Preliminary Study On Relationship Between Macrophages In Mouse Uterus And Angiogenesis In Endometrium During Peri-implantation Period

BACKGROUNDAngiogenesis refers to the new capillaries from existing blood vessels in the generation and development process,which is the basic features of regeneration including specific steps:vascular endothelial cells migrating to the area after the the original vascular grassroots cracking, proliferation forming capillaries buds,growth and reconstruction of the formation of new artery or vein. The vast majority of angiogenesis in adult exists in pathological conditions (such as tumor formation and invasion, wound healing, inflammation and immune response), and the cyclically changed angiogenesis in the endometrium in the normal female reproductive system cyclical changes is the only physiological angiogenesis process in adult. This organ-specific physiological angiogenesis throughout the entire menstrual cycle is always critical to the growth and development of the female reproductive system and its function. But its occurrence and regulation mechanism is still not clear.Peri or implantation window period is a very short but critical period in normal endometrium.It lasts only from Day7to Day8in humans after ovulation(the mid-luteal period),allowing blastocyst implantation. This period is the critical step for pregnancy occurrence. Histological studies on endometrium have shown that local angiogenesis of endometrium is in peak condition around inplantation period, which is one of the most significant signs. This normal angiogenic microenvironment is the premise for embryo implantation, placentation and ongoing-pregnancy.In recent years, the status and the physiological role of macrophages in the intimal angiogenesis has begun to attract a growing number of scholars. Macrophages number in endometruim has a cyclical change.Macrophages migrate from the hematopoietic tissue and settle in uterus and mammals. Macrophages are found in humans and other mammals endometrial stromal and muscle connective tissue, and can demonstrate active migratory. They display appropriate changes on number and distribution in cycle of endometrium and during pregnancy. Activated macrophages release cytokines and nitric oxide and VEGF, regulate the cyclical changes in the endometrium, implantation, and pregnancy process. F4/80is the specific antigen on mouse mature macrophages, positioned in the cell membrane, cytoplasm. Macrophage colony-stimulating factor (M-CSF) is the most important Mcp chemoattractant substances. Decidual stromal cells synthesize and secrete large amounts of M-CSF under the stimulation of estradiol, progesterone and human chorionic gonadotropin. M-CSF raise a large number of Mφ in local site of endometrium and promote Mφ activation, functional differentiation and maturation. Robertson and other researchers have shown that in the proportion of endometrial Mφ surge sharply after fertilization (embryo preimplantation).Invasion of malignant tumors to a certain extent is similar with embryo adhesion and implantation. Peripheral circulating blood monocytes differentiate into tumor-associated macrophage cells (TAM) when they migrate to the tumor tissue microenvironment. TAM has become a hot issue in the study of tumor foundation for its status and target therapeutic in the tumor angiogenesis microenvironment. The TAM research shows bi-directional regulation for angiogenesis of NO may be mainly indirected through the effects of VEGF synthesis achieved. And decidual Mφ is one of the important sources of NO and VEGF.Nitric oxide (NO), an important messenger molecule and biologically active substance with extremely short half-life, is now known bidirectionalyy regulating angiogenesis and development of the most important cytokine. And M<p is the important synthesis, storage and release sources of endogenous NO. Generation of endogenous NO is catalyzed from L-arginine (L-ARG) and oxygen by nitric oxide synthase (NOS) and L-hydroxy-arginine, and then after further oxidated in stable in the final NO product. iNOS(macrophage NOS) is synthetized by activated macrophages induced by the immune or inflammatory stimuli.Interferon-gamma and LPS induce iNOS production of endogenous NO. Endogenous NO significantly inhibit the adhesion of leukocytes and endothelial cells. NOS inhibitors increase the expression of neutrophil adhesion molecules CD11/CD18and promote its adhesion to the vascular endothelial cells, thus promoting its vasculature. Implantation is considered as a kind of inflammatory response which may be associated with leukocyte. And decidual reaction and appropriate implantation is NO dependent.Endometrium angiogenesis involves various cytokines.The strongest pro-angiogenic cytokine is vascular endothelial growth factor (VEGF) family members. VEGF (also called VEGF-A) plays a key role to promote angiogenesis in different physiological and pathological states. VEGF is a pro-angiogenic endothelial cells mitosis original regulation and vascular permeability factor.The TAM research shows NO-bidirectional regulation of angiogenesis may be mainly indirected through the effects of VEGF synthesis to achieve, that is to say NO and VEGF compose a functional protein complex regulating angiogenesis. NO plays as upstream regulator of VEGF synthesis, and that is adjusted with local NO concentration gradient, the partial pressure of oxygen, NO donor and other factors. They are closely related. A small amount of NO may raise the VEGF gene the expression through wire mitogen protein activated kinase (MAPK) or p38kinase pathway which there may be hypoxia-inducible protein (HIF)-1and heme oxidase (HO)-1participation in hypoxic environment. But the pathway that a large number of NO inhibit of the VEGF gene expression is not yet clear. In addition, scholars have found that the trophoblast cells can be detected of iNOS. Embryo implantation is therefore another important source of NO. According to the findings of TAM in tumor angiogenesis microenvironment, we initially speculate that the synthesis and release of NO-VEGF functional protein complex under certain conditions may be the core that macrophages dynamicly adjust Peri-angiogenic microenvironment in bidirection.So far, there are few comprehensive researchs to dynamicly observer relationship between macrophages, iNOS and VEGF on angiogenesis in endometrium. Subjected to ethics factors and technical limitations, endometrium angiogenesis research in human beings in peri-implantation period is difficult. Mouse embryo implantation type is the surface implantation mode that is consistent with human. Therefore mouse is an excellent model to study human implantation. It is generally believed that4.5days after mice mating is the starting time of implantation. Endometrium angiogenesis process is divided into two stages by trophoblast adhesion to the endometrial epithelium (D4.5) to study angiogenesis-related factors. The former one is called preimplantation stage (D2.5-4.5), and the later one is implantation (D4.5-6.5) stage. The main difference between the two stages in endocrine environment is that the estradiol and progesterone overwhelms in the former, while gonadotropic of hormones advantage due to the trophoblast cells invading and functional maturation in the latter stage. So we applied peri-implantation D1.5-D6.5mice to observe F4/80+macrophages, iNOS and VEGF expression in the changes of number as well as distribution, to make comparison between implantation sites and non-implantation sites about the expression of macrophages, iNOS and VEGF as well as distribution differences.Further research about endometrium angiogenesis regulation mechanism of the micro-environment help to understand the connotation of endometrial receptivity, and provide more adequate evidence to explore new targets for intervention endometrial receptivity in order to solve the implantation dysfunction (repeated in vitro fertilizationembryo transfer and habitual abortion), unexplained infertility pro-angiogenic therapy experimental basis.And it also provides new ideas for the abnormal uterine bleeding, immune contraception and organ transplantation research.OBJECTIVEThe central issue in all these studies is to study macrophages changes before and after embryo implantation in the uterine tissue, as well as iNOS and VEGF expression levels. And Macrophages in peripheral blood was observed. The relationship between macrophages, iNOS and VEGF expression was analysed to explore the mechanism of macrophage in endometrium angiogenesis for embryo implantation.METHODSMatedals divided into three groups:pregnancy group, pseudopregnancy group (the male mice was ligated the vasdeferens) and Estrous group (without mating).Using HE staining and microscopy to observe the uterine morphology and structure.Detection the macrophages, iNOS and VEGF by the methods of flow cytometry and immunohistochemistry in the blood and uterus to study their change and distribution during early pregnancy.RESULTS 1. The results of morphology and HE staining of mouse’uterus showed that the pregnancy and pseudopregnancy model was successful.2. The date of FCM found that the number of peripheral blood macrophages in pregnancy group was significantly higher than in other groups.(P<0.05). In pregnancy group, the number of macrophages maintain high level after implantation, and macrophages ratio was down and then up. It began to decline to reach a minimum value (19.64±9.45%) on D2.5. And then it increased again and maintained at a high level state (D6.5:29.24±16.9%).3. The result of immunohischemistry showed that the positive macrophage not only change the amount but also the distribution. In pregnancy group, most macrophages lied in endometrium and myometrium. The number of macrophage was significantly reduced (P<0.05) and gradually transferred to perimetrium on D4.5. In pregnancy group, the number of macrophages changed over time, reached the highest value (32.15±4.86) on D1.5number of macrophages, fell dramatically (14.54±4.00) on D2.5, On D4.5after implantation, macrophage cells decreased to a low level (D6.5:4.06±4.15). Expression of iNOS in pregnancy group (25785.51±21708.47) and pseudo-pregnant group (14336.41±6442.72) was higher than the blank group (993.23±700.97) in the mouse uterus. The difference on D1.5～D4.5was statistically significant (P=0.001, P=0.004, P=0.042, P=0.041); The expression of iNOS after embryo reached high value (D5.5:56823.88±86747.64; D6.5:48916.69±75108.58), with high expression (D5.5:76618.41±86747.64; D6.5:79758.47±99641.83) in implantation site. Pregnancy group (36779.85±45806.05) and pseudo-pregnant group (7242.97±6540.59) mouse uterus expressed higher levels of VEGF than blank group (579.84±596.00). And the difference was statistically significant (P=0.012, P=0.012, P=0.007, P=0.024) on D1.5, D3.5, D4.5, D6.5; VEGF in the pregnancy endometrium on D4.5(55572.24±60725.29) and D5.5(64515.03±11082.60) was highly expressed and gathering expressed in the implantation point.4. Endometrial macrophages stayed away from the implantation point. iNOS and VEGF gathering expressed in embryo implantation site, but their expression in the implantation site(iNOS:76618.41±114,986.9, VEGF:94692.84±147724.9) and non-implantation site(iNOS:37029.35±52249.88, VEGF:34337.23±59464.09) has no significant difference (P>0.05).5. Peritoneal macrophage proportion and uterine macrophage number was negatively related (r=-0.496, P=0.005). Macrophages number and iNOS (r=0.546, P=0.002) as well as VEGF (r=0.498, P=0.000) expression in endometrium were positively correlated. The expression of iNOS and VEGF in endometrium also displayed a positive correlation (r=0.779, P=0.000), and the correlation is very strong.CONCLUSIONS1. Peripheral macrophages increased the ratio of decrease in macrophages in utero when embryo implants, suggesting Peri macrophages may migrate into the peripheral blood and uterine and adapt to pregnancy.2. The uterine macrophages distributed away from the implantation sites reducing immune defenses to embryo.3. The expression of iNOS, VEGF expression levels in the implantation window expression increased, and gathered in embryo implantation, suggesting that they may play an important role in promoting embryo implantation.4. The number of macrophages in the uterus and uterine iNOS and VEGF are positively correlated, suggesting that macrophages might be a downstream signaling molecules and regulate of iNOS and VEGF during implantation process. It’s sensible to speculate that the synthesis and release of NO-VEGF functional protein complex under certain conditions may be the core that macrophages dynamicly regulate Peri-angiogenic microenvironment in bidirection.