sVEGF-R1 as well as the TNF- content were increased. CONCLUSIONS: This is the first study to show abnormalities of factors important for endothelial cell behaviour in the endometrium of women with menorrhagia. This may underlie the disordered vessel structure and/or function in this condition.
Key words: blood vessels/endometrium/matrix metalloproteinase/menorrhagia/VEGF-A
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Heavy, regular periods (idiopathic menorrhagia) are an important cause of ill health and gynaecology referrals in women, being the single largest reason for performing hysterectomy (Carlson et al., 1993
). Menorrhagia is the loss of greater than 80 ml of blood at the time of menstruation (Rybo, 1966). The term is derived from the Greek language and means ‘to burst forth monthly’ – (mene, ‘the moon’ and rhegnymi, ‘to burst forth’). The cause of this condition is still poorly understood but is not associated with gross histological abnormalities of the endometrium (Rees et al., 1984) nor does it arise from disturbances of the hypothalamic-pituitary-ovarian axis. Attention has therefore focused on functional aspects within the endometrium.
Markee et al. (1940) demonstrated that menstruation was initiated by constriction of specialized spiral arterioles. These vessels constrict before the onset of bleeding and whilst the superficial two thirds of the endometrium is shed, continues to undergo constriction in the basal part of the endometrium. As 75% of the blood loss is arteriolar, and in the absence of obvious platelet-fibrin plugs in the cut ends of the vessels, it is assumed that vascular constriction is responsible for controlling the menstrual loss. Thinning of the vascular smooth muscle cell (SMC) layer of these vessels is present in women with heavy periods. Changes in prostaglandin (PG) secretion towards vasodilator PGs is found in endometrium of women with heavy periods when compared with those with a normal measured menstrual blood loss (MBL) (Smith et al., 1981a,b), and agents that reduce the concentration of PGs in endometrium reduce MBL by about 20% (Cameron et al., 1990; Bonnar and Sheppard, 1996).
Alternatively, disturbances may exist in the endometrial mechanisms of coagulation as agents that reduce fibrinolysis (e.g. tranexamic acid) also reduce MBL but by a larger volume of about 50% (Preston et al., 1995). Finally, the structure of the tissue may play a part as MBL is halved in women taking oral contraception (Nilsson and Rybo, 1971).
A critical agent in the regulation of blood vessel growth and development is the vascular endothelial cell growth factor (VEGF) family of genes. VEGF-A is expressed in endometrium in epithelial cells and stroma in the proliferative phase of the cycle and in epithelial cells in the secretory phase. Highest levels of expression are found at menstruation and are probably elevated by the hypoxia arising in shed endometrium. Brenner et al. have shown peaks of VEGF-A mRNA expression in the surface epithelium glands and stroma of macaque endometrium in both menstrual and early proliferative phases (Brenner et al., 2002). Additionally, VEGF-A protein is expressed in endometrial vascular epithelium (Bausero et al., 1998), although this does not necessarily imply production in these cells as the antibody used detects receptor-bound VEGF-A. VEGF-A has several actions that if altered could give rise to defective control of the menstrual process. Firstly, VEGF-A induces endothelial cell expression of platelet-derived growth factor (PDGF) and heparin-binding epidermal growth factor (HB-EGF) (Arkonac et al., 1998), which promotes the migration and differentiation of vascular SMCs. Secondly, it stimulates vasodilating nitric oxide (NO) release (Papapetropoulos et al., 1997) through activation of the kinase insert domain receptor (KDR) receptor (Kroll and Waltenberger, 1997). Thirdly, it stimulates expression of tissue factor (TF), the major cellular initiator of coagulation (Blum et al., 2001). Thus altered expression of VEGF-A could alter the structure of the blood vessels, promote spiral arteriolar vasodilatation and/or inhibit coagulation.
Abnormalities in VEGF-A expression may also have direct consequences on endometrial endothelial cell proliferation and function. Kooy et al. (Kooy et al., 1996) have shown enhanced endothelial cell proliferation in the endometrium of women with menorrhagia. Moreover, Gargett et al. (2001) showed a highly significant correlation between the percentage of VEGF expressing vessels and vessels containing proliferating endothelial cells. The VEGF was localized to neutrophils in the subepithelial capillary plexus and functionalis microvessels, particularly in the proliferative phase, when angiogenis occurs.
A soluble form of the Flt 1 receptor, (sVEGFR-1 or sflt-1) generated by alternative splicing of Flt 1 pre-mRNA (Kendall and Thomas, 1993), acts as a specific high-affinity antagonist of VEGF-A function by competitively binding VEGF-A and thus affecting its bio-availability in vivo (He et al., 1999). Human endothelial cells produce high levels of this soluble receptor, and a hypoxia-induced increase in its expression has been shown in cells cultured from placenta (Hornig et al., 2000). Jelkmann (2001) has suggested that sVEGFR-1 levels should be presented alongside VEGF-A levels in experimental and pathological conditions. Certainly, an alteration in their balance may interfere with ligand-signal transduction and consequently endothelial cell function.
An alternative cytokine that is highly expressed in endometrium and is angiogenic is tumour necrosis factor- (TNF-) (Tabibzadeh et al., 1995). Trans-membrane TNF is required for VEGF-mediated endothelial cell hyper-permeability in vitro and in vivo (Clauss et al., 2001). This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablate the VEGF-induced permeability but not proliferation of cultivated human endothelial cells (Frater-Schroder et al., 1987).
Finally, matrix metalloproteinases (MMPs) are responsible for the cleavage of extra-cellular matrix in the upper functional two thirds of the endometrium and are key factors involved in the process of menstruation (Rodgers et al., 1994; Zhang and Salamonsen, 2002). Moreover, two MMPs, MMP-2 and MMP-9 (gelatinase A and B), are expressed in endometrial blood vessels throughout the cycle (Freitas et al., 1999). They are also expressed in endometrial epithelial cells, and stromal cells, with a major increase in MMP-9 mRNA expression at menstruation (Soini et al., 1997; Cornet et al., 2005). In addition, VEGF-A has been shown to stimulate MMP-2 activation in endothelial cells (Wary et al., 2003) and in an in vitro model of vessel formation (Burbridge et al., 2002). Mice with a null mutation in the MMP-9 gene exhibit deficient growth-plate angiogenesis (Vu et al., 1998) and VEGF-A causes an up-regulation of MMP-9 production in splenic T-cells of mammary tumour mice bearers (Owen et al., 2003). TNF has also been shown to increase the expression of MMP-9 in a murine model of retinal neo-vascularization (Majka et al., 2002).
As the damaged endometrial vessels are repaired within 5 days of menstrual onset (Ludwig et al., 1976), events during menstruation and the early proliferative phase are key to the angiogenic process and endothelial repair. We therefore chose to study the menstrual effluent of women with menorrhagia. Our hypothesis was that disordered expression of the above factors which are involved in the tissue breakdown at menstruation, vessel constriction and the angiogenesis required for tissue growth and repair in the cyclical regeneration of endometrium, lead to excessive MBL. To study events at menstruation, desquamated endometrium was collected in cervical cups and VEGF-A mRNA measured. Protein levels for VEGF-A, VEGFR-1 and TNF- along with both the latent and active forms of MMP-2 and MMP-9 were measured in the menstrual effluent.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Patient selection
The Ethics Committee of Addenbrooke’s Hospital University NHS Trust, Cambridge, UK, approved this study. Written informed consent was obtained from all recruits. The study group women were recruited from the gynaecology clinics at the Rosie Hospital, Cambridge. All the women in this study had regular menstrual cycles, of between 21 and 35 days. None of them were using any hormonal preparations, an intrauterine device, nor complaining of either inter-menstrual or post-coital bleeding. Their MBL was measured over two cycles (see method below) and taken to be the average of the two measurements.
The patient characteristics are summarized in Table I. In the normal group, 11 women were volunteers who fulfilled all the above criteria, 9 had subjectively complained of menorrhagia but were found to have a normal MBL, and 5 were recruited from outpatient clinics with symptoms such as unexplained pelvic pain (but had a normal pelvis at laparoscopy). Of the 23 patients, 16 had undergone surgical (laparoscopy and/or hysteroscopy) assessment of their pelvis, showing normal findings. The remaining had a completely normal pelvic examination and a normal MBL. In the menorrhagic group of patients, 11 had subjective menorrhagia confirmed by objective measurement and the remaining 3 were women who had undergone laparoscopic sterilization and found to have an MBL >80 ml. Women found to have fibroids were excluded from the study. Of the 14 study patients, 12 had undergone a surgical assessment of their pelvis showing normal findings (laparoscopy and/or hysteroscopy and one hysterectomy). Of the remaining two, one had a normal ultrasound scan of the pelvis, and the other had a completely normal pelvic examination. All had regular menstrual cycles. Thus, none of the menorrhagic patients had an obvious hormonal or physical cause for their menorrhagia (idiopathic menorrhagia).
|
MBL measurement
MBL was measured using a modification of the alkaline haematin technique (Gannon et al., 1996
) (This method was validated in our own laboratory using time expired blood measured and applied to the same sanitary ware given to the study group. A standard curve was generated showing the optical density (OD) to be directly proportional to the blood volume.). Each patient was given the same brand of tampon and/or pad (Lillets tampons and Secrets towels, Smith and Nephew, Birmingham, UK) along with clear verbal and written instructions on collection to ensure maximal saving of menstrual blood. Venous haemoglobin levels were measured in each patient. On receipt at the laboratory, the pad and tampon collection was frozen at –20°C. The sanitary ware was then washed in 10 l of deionized water and a non-ionic detergent (Triton X-100, BDH Chemicals, Poole, UK) for 10 min. A 10 ml sample of elute was centrifuged at 600 g for 3 min; 0.5 ml of the supernatant was aspirated and mixed with an equal volume of 0.85 mol/l sodium carbonate. Ten minutes later, absorbance was measured at 550 nm in a spectrophotometre against a blank of sodium carbonate and the volume of MBL calculated.
Collection of menstrual effluent
Menstrual blood was collected with a menstrual cup in the second 24 h of bleeding, as this is the heaviest day of bleeding (Haynes and Wolfe, 1970). The menstrual cup used was a soft natural rubber cup, shaped like a cone, size depending on parity (The Keeperä, Eco Logique Inc., Ontario, Canada). It was inserted into the upper vagina by the same researcher, with the mouth directed towards the cervix. The cup was left in situ for 4 h in all patients. The fluid sample was collected, the volume measured and washed with an equal volume of phosphate-buffered saline (PBS). This was centrifuged at 600 g for 10 min at 4°C. The supernatant was removed and frozen at –70°C, as was the cellular material. The supernatant was used to measure protein levels of VEGF-A, sVEGF-R1 and TNF-. RNA was isolated (as detailed below) from the menstruated endometrium in the cellular pellet obtained at centrifugation. Thus mRNA levels reflect those in the shed endometrium (the functionalis layer shed during menstruation).
Measurement of VEGF-A levels in menstrual effluent
This assay measures free VEGF-A. Matched pairs of antibodies were used for this assay. Nunc-Immuno Maxisorp hard plates were coated with 100 ml/well human VEGF-specific goat IgG antibody (R&D Systems, Abingdon, UK; AF-293-NA), diluted to 1 mg/ml. For the standard curve, recombinant human VEGF-165 (R&D Systems, 293-VE-010) was diluted in Tris-buffered saline with Tween (TBS-T) and 0.1% bovine serum albumin (TBS-T/BSA) to span a VEGF-A concentration upto 2000 pg/ml.
Hundred millilitres of samples and standards were added to each well and measured in duplicate. Some samples had to be diluted in TBS-T/BSA as the VEGF levels were found to be above the top of the standard curve (i.e. greater than 2000 pg/ml). Plates were incubated at room temperature for 2 h and then washed five times with PBS-T. Secondary antibody at a concentration of 200 ng/ml was added and incubated at room temperature for 2 h (biotinylated anti-human VEGF-specific goat IgG, R&D Systems), followed by a further five washes. HRPO-streptavidin conjugate (Zymed, San Francisco, CA, USA) diluted to 1 in 20 000 in PBS was then added and incubated at room temperature for 20 min. After adding 100 ml tetramethylbenzidine (TMB) microwell peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), plates were incubated at room temperature for 30 min in the dark. The reaction was stopped with 50 ml/well of 0.5 M H2SO4. The plates were read on filter 450 nm with a plate reader, using a secondary filter of 540 nm.
TNF- ELISA
A commercial ELISA kit (R&D Systems) was used to estimate levels of TNF- in the menstrual effluent, according to the manufacturer’s instructions.
sVEGF-R1 ELISA
A commercial ELISA kit (R&D Systems) was used to estimate levels of sVEGF-R1 in the menstrual effluent, according to the manufacturer’s instructions.
Measurement of MMP-2 and MMP-9
Proteolytic activities of the gelatinase MMPs were determined by zymography. This procedure results in the activation of both the inactive proforms and the active forms, which are seen as separate bands on the gel. MMP-2 (proform, 72 kDa; active form, 66 kDa) and MMP-9 (proform, 92 kDa; active form, 83 kDa) proteolytic activities were determined by gelatin zymography on 10% sodium dodecyl sulphate (SDS) gels incorporating 1 mg/ml gelatin (Novex-Novel Experimental Technology, San Diego, CA, USA). About 10.5 ml samples (containing 1.5 ml of sample plus 9 ml of loading buffer) were loaded onto the gels. These were then subjected to electrophoresis under non-reducing conditions for 4–5 h at 75 mV. After electrophoresis, the gels were incubated for 45 min in 2.5% Triton X-100 (Union Carbide Corporation, Danbury, CT, USA) and then incubated at 37°C overnight in 50 mol/l Tris buffer (pH 7.6) containing 0.5 mol/l sodium chloride and 10 mmol/l calcium chloride or 10 mmol/l ethylenediaminetetraacetic acid. Gels were stained with Coomassie blue; clear bands indicated active enzymes. Pre-stained molecular weight markers were included along with conditioned media from HT1080 fibrosarcoma cell line or amniotic fluid samples, which represented positive control preparations for the pro- and active-MMP forms. Enzymatic activity was estimated by densitometry of negative-image zymographic gels and measured in OD units.
Menstruated endometrium and real-time quantitative PCR
Menstruated endometrial tissue was collected from the effluent and frozen. A single step acid-phenol method of RNA isolation (Chomczynski and Sacchi, 1987) was used for this tissue and cDNA made using random hexamers. Briefly, 2 µl of RNA was added to 1.5 µl of N6 primer (Pharmacia, Little Chalfont, UK) made up to 20 µl with deionized water and left on ice for 15 min. To this was added 3 µl of 10 mM dNTP’a, 3 µl of RT buffer, 1.5 µl super RT and 1.5 µl RNAse inhibitor. This mixture was incubated at 42°C for 1 h, then 80°C for 10 min The quantitation of relative mRNA abundance was performed using a real-time fluorescence detection method using Taqman chemistry and analysed on a Model 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Primer and probe optimization was performed for VEGF-A using a commercial kit from Applied Biosystems. The optimal concentrations for primers and probe were 50 nM for the probe, 300 nM forward primer and 900 nM reverse primer. Relative quantitation was performed compared to Cyclophilin A mRNA. This was chosen as the housekeeping gene as it is stable in hypoxic tissues, whereas 18S RNA is not. The results were expressed in arbitrary units. The Ct value indicates the amount of message present. A higher amount of mRNA means that the signal will appear earlier and show as a lower number of cycles (i.e. as a lower Ct). The delta Ct (DCt) was calculated by subtracting the Ct for Cyclophilin A (as this is the control) from that of VEGF-A for each patient. As the Ct values for Cyclophilin are greater than those for VEGF-A, the results are presented as a negative value. The higher a Ct value, the lower the mRNA levels of the factor measured (as more amplification cycles are needed to reach the threshold cycle). Thus, a higher numerical value of the DCt (meaning a smaller difference between the Ct for Cyclophilin and the Ct for VEGF-A) indicates lower message levels of VEGF-A. All experiments were performed in triplicate, with a negative control-lacking template RNA included in each experiment. The following oligonucleotides were used for real-time RT-PCR (Applied Biosystems): (i) forward VEGF-A primer, 5'-CTGGAGTGTGTGCCCACTGA; reverse VEGF-A primer, 5'-TCCTATGTGCTGGCCTTGGT; VEGF-A probe, 5'-FAMACAT CACCATGCAGATTATGCGGATCAAA. The primers for VEGF-A span exon 1 to exon 3 and thus detect all isoforms of VEGF-A but will not amplify genomic DNA. (ii) The primers and probe for Cyclophilin A are provided as a kit (Applied Biosystems).
Statistical analysis
MBL was taken as the mean over two menstrual cycles. The data did not demonstrate a normal Gaussian distribution. Measurements are therefore recorded as the median with the range in parenthesis. Comparison is made by using the non-parametric Mann–Whitney U test (one-tailed P-value). Results with a P-value of less than 0.05 were considered significant. These tests were done using Instat software (2.01 version).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Levels of VEGF-A in menstrual effluent and mRNA levels encoding VEGF-A in menstruated endometrium
The total amount of VEGF-A protein released into the menstrual effluent was not significantly different between the two groups, being 4141 pg/4 h (range 62–76 588) for the control women and 4812 pg/4 h (range 1990–17 294) in the study group (Table II). However, the concentration in the effluent of women with an MBL >80 ml was significantly lower (P < 0.002). The median concentration of VEGF-A in the menstrual effluent was 1031 pg/ml in the control women (range 125–6660) and 407 pg/ml in the study group (range 199–1103) (Figure 1). The levels of mRNA encoding VEGF-A in the menstruated endometrium from women with menorrhagia was significantly reduced, the median DCt being –7.87 in the normal group (range –13.17 to –6.70) and –7.31 in the menorrhagics with a P < 0.016 (range –8.10 to –5.33). The mRNA levels were measured in 19 control patients and all 14 menorrhagics (Figure 1).
|
|
TNF-
ELISAAn estimation of TNF-
using ELISA was performed in 10 patients from each group. Total TNF- protein over 4 h was significantly higher in the study group at 21 259 pg/4 h (range 7935–101 862) compared to 9013 pg/4 h (range 53–48 903) in the controls (P < 0.04) (Table II; Figure 2). There was no significant difference in effluent concentration of TNF- with a median of 2254 pg/ml (range 613–7612) in the study group and 1996 pg/ml (range 1241–5668) in the controls, (P < 0.34).
|
sVEGF-R1 ELISA
An estimation of sVEGF-R1 was performed in 22 controls and all 14 menorrhagic patients (Table II). Total sVEGF-R1 protein over 4 h was significantly higher in women with menorrhagia compared to those with a normal MBL (P < 0.007). The median for the menorrhagics was 30 503 pg/4 h (range 10 339–94 389) compared to 11 734 pg/4 h in the normal group (range 254–91 276). The concentration of sVEGF-R1, however, was not significantly different between the two groups (P < 0.12), being 2841 pg/ml in the menorrhagics (range 682–8762 pg/ml) compared to 3709 pg/ml in the controls (range 823–15 213).
MMP-2 and MMP-9
Following zymography of the menstrual effluent samples (n = 20 normals, n = 12 menorrhagics), densitometry was used to estimate the levels of the active and latent forms of MMP-2 and MMP-9 (Table II). This showed that the latent forms of both MMP-2 (570 versus 572 OD; P < 0.49) and MMP-9 (1416 versus 1364 OD; P < 0.29) were similar in women who had a normal or excessive MBL. However, the active forms were markedly reduced in women with objective menorrhagia. The median for women with menorrhagia was 0 OD compared to 261 OD in the normal group (P < 0.014) for MMP-2 and 0 OD compared to 129 OD for MMP-9 (P < 0.04) as shown in Figure 3.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
These findings provide the first evidence for a potential role of VEGF-A, MMP-2 and MMP-9, and TNF-
in the mechanism of idiopathic excessive uterine bleeding. Women’s perceptions of an abnormal MBL may sometimes be a result of increased secretions rather than a genuine abnormality in menstrual blood flow. However, as the entire study group had an objective measurement of MBL, this is unlikely to be a cause of our findings. Menstrual effluent collected from women with objectively assessed heavy periods (MBL > 80 ml) contained significantly lower concentrations of VEGF-A than similar fluid collected from women with normal periods. In addition, mRNA encoding VEGF-A was also suppressed in the desquamated endometrium of the same women.
It is not clear from our study why this change in VEGF-A expression arises. As the total amount of VEGF-A protein was similar between controls and menorrhagics, the reduced concentration may just be a dilutional effect because of the increased rate of blood loss in menorrhagic women. With an increase in sVEGFR-1 protein over the same time-frame, it may even be that the amount of secreted VEGF-A protein is increased in menorrhagia but our assay for free VEGF-A would not reflect this. The increased VEGF-A protein could be bound by the sVEGFR-1 receptor being shed from the endometrium. This may have either a direct effect on the cells producing VEGF-A, by switching off mRNA production, or an indirect effect. For example, the oedematous and proliferative effects of estradiol (E2) in the uterus, mediated by VEGF-A, are significantly reduced by the administration of sVEGFR-1 (Hastings et al., 2003). Brenner et al. (2002) localized VEGF-A receptors 1 and 2 to the capillaries just below the luminal epithelium in the post-menstrual repair phase. Thus, a reduction in VEGF-A levels and receptor binding might well impair the early angiogenic processes involved in endometrial repair and regeneration. sVEGFR-1 is known to be a dominant negative regulator of VEGF-A (Clark et al., 1998) and may act by reducing the bioavailability of VEGF-A at a crucial time in endometrial repair. Graubert et al. (Graubert et al., 2001) showed a high level of sVEGFR-1 in early menstrual samples and also that immunoprecipitated VEGF-A was bound to the soluble receptor, again in early menstrual samples. They suggest that VEGF-A receptor activation and subsequent modulation of sVEGFR-1 in the late menstrual phase contributes to the onset of angiogenesis and endothelial repair in human endometrium. Interestingly, the sVEGFR-1 was almost undetectable in the late menstrual phase, when KDR (VEGFR-2) activation was shown to occur along with a peak in endothelial cell proliferation. We have shown that in women with menorrhagia, the endometrium is exposed to a significantly greater total amount of sVEGFR-1 over a given period. It may therefore play an important role in the aetiology of menorrhagia. In addition, TNF- is able to inhibit the expression of KDR in cultured endothelial cells (Patterson et al., 1996), and the increased levels found in our study may have a further negative effect on signalling pathways.
Furthermore, secreted endometrial TNF- peaks in the menstrual phase (Tabibzadeh et al., 1995). It has been suggested as a key local signal contributing to the process of menstrual shedding and bleeding, by the induction of apoptosis and by acting on cell–cell dissociation (Tabibzadeh, 1996). In fact, in the mouse, administration of TNF- induces vascular damage and haemorrhage in endometrium, indistinguishable from the bleeding that occurs at menstruation in the human (Shalaby et al., 1989). Chen et al. (1995) have shown that TNF- stimulates release of PG F2 and PGE2 in cultured human luteal phase endometrial cells. It is plausible then that an abnormally high level of TNF- in menorrhagic endometrium at menstruation can contribute significantly to increasing blood loss from vessels by all of the above pathways.
We have shown previously that hypoxia (independent of steroids) markedly increases VEGF-A expression and release from both human endometrial glandular epithelium and stroma by increasing steady-state levels of mRNA levels (Sharkey et al., 2000). The superficial endometrium that is shed at menstruation is presumably hypoxic having been shed. Since steroid levels are low at menstruation, failure to respond adequately to the hypoxic stimulus of shedding may well lead to the reduced levels of VEGF-A. Whilst glandular VEGF-A is unlikely to contribute directly to angiogenesis [it is secreted apically in the glands (Hornung et al., 1998)], focal-bound VEGF-A expressed in stromal neutrophils associated with endometrial micro-vessels correlates strongly with endothelial cell proliferation (Gargett et al., 2001). Decreased expression of VEGF-A mRNA may reflect an abnormality at this level and warrants further research.
Reduced levels of VEGF-A on the 2nd day of bleeding could influence several of the mechanisms that regulate MBL. As menstrual blood passes through the spiral arterioles into the uterine cavity, it undergoes coagulation and rapid fibrinolysis (Cederholm-Williams et al., 1984a,b). One of the most potent initiators of coagulation is TF. Because VEGF-A stimulates TF production by PKC-dependent activation of EGR-1 (Mechtcheriakova, 2001), reduced levels could impair coagulation by failing to stimulate TF expression. Further evidence to support this view is contained in our finding that not only were VEGF-A levels reduced in endometrium of women with menorrhagia, but also significant reductions were found in levels of activated MMP-2 and MMP-9 though not of their pro-MMP precursors. Human endometrial stromal cells express high levels of TF under steroidal control (Lockwood et al., 1994) and the principal consequence of the action of TF is to release thrombin, a highly effective protease that converts latent pro-MMP-2 to its active form (Zucker et al., 1998). Reduced levels of TF would also impair coagulation further increasing the propensity to heavy menstrual loss.
A striking feature of this study was the almost complete absence of the active forms of MMP-2 and 9 in the menstrual effluent. However, the inactive latent forms of both MMPs were not altered in the women with heavy periods, arguing against a direct transcriptional effect. Instead, the absence of the cleaved forms might suggest a significant reduction in the levels or activity of enzymes that cleave the latent form of the MMPs into their active states. The presence of active MMP-9 seems to be restricted and linked to the peri-menstrual phase in the endometrium and is activated by MMP-3 (Rigot et al., 2001) and is mediated in part by LEFTY-A (Cornet et al., 2005). MT1-MMP is perhaps the best-known molecule to cleave MMP-2 (English et al., 2001), but unfortunately LEFTY-A and MT1-MMP were not measured in this study. MMPs can have an effect on vessel function at several levels. The activation of VEGF-A by MMP-2 is blocked by synthetic MMP inhibitors and tissue inhibitors of metalloproteinase-2 (TIMP) and also abolished by partial deletion of the MT1-MMP gene (Sounni et al., 2004). The vessels of women with heavy periods are defective when compared to similar vessels in women with normal blood loss. Vascular SMCs in the spiral arterioles of these women show significantly reduced proliferation in the mid to late secretory phase of the cycle (Abberton et al., 1999). MMPs may play a role in smooth muscle proliferation and migration, because non-selective MMP inhibitors diminish rabbit vascular SMC proliferation in vitro (Galis and Khatri, 2002) and SMC migration in the rat carotid injury model (Bendeck et al., 1994). Furthermore, MMP-2 promotes vasoconstriction in the rat [by cleaving big Endothelin-1 (big ET-1) and yielding a potent vasoconstrictor ET-1 (1–32)], and selective pharmacological inhibition of vascular MMP-2 induces vasodilatation (Fernandez-Patron et al., 2000). Intravascular thrombi functioning as haemostatic plugs contain platelets and fibrin in shed normal menstrual tissue (Christiaens et al., 1980). The release of active MMP-2 during platelet activation has been shown to mediate platelet aggregation (Sawicki et al., 1997). A reduction in active gelatinase A could thus enhance blood loss by acting at several levels. In addition, cleavage of big ET-1 by MMP-2 has also been shown to activate neutrophils and promote leukocyte-endothelial cell adhesion and neutrophil trafficking into inflamed tissues (Fernandez-Patron et al., 2001). MT1-MMP, which activates MMP-2, is present in the membrane of neutrophils (Salamonsen et al., 2000). It is tempting to speculate that in menorrhagic endometrium, a defect in microvessel-associated neutrophil activation occurs as a result of defective MMP-2 activation, which then has an impact on local VEGF-A levels and thus on vessel growth and/or function.
Alternatively, MMPs play a critical part in the process of angiogenesis itself, and their absence from the wound site might further impair the angiogenesis arising from the lack of VEGF-A. A study in mice has shown that MMP-9 is required for adequate angiogenic re-vascularization of ischemic tissues and its source was shown to be bone marrow-derived macrophages (Johnson et al., 2004). Activation of MMP-2 and 9 is critical in the VEGF-mediated mouse hind-limb angiogenesis induced by hypoxia (Silvestre et al., 2001), where the angiogenic process was profoundly impaired in the absence of MMP activation even with sustained VEGF-A levels. This group’s finding that MMPs are activated before VEGF-A expression suggests that matrix degradation is a rate-limiting event that is required for triggering the angiogenic process and sensitizing tissue to the effects of VEGF. Lack of this activation, as in our study, could then lead to impaired angiogenesis and remodeling in the endometrium of women with menorrhagia.
The regulation of endometrial bleeding is complex, and although the mechanisms are unclear, the findings from this study provide for the first time convincing evidence that significant changes in endometrial VEGF-A expression and altered levels of active MMP-2 and 9 with increased total concentrations of sVEGFR-1 and TNF- are present in the menstrual effluent of women who have excessively heavy periods. These changes suggest a link between angiogenesis, vascular tone and menstrual shedding with the volume of the MBL and add further to our understanding of this process.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank John McLaren for the gelatin zymography. We thank the consultants and staff of Addenbrooke’s Hospital for their assistance in recruiting patients for this study and the patients and volunteers who kindly participated in it. This work was funded by an MRC programme grant (G9623012) awarded to SKS and DSCJ.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Abberton KM, Taylor NH, Healy DL, Rogers PA. (1999) Vascular smooth muscle cell proliferation in arterioles of the human endometrium. Hum Reprod 14:1072–1079.
Arkonac BM, Foster LC, Sibinga NES, Patterson C, Lai K, Tsai JC, Lee ME, Perrella MA, Haber E. (1998) Vascular endothelial growth factor induces heparin-binding epidermal growth factor-like growth factor in vascular endothelial cells. J Biol Chem 273:4400–4405.
Bausero P, Cavaille F, Meduri G, Freitas S, Perrot-Applanat M. (1998) Paracrine action of vascular endothelial growth factor in the human endometrium: production and target sites, and hormonal regulation. Angiogenesis 2:167–182.[Medline]
Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. (1994) Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res 75:539–545.
Blum S, Issbruker K, Willuweit A, Hehlgans S, Lucerna M, Mechtcheriakova D, Walsh K, von der Ahe D, Hofer E, Clauss M. (2001) An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression. J Biol Chem 276:33428–33434.
Bonnar J and Sheppard BL. (1996) Treatment of menorrhagia during menstruation: randomised controlled trial of ethamsylate, mefenamic acid, and tranexamic acid. BMJ 313:579–582.
Brenner RM, Nayak NR, Slayden OD, Critchley HO, Kelly RW. (2002) Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis. Ann N Y Acad Sci 955:60–74 Discussion 86–88,396–406.[Web of Science][Medline]
Burbridge MF, Coge F, Galizzi JP, Boutin JA, West DC, Tucker GC. (2002) The role of the matrix metalloproteinases during in vitro vessel formation. Angiogenesis 5:215–226.[CrossRef][Medline]
Cameron IT, Haining R, Lumsden MA, Reid Thomas V, Smith SK. (1990) The effects of mefenamic acid and norethisterone on measured menstrual blood loss. Obstetrics Gynecol 76:85–88.[Web of Science][Medline]
Carlson KJ, Nichols DH, Schiff I. (1993) Indications for hysterectomy. N Engl J Med 328:856–860.
Cederholm-Williams SA, Rees MC, Turnbull AC. (1984a) Consumption of fibrinolytic proteins in menstrual fluid from women with normal menstrual blood loss. J Clin Pathol 37:879–881.
Cederholm-Williams SA, Rees MCP, Turnbull AC. (1984b) Examination of certain coagulation factors in menstrual fluid from women with normal blood loss. Thromb Haemost 52:224–225.[Web of Science][Medline]
Chen DB, Yang ZM, Hilsenrath R, Le SP, Harper MJ. (1995) Stimulation of prostaglandin (PG) F2 alpha and PGE2 release by tumour necrosis factor-alpha and interleukin-1 alpha in cultured human luteal phase endometrial cells. Hum Reprod 10:2773–2780.
Chomczynski P and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.[Web of Science][Medline]
Christiaens G, Sixma JJ, Haspels AA. (1980) Morphology of haemostasis in menstrual endometrium. Br J Obstet Gynaecol 87:425–439.[Web of Science][Medline]
Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps AN, Lammoglia R, Charnock-Jones DS. (1998) A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol Reprod 59:1540–1548.
Clauss M, Sunderkotter C, Sveinbjornsson B, Hippenstiel S, Willuweit A, Marino M, Haas E, Seljelid R, Scheurich P, Suttorp N, et al. (2001) A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability. Blood 97:1321–1329.
Cornet PB, Galant C, Eeckhout Y, Courtoy PJ, Marbaix E, Henriet P. (2005) Regulation of matrix metalloproteinase-9/gelatinase B expression and activation by ovarian steroids and LEFTY-A/endometrial bleeding-associated factor in the human endometrium. J Clin Endocrinol Metab 90:1001–1011.
English WR, Holtz B, Vogt G, Knauper V, Murphy G. (2001) Characterization of the role of the "MT-loop": an eight-amino acid insertion specific to progelatinase A (MMP2) activating membrane-type matrix metalloproteinases. J Biol Chem 276:42018–42026.
Fernandez-Patron C, Stewart KG, Zhang Y, Koivunen E, Radomski MW, Davidge ST. (2000) Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res 87:670–676.
Fernandez-Patron C, Zouki C, Whittal R, Chan JS, Davidge ST, Filep JG. (2001) Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1–32]. Faseb J 15:2230–2240.
Frater-Schroder M, Risau W, Hallmann R, Gautschi P, Bohlen P. (1987) Tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci USA 84:5277–5281.
Freitas S, Meduri G, Le Nestour E, Bausero P, Perrot-Applanat M. (1999) Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biol Reprod 61:1070–1082.
Galis ZS and Khatri JJ. (2002) Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res 90:251–262.
Gannon MJ, Day P, Hammadieh N, Johnson N. (1996) A new method for measuring menstrual blood loss and its use in screening women before endometrial ablation. Br J Obstet Gynaecol 103:1029–1033.[Web of Science][Medline]
Gargett CE, Lederman F, Heryanto B, Gambino LS, Rogers PA. (2001) Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils. Hum Reprod 16:1065–1075.
Graubert MD, Ortega MA, Kessel B, Mortola JF, Iruela-Arispe ML. (2001) Vascular repair after menstruation involves regulation of vascular endothelial growth factor-receptor phosphorylation by sFLT-1. Am J Pathol 158:1399–1410.
Hastings JM, Licence DR, Burton GJ, Charnock-Jones DS, Smith SK. (2003) Soluble vascular endothelial growth factor receptor 1 inhibits edema and epithelial proliferation induced by 17beta-estradiol in the mouse uterus. Endocrinology 144:326–334.
Haynes DM and Wolfe WM. (1970) Tubal sterilization in an indigent population. Report of fourteen years’ experience. Am J Obstet Gynecol 106:1044–1053.[Web of Science][Medline]
He Y, Smith SK, Day KA, Clark DE, Licence DR, Charnock-Jones DS. (1999) Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol 13:537–545.
Hornig C, Barleon B, Ahmad S, Vuorela P, Ahmed A, Weich HA. (2000) Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids. Lab Invest 80:443–454.[Web of Science][Medline]
Hornung D, Lebovic DI, Shifren JL, Vigne JL, Taylor RN. (1998) Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil Steril 69:909–915.[CrossRef][Web of Science][Medline]
Jelkmann W. (2001) Pitfalls in the measurement of circulating vascular endothelial growth factor. Clin Chem 47:617–623.
Johnson C, Sung HJ, Lessner SM, Fini ME, Galis ZS. (2004) Matrix metalloproteinase-9 is required for adequate angiogenic revascularization of ischemic tissues: potential role in capillary branching. Circ Res 94:262–268.
Kendall RL and Thomas KA. (1993) Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci USA 90:10705–10709.
Kooy J, Taylor NH, Healy DL, Rogers PA. (1996) Endothelial cell proliferation in the endometrium of women with menorrhagia and in women following endometrial ablation. Hum Reprod 11:1067–1072.
Kroll J and Waltenberger J. (1997) The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem 272:32521–32527.
Lockwood CJ, Krikun G, Papp C, Toth-Pal E, Markiewicz L, Wang EY, Kerenyi T, Zhou X, Hausknecht V, Papp Z, et al. (1994) The role of progestationally regulated stromal cell tissue factor and type-1 plasminogen activator inhibitor (PAI-1) in endometrial hemostasis and menstruation. Ann N Y Acad Sci 734:57–79.[Web of Science][Medline]
Ludwig H, Metzger H, Frauli M. (1976) The re-epitheliazation of endometrium after menstrual desquamation. Arch Gynakol 221:51–60.[CrossRef][Web of Science][Medline]
Majka S, McGuire PG, Das A. (2002) Regulation of matrix metalloproteinase expression by tumor necrosis factor in a murine model of retinal neovascularization. Invest Ophthalmol Vis Sci 43:260–266.
Markee JE. (1940) Menstruation in intraocular endometrial transplants in the rhesus monkey. Contributions Embryol 28:219–308.
Mechtcheriakova DSG, Lucerna M, Clauss M, De Martin R, Binder BR, Hofer E. (2001) Specificity, diversity, and convergence in VEGF and TNF-alpha signaling events leading to tissue factor up-regulation via EGR-1 in endothelial cells. FASEB J 15:230–242.
Nilsson L and Rybo G. (1971) Treatment of menorrhagia. Am J Obstet Gynecol 110:713–720.[Web of Science][Medline]
Owen JL, Iragavarapu-Charyulu V, Gunja-Smith Z, Herbert LM, Grosso JF, Lopez DM. (2003) Up-regulation of matrix metalloproteinase-9 in T lymphocytes of mammary tumor bearers: role of vascular endothelial growth factor. J Immunol 171:4340–4351.
Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. (1997) Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 100:3131–3139.[Web of Science][Medline]
Patterson C, Perrella MA, Endege WO, Yoshizumi M, Lee M-E, Haber E. (1996) Downregulation of vascular endothelial growth factor receptors tumor necrosis factor- in cultured human vascular endothelial cells. J Clin Invest 98:4900–4906.
Preston JT, Cameron IT, Adams EJ, Smith SK. (1995) Comparative study of tranexamic acid and norethisterone in the treatment of ovulatory menorrhagia. Br J Obstet Gynaecol 102:401–406.[Web of Science][Medline]
Rees MCP, Dunnill MS, Anderson ABM, Turnbull AC. (1984) Quantitative uterine histology during the menstrual cycle in relation to measured menstrual blood loss. Br J Obstet Gynaecol 91:662–660.[Web of Science][Medline]
Rigot V, Marbaix E, Lemoine P, Courtoy PJ, Eeckhout Y. (2001) In vivo perimenstrual activation of progelatinase B (proMMP-9) in the human endometrium and its dependence on stromelysin 1 (MMP-3) ex vivo. Biochem J 358:275–280.[CrossRef][Web of Science][Medline]
Rodgers WH, Matrisian LM, Giudice LC, Dsupin B, Cannon P, Svitek C, Gorstein F, Osteen KG. (1994) Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones. J Clin Invest 94:946–953.[Web of Science][Medline]
Rybo G. (1966) Clinical and experimental studies on menstrual blood loss. Acta Obstet Gynecol Scand 45:Suppl. 7, 1–23.[Web of Science][Medline]
Salamonsen LA, Zhang J, Hampton A, Lathbury L. (2000) Regulation of matrix metalloproteinases in human endometrium. Hum Reprod 15:Suppl. 3, 112–119.
Sawicki G, Salas E, Murat J, Miszta-Lane H, Radomski MW. (1997) Release of gelatinase A during platelet activation mediates aggregation. Nature 386:616–619.[CrossRef][Medline]
Shalaby MR, Laegreid WW, Ammann AJ, Liggitt HD. (1989) Tumor necrosis factor-alpha-associated uterine endothelial injury in vivo. Influence of dietary fat. Lab Invest 61:564–570.[Web of Science][Medline]
Sharkey AM, Day K, McPherson A, Malik S, Licence D, Smith SK, Charnock-Jones DS. (2000) Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J Clin Endocrinol Metab 85:402–409.
Silvestre JS, Mallat Z, Tamarat R, Duriez M, Tedgui A, Levy BI. (2001) Regulation of matrix metalloproteinase activity in ischemic tissue by interleukin-10: role in ischemia-induced angiogenesis. Circ Res 89:259–264.
Smith SK, Abel MH, Kelly RW, Baird DT. (1981a) A role for prostacyclin (PGi2) in excessive menstrual bleeding. Lancet 1:522–524.[Web of Science][Medline]
Smith SK, Abel MH, Kelly RW, Baird DT. (1981b) Prostaglandin synthesis in the endometrium of women with ovular dysfunctional uterine bleeding. Br J Obstet Gynaecol 88:434–442.[Web of Science][Medline]
Soini Y, Alarakkola E, Autio-Harmainen H. (1997) Expression of messenger RNAs for metalloproteinases 2 and 9, type IV collagen, and laminin in nonneoplastic and neoplastic endometrium. Hum Pathol 28:220–226.[CrossRef][Web of Science][Medline]
Sounni NE, Roghi C, Chabottaux V, Janssen M, Munaut C, Maquoi E, Galvez BG, Gilles C, Frankenne F, Murphy G, et al. (2004) Up-regulation of vascular endothelial growth factor-A by active membrane-type 1 matrix metalloproteinase through activation of Src-tyrosine kinases. J Biol Chem 279:13564–13574.
Tabibzadeh S. (1996) The signals and molecular pathways involved in human menstruation, a unique process of tissue destruction and remodelling. Mol Hum Reprod 2:77–92.
Tabibzadeh S, Zupi E, Babaknia A, Liu R, Marconi D, Romanini C. (1995) Site and menstrual cycle-dependent expression of proteins of the tumour necrosis factor (TNF) receptor family, and BCL-2 oncoprotein and phase-specific production of TNF alpha in human endometrium. Hum Reprod 10:277–286.
Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. (1998) MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422.[CrossRef][Web of Science][Medline]
Wary KK, Thakker GD, Humtsoe JO, Yang J. (2003) Analysis of VEGF-responsive genes involved in the activation of endothelial cells. Mol Cancer 2:25.[CrossRef][Medline]
Zhang J and Salamonsen LA. (2002) In vivo evidence for active matrix metalloproteinases in human endometrium supports their role in tissue breakdown at menstruation. J Clin Endocrinol Metab 87:2346–2351.
Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, Jesty J. (1998) Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 75:780–786.[CrossRef][Web of Science][Medline]
Submitted on May 11, 2005; resubmitted on September 28, 2005; resubmitted on February 20, 2006; accepted on March 6, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Rae, A. Mohamad, D. Price, P. W. F. Hadoke, B. R. Walker, J. I. Mason, S. G. Hillier, and H. O. D. Critchley Cortisol Inactivation by 11{beta}-Hydroxysteroid dehydrogenase-2 May Enhance Endometrial Angiogenesis via Reduced Thrombospondin-1 in Heavy Menstruation J. Clin. Endocrinol. Metab., April 1, 2009; 94(4): 1443 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 The ESHRE Capri Workshop Group Endometrial bleeding Hum. Reprod. Update, September 1, 2007; 13(5): 421 - 431. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||