Angiogenetic Study And Anti-human VEGF Antibody As A New Target Therapy On Nude Mice Model On Endometriosis

Endometriosis is a common gynecologic syndrome of unknown etiology and pathogenesis. It is well known that endometriosis is often associated with infertility, dysmenorrhea and pelvic pain, even it can make urethrostenosis, and characterized by the presence of ectopic endometrial implants. It affects about 1 in 10 women of reproductive age with developing numbers in these years. It has some malignant character such as invasive and plant to the ectopic part out of the uterine endometrium, transform to the remote part of the body, recurrence after operation or drug use. About 1% endometriotic disease will develop to special type ovary cancer. It is not satisfied in treatment on drugs because low effect and recurrence when stop drug administration and low sex hormone level caused by GnRHa.It is well accepted that endometrium, shed at the time of menstration, passes backward along the fallopian tubes and into the abdominal cavity. Here the explants establish a blood supply and grow. Although most women have retrograde menstruation, only 10% of women develop endometriosis. Adhesion, implant and angiogenesis are the three key steps in endometriosis.The development of new blood vessels is a closely regulated process that involves the coordinated interaction among many gene families. The VEGF family is particularly important. VEGF-A, acting through the kinase domain receptor (VEGFR2), induces differentiation, proliferation, and migration of endothelial cells. But if VEGF-A support is withdrawn, endothelial cells undergo apoptosis, and the vessels regress. However, endothelial cells are only responsive to VEGF-A and its withdrawn if they are not surrounded by pericytes. Pericytesare smooth muscle α -actin (α -SMA)-containing, mesenchymal cells that areattracted to immature blood vessels by platelet-derived growth factor synthesized by endothelial cells. Pericytes have common basement membrane with endothelial cells and have complex interaction with endothelial cells. New studyon pericytes show that it can increase levels of transforming growth factor β ,then stimulate VEGFR1 expression on endothelial cells. Meanwhile, pericyte-derived angiopoetin-1 prevents endothelial cell apoptosis and maintains vessel integrity. When endothelial cells are surrounded by pericytes, they become resistance to the withdrawal of VEGF-A.In the view of the importance of blood vessels to the establishment and subsequent growth of endometriotic lesions, we sought to test the hypothesis that antiangiogenetic agents may be effective in preventing ectopic endometrial growth. We choose specifically VEGF-A antibody because of the elevated levels of VEGF-A in peritoneal fluid and ectopic endometrium of women with endometriosis and its importance in other angoigenesis-dependent conditions. Antihuman VEGF-A antibody can easily combine with VEGF-A on the endometrial cells without pericyte, such competitive inhibit its combination to VEGFR2, then inhibit the action of VEGF, lead to apoptosis and regress in immature micro vessels, thus microvessels in eutopic endometrium become regular as normal. So anti-human VEGF antibody will take its effect in early phase of the disease or decrease the recurrence after operation or drug stopping.PARTI Clinical and Laboratory Study on Angiogenesis of EndometriosisOBJECTIVE: To investigate the angiogenetic character between endometriosis on ovary and myometrium (Adenomyosis) which occurred simultaneously in one woman by immunohistochemistry method. Compare VEGF expression and MVD in different site of ectopic lesions and patterns endothelial cells surrounded with pericyte will lead to a clear clue on angiogenesis of endometriosis. Thus offer a theoretic basement on the new treatment of anti-angiogenetic agent on endometriosis.METHOD: Endometriosis women with ectopic lesions on both ovary and myometrium (Adenomyosis) were chosen to our study. Ectopic endometruim and eutopic endometrium tissues were obtained from Hysterectomy or endometrial suction curettage ahead of the operation. To insure both endometrial gland and stroma are all in the ectopic lesions in ovary endometriosis, we invited one sophisticated pathologic doctor to read the HE sections to delete unaproppriate cases. Meanwhile, we also avoid of uterine myoma and history of steroid drug use in the 3 months before the operation. 39 cases were selected. Twenty were in the proliferative phase, the others were in secretory phase. 31 cases of normal endometrium were obtained as control group, eighteen were in proliferative phase, the others were in the secretory phase. The expression ofVEGF-A was measured by immunohistochemistry method. CD34 and α -SMAwere double stained in one section which can locate endothelial cells and pericytes simultaneously. Endothelial cytoplasm stained pink-red while pericytestained purple.RESULT: (1) VEGF level is significantly higher both in the ectopic site in ovary and uterine myometrium than normal control group. Positive rate in ectopic epithlial gland in adenomyosis and ovary endometriosis is 34.21%, 50%, respectively. While in eutopic and normal endometrium, positive rate of VEGF expression is 44.74% and 16.13%. (2) Eutopic endometrium has abnormal increase in VEGF expression than normal control group. (3) Ovary endometriosis and adenomyosis have different character in VEGF expression and microvessel density. Ovary endometriosis has a sustaining high level of VEGF expression in all period of menstruation with numerous macrophages exhibited highest frequency of diffuse immunoreactivity in stroma and has a little high level of MVD than normal. MVD in Adenomyosis is significantly higher than Ovary endometriosis. VEGF expression in adenomyosis is only increase in secretory phase. (4) Microvessel pericytes coverage index (MPI) is decreased in ectopic endometrium, especially in adenomyosis. The superficial eutopic endometrial MPI is significantly decreased in proliferative phase. These indicate that interaction between endothelial cells and pericyte changed to abnormal state in endometriotic lesion and their eutopic origin. Thus make the use of anti-angiogenetic agent as new targets in endometriosis treatment become reasonable.CONCLUSION: Angiogenesis take very important role in endometriotic pathogenesis. Eutopic endometrium of endometriosis has some abnormal angiogenetic activity, this may be the decisive factor in ectopic lesion formation. Endometriosis in ovary and myometrium may have common origin, but they have different phenotype in VEGF expression and microvessel density and pericyte diffusion. This may be the result of different environmental effect.PART 2Establish nude mice model on endometriosis and theraputic effect study on anti-human VEGF antibody in vivoOBJECTIVE: (1) To establish an experimental endometriosis nude mice model using eutopic endometrium from endometriosis woman. Compare the effect by different tissue culture condition with progesterone or not before implantation in order to probe a more appropriate manner in this model. (2) To evaluate the inhibition effect on the ectopic lesion on this nude mice model of endometriosis using purified affinity anti-human VEGF antibody.METHOD: (1) 13 nude mice were divided into two groups implanting 8-10 pieces of eutopic endometrial debris obtained from emdometriosis woman which cultured with estrogen or estrogen plus progesterone before implanting, respectively. After 7 days from implanting, the mice were sacrified. Numbers and maxim diameter of ectopic lesion were compared in this two group. Then all...