Department of Obstetrics and Gynecology, Nagoya City University, Graduate School of Medicine, 1 Kawasumi, Mizuho-ku, Mizuho-cho, Nagoya 467-8601, Japan
1 To whom correspondence should be addressed. e-mail: og.n.suz{at}med.nagoya-cu.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: angiogenesis/menstrual cycle/neovascularization/PlGF/red lesions
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endometriosis features progressive growth that is estrogen-dependent (Rock et al., 1992), and several lines of evidence suggest that the angiogenic factor, VEGF, is also involved in the maintenance and development of endometriotic lesions (McLaren et al., 1996
; Li et al., 2001
). An important condition for endometriotic tissues following adhesion and implantation is the establishment of neovascularization or angiogenesis (Folkman, 1995
; Kats et al., 2002
). Recently, several angiogenic molecules have been shown to demonstrate elevated concentrations in the peritoneal fluid of endometriosis patients, examples being VEGF, interleukin (IL)-8 and ENA-78 (Arici et al., 1996
; McLaren et al., 1996
; Mahnke et al., 2000
; Calhaz-Jorge et al., 2003
; Mueller et al., 2003
). Recent data suggest that gene therapy with angiogenic inhibitors might be highly effective for the control of endometriosis, even in a host with preserved estrogen levels (Dabrosin et al., 2002
). Here we speculated that the concentration of PlGF, an angiogenic factor, might be elevated in the peritoneal fluid of women with endometriosis. To our knowledge, no such report of PlGF levels in endometriosis patients has been published previously.
Since endometriosis is influenced by the cyclic change in ovarian steroids, we also examined here whether the PlGF concentration varies with the phase of the menstrual cycle. In addition, several findings suggest greater neovascularization within endometriotic implants in red as compared with black or white lesions (Nisolle et al., 1993). Therefore, the present study was designed to assess variation with the presence of red lesions as well as with menstrual cycle and endometriosis stage.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The patients with endometriosis were scored according to the American Society of Reproductive Medicine classification 1996 (American Society for Reproductive Medicine, 1997), and included individuals with or without red lesions. Samples of peritoneal fluid were obtained during laparoscopic surgery or laparotomy (for cystectomy, adnexectomy or total abdominal hysterectomy). There was no contradiction between BBT and follicle or corpus luteum. We studied eight cases of laparotomy [study group, n = 4 (proliferative phase, n = 2; secretory phase, n = 2), all of them stage IV; control group, n = 4 (proliferative phase, n = 2; secretory phase, n = 2)] and 51 cases of laparoscopy (study group, n = 31; control group, n = 20) for this study.
Collection of peritoneal fluid
Aspiration of the peritoneal fluid during surgery was performed under direct visualization from the posterior cul de sac and anterior vesico-uterine fold. Patients with bleeding into the peritoneal cavity from the abdominal walls were excluded. The volume of peritoneal fluid was then measured. After clarification by centrifugation at 2000 g for 10 min, the supernatants were stored at 80°C until assayed.
Measurement of PlGF concentrations
Amounts of PlGF in the peritoneal fluid were determined with a PlGF enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturers protocol. Samples from all patients were measured in parallel and in duplicate to control for interassay variance. The optical density of each well was measured at dual wavelengths of 450 and 570 nm. Concentrations of PlGF were calculated by interpolation from a standard curve. The sensitivity of the PlGF ELISA was 7 pg/ml.
Statistical analysis
Calculated values were expressed as medians and interquartile ranges, and comparisons were made with the MannWhitney and KruskalWallis non-parametric tests. A P-value <0.05 was regarded as statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The endometriosis patients were staged from I to IV depending on the severity of disease based on the Revised American Society for Reproductive Medicine classification (American Society for Reproductive Medicine, 1997). The stage distribution at surgery was as follows: stage I, n = 8 (proliferative phase, n = 4; secretory phase, n = 4; 101 pg/ml), 84235 pg/ml; stage II, n = 7 (proliferative phase, n = 4; secretory phase, n = 3), 357 pg/ml, 219475 pg/ml; stage III, n = 12 (proliferative phase, n = 7; secretory phase, n = 5), 173 pg/ml, 105235 pg/ml; and stage IV, n = 8 (proliferative phase, n = 5; secretory phase, n = 3), 192 pg/ml, 122346 pg/ml. There was a significant difference in PlGF levels among the endometriosis stage IIV groups (P < 0.001, KruskalWallis test) (Figure 1A). Red lesions were identified in 22 patients, and appeared to be lacking in the other 13. Patients with peritoneal implants classified as red lesions (stage I, n = 3; stage II, n = 6; stage III, n = 7; stage IV, n = 6) had significantly higher concentrations of PlGF [n = 22 (proliferative phase, n = 13; secretory phase, n = 9), 181 pg/ml, 133475 pg/ml) in their peritoneal fluid than in those without [n = 13 (proliferative phase, n = 7; secretory phase, n = 6), 134 pg/ml, 84307 pg/ml; P< 0.001]. There were notable differences in volume of the peritoneal fluid between cases with (mean ± SEM 22 ± 2.7 ml) and without (10 ± 1.8 ml) endometriosis (P < 0.001).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VEGF and its receptors are overexpressed in several inflammatory diseases (Dvorak et al., 1995). Recent data indicate that expression of PlGF is up-regulated during inflammation and with inflammatory human skin diseases (Oura et al., 2003
). Endometriosis causes an inflammatory reaction in the pelvis, changing the characteristics of the peritoneal fluid and its cellular components (Halme, 1991
; Ramey et al., 1993
; Hill et al., 1988
). Our results suggest that the inflammation associated with endometriosis, through increased levels of peritoneal fluid PlGF, may promote angiogenesis and support the progressive growth and activity of endometriosis.
PlGF and VEGF have been shown to be equipotent in stimulating tissue factor production and chemotaxis in monocytes (DiSalvo et al., 1995). A recent report suggested that PlGF stimulated angiogenesis and collateral growth in ischaemic heart and limb with efficiency at least comparable to VEGF (Luttun et al., 2002
). Peritoneal VEGF concentrations in women with endometriosis are reported to be significantly higher in the proliferative phase than in the secretory phase (McLaren et al., 1996
). However, in our study both the endometriosis group and controls with cystadenomas demonstrated significantly higher PlGF levels in the secretory phase. A recent report suggested that expression of PlGF mRNA occurs in uterine natural killer cells, with highest levels found in the midsecretory phase of the cycle (Li et al., 2001
), coincident with increase of lymphocytes in the human endometrium (King et al., 1998
). Our data are in line with PlGF production by lymphocytes in the peritoneal cavity and regulation by ovarian steroids. PlGF is thought to play a role in regulating trophoblast function and the early process of placentation (Tjoa et al., 2001
), so that our findings may point to a potential contribution of PlGF to implantation and invasion of endometriotic tissues.
Several reports suggest that red lesions represent active endometriosis because they feature more pronounced angiogenesis than their black or white counterparts (Wiegerinck et al., 1993; Iwabe et al., 1998
). Early red lesions have been reported to be characterized by invasion of the extracellular matrix (Spuijbroek et al., 1992
). Our findings thus suggest that PlGF in the peritoneal fluid may be involved in the neovascularization of endometriotic tissues.
We conclude that PlGF is an important factor that may contribute to the pathogenesis of endometriosis, especially with red lesions, possibly promoting neovascularization. Further studies are now needed to define the actual role of PlGF in regulation of angiogenesis in endometriosis and how its production may be regulated by ovarian steroids.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arici, A., Tazuke, S.I., Attar, E., Kliman, H.J. and Olive, D.L. (1996) Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol. Hum. Reprod., 2, 4045.[Abstract]
Calhaz-Jorge, C., Costa, A.P., Santos, M.C. and Palma-Carlos, M.L. (2003) Peritoneal fluid concentrations of interleukin-8 in patients with endometriosis depend on the severity of the disorder and are higher in the luteal phase. Hum. Reprod., 18, 593597.
Cao, Y., Chen, H., Zhou, L., Chiang, M.K., Anand-Apte, B., Weatherbee, J.A., Wang, Y., Fang, F., Flanagan, J.G. and Tsang, M.L. (1996) Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR. J. Biol. Chem., 271, 31543162.
Dabrosin, C., Gyorffy, S., Margetts, P., Ross, C. and Gauldie, J. (2002) Therapeutic effect of angiostatin gene transfer in a murine model of endometriosis. Am. J. Pathol., 161, 909918.
DiSalvo, J., Bayne, M.L., Conn, G., Kwok, P.W., Trivedi, P.G., Soderman, D.D., Palisi, T.M., Sullivan, K.A. and Thomas, K.A. (1995) Purification and characterization of a naturally occurring vascular endothelial growth factor/placenta growth factor heterodimer. J. Biol. Chem., 270, 77177723.
Dvorak, H.F., Detmar, M., Claffey, K.P., Nagy, J.A., van de Water, L. and Senger, D.R. (1995) Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int. Arch. Allergy Immunol., 107, 233235.[ISI][Medline]
Folkman, J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med., 1, 2731.[ISI][Medline]
Halme, J. (1991) Role of peritoneal inflammation in endometriosis-associated infertility. Ann. N. Y. Acad. Sci., 622, 266274.[Abstract]
Hauser, S. and Weich, H.A. (1993) A heparin-binding form of placenta growth factor (PlGF-2) is expressed in human umbilical vein endothelial cells and in placenta. Growth Factors, 9, 259268.[ISI][Medline]
Hill, J.A., Faris, H.M., Schiff, I. and Anderson, D.J. (1988) Characterization of leukocyte subpopulations in the peritoneal fluid of women with endometriosis. Fertil. Steril., 50, 216222.[ISI][Medline]
Iwabe, T., Harada, T., Tsudo, T., Tanikawa, M., Onohara, Y. and Terakawa, N. (1998) Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil. Steril., 69, 924930.[CrossRef][ISI][Medline]
Kats, R., Metz, C.N. and Akoum, A. (2002) Macrophage migration inhibitory factor is markedly expressed in active and early-stage endometriotic lesions. J. Clin. Endocrinol. Metab., 87, 883889.
King, A., Burrows, T., Verma, S., Hiby, S. and Loke, Y.W. (1998) Human uterine lymphocytes. Hum. Reprod. Update, 4, 480485.
Li, X.F., Charnock-Jones, D.S., Zhang, E., Hiby, S., Malik, S., Day, K., Licence, D., Bowen, J.M., Gardner, L., King, A. et al. (2001) Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells. J. Clin. Endocrinol. Metab., 86, 18231834.
Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., Nagy, J.A., Hooper, A., Priller, J., De Klerck, B. et al. (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat. Med., 8, 831840.[ISI][Medline]
Maglione, D., Guerriero, V., Viglietto, G., Delli-Bovi, P. and Persico, M.G. (1991) Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc. Natl Acad. Sci. USA, 88, 92679271.[Abstract]
Maglione, D., Guerriero, V., Viglietto, G., Ferraro, M.G., Aprelikova, O., Alitalo, K., Del Vecchio, S., Lei, K.J., Chou, J.Y. and Persico, M.G. (1993) Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14. Oncogene, 8, 925931.[ISI][Medline]
Mahnke, J.L., Dawood, M.Y. and Huang, J.-C. (2000) Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil. Steril., 73, 166170.[CrossRef][ISI][Medline]
McLaren, J., Prentice, A., Charnock-Jones, D.S. and Smith, S.K. (1996) Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum. Reprod., 11, 220223.[Abstract]
Mueller, M.D., Mazzucchelli, L., Buri, C., Lebovic, D.I., Dreher, E. and Taylor, R.N. (2003) Epithelial neutrophil-activating peptide 78 concentrations are elevated in the peritoneal fluid of women with endometriosis. Fertil. Steril., 79, 815820.[CrossRef][ISI][Medline]
Nisolle, M., Casanas-Roux, F., Anaf, V., Mine, J.M. and Donnez, J. (1993) Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil. Steril., 59, 681684.[ISI][Medline]
Oura, H., Bertoncini, J., Velasco, P., Brown, L.F., Carmeliet, P. and Detmar, M. (2003) A critical role of placental growth factor in the induction of inflammation and edema formation. Blood, 101, 560567.
Ramey, J.W. and Archer, D.F. (1993) Peritoneal fluid: its relevance to the development of endometriosis. Fertil. Steril., 60, 114.[ISI][Medline]
Rock, J.A. and Markham, S.M. (1992) Pathogenesis of endometriosis. Lancet, 340, 12641271.[CrossRef][ISI][Medline]
Spuijbroek, M.D., Dunselman, G.A., Menheere, P.P. and Evers, J.L. (1992) Early endometriosis invades the extracellular matrix. Fertil. Steril., 58, 929933.[ISI][Medline]
Tjoa, M.L., van Vugt, J.M.G., Mulders, M.A.M., Schutgens, R.B.H., Oudejans, C.B.M. and van Wijk, I.J. (2001) Plasma placenta growth factor levels in midtrimester pregnancies. Obstet. Gynecol., 98, 600607.
Wiegerinck, M.A., Van Dop, P.A. and Brosens, I.A. (1993) The staging of peritoneal endometriosis by the type of active lesion in addition to the revised American Fertility Society classification. Fertil. Steril., 60, 461464.[ISI][Medline]
Submitted on May 13, 2003; resubmitted on June 30, 2003; accepted on August 14, 2003.