Andrea Schanz

Nitric Oxide Synthase Expression and Functional Response to Nitric Oxide Are Both Important Modulators of Circulating Angiogenic Cell Response to Angiogenic Stimuli

Objective—Circulating angiogenic cells (CACs), also termed endothelial progenitor cells, play an integral role in vascular repair and are functionally impaired in coronary artery disease (CAD). The role of nitric oxide (NO) in CAC function is poorly understood. We hypothesized that CAC migration toward angiogenic signals is modulated by both NO synthase (NOS) expression and functional response to NO. Methods and Results—Similar to endothelial cells, CAC chemotaxis to vascular endothelial growth factor (VEGF) was blocked by inhibition of NOS, phosphatidylinositol 3-kinase, or guanylyl cyclase or by treatment with an NO scavenger. Addition of an NO donor (S-nitroso-N-acetylpenicillamine) and the NOS substrate l-arginine increased random cell migration (chemokinesis) and enhanced VEGF-dependent chemotaxis. Healthy CACs expressed endothelial NOS, but endothelial NOS was not detected in CAD patient CACs. Both chemokinesis and chemotaxis to VEGF of patient CACs were decreased compared with healthy CACs but were restored to healthy values by S-nitroso-N-acetylpenicillamine. In parallel, CAD patients exhibited lower flow-mediated vasodilation and plasma NO source nitrite than young, healthy subjects, indicating endothelial dysfunction with reduced NO bioavailability. Conclusion—NOS activity is required for CAC chemotaxis. In CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity.

Habitual smoking causes an abnormality in platelet thromboxane A 2 metabolism and results in an altered susceptibility to aspirin effects

The present study investigates the effects of aspirin (100 mg every second day for 14 days) on platelet function in nine healthy non-smokers and in nine healthy habitual smokers. There was a significantly (P < 0.05) stronger inhibition of collagen (0.6 microgram/ml)- and ADP (2 microM)-induced platelet aggregation by aspirin in smokers as compared to non-smokers. This difference occurred in the presence of an almost complete (> 95%) inhibition of thromboxane A2 (TXA2) synthesis in both groups. The platelet capacity to generate TXA2 in vitro was significantly reduced in smokers, urinary excretion of TXA2, however, was significantly increased. Thus, the better susceptibility of smokers to anti-aggregatory effects of aspirin is very likely to be related to a chronic smoking-induced alteration of platelet TXA2 system. Cessation of smoking should, therefore, be encouraged.The present study investigates the effects of aspirin (100 mg every second day for 14 days) on platelet function in nine healthy non-smokers and in nine healthy habitual smokers. There was a significantly (P<0.05) stronger inhibition of collagen (0.6 mu g/ml)- and ADP (2 mu M)-induced platelet aggregation by aspirin in smokers as compared to non-smokers. This difference occurred in the presence of an almost complete (>95%) inhibition of thromboxane A 2 (TXA 2 ) synthesis in both groups. The platelet capacity to generate TXA 2 in vitro was significantly reduced in smokers, urinary excretion of TXA 2 , however, was significantly increased. Thus, the better susceptibility of smokers to anti-aggregatory effects of aspirin is very likely to be related to a chronic smoking-induced alteration of platelet TXA 2 system. Cessation of smoking should, therefore, be encouraged.

CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts

The placenta forms the interface between the mother and the fetus. During placental development cytotrophoblasts differentiate to form the syncytium or to invade the decidual wall to breach maternal vessels and establish the blood flow in the intervillous space. This process is still not well understood but it is proposed that chemokines and their receptors are involved in guiding cytotrophoblasts to the decidua and maternal vessels as well as attracting immunocompetent cells to the implantation site. CXCL12 is a chemokine expressed by cytotrophoblasts and is involved in cytotrophoblast invasion, differentiation and survival. One of its receptors, CXCR4, has been detected on cytotrophoblasts. Recent data show that CXCR7 and syndecan-4 might partially mediate CXCL12 function in other cell types. In this study, we examined CXCR7 and syndecan-4 expression at the maternal-fetal interface via immmunolocalization in placental tissue sections and in isolated cytotrophoblasts. We further used immunoblot analyses to confirm the data. We were able to show that cytotrophoblasts express both receptors and that upregulation occurs during the differentiation process of cytotrophoblasts towards the invasive phenotype. On a functional level CXCR7 seems not to be involved in JAR cell chemotaxis, suggesting a different function of this receptor. In conclusion, we propose that CXCL12 binds to CXCR4, but also to CXCR7 and syndecan-4. These three receptors could mediate different functions of CXCL12, such as cell migration, directed invasion, proliferation and survival. The latter molecules might also be involved in the development of placental pathologies, such as preeclampsia or choriocarcinoma growth.

Expression of the vascular endothelial growth factor receptor neuropilin-1 at the human embryo–maternal interface

OBJECTIVE Angiogenesis is required for successful implantation of the invading blastocyst. Vascular endothelial growth factor (VEGF) is an important key player in angiogenesis and vascular remodeling during the implantation process. Besides its well-characterized receptors VEGFR1 and VEGFR2, neuropilin-1 (NRP-1) has been shown to play an additional role in the signaling process of angiogenesis in human endometrium during the menstrual cycle, as a co-receptor of VEGF. These findings led to the hypothesis that NRP-1 might play a role in the vascular remodeling process during embryo implantation and the establishment of a pregnancy. STUDY DESIGN NRP-1 mRNA transcript and protein expression were investigated in human choriocarcinoma cell lines (JEG-3, Jar and BeWo) aiming to evaluate the expression of NRP-1 in vitro, as well as in human decidua of all three trimesters of pregnancy, by western blot analysis (three samples of each trimester of pregnancy). The localization of NRP-1 in human decidua of all three trimesters of pregnancy was analyzed by immunohistochemistry (five samples of each trimester of pregnancy). RESULTS NRP-1 transcript and protein were expressed in all cell lines examined. Corresponding to the analysis of human tissue by western blot and the localization by immunohistochemistry, NRP-1 protein higher expressed in samples of early pregnancy in comparison to the end of pregnancy. NRP-1 was expressed in the decidua, villi and invading cytotrophoblast of all samples investigated. CONCLUSIONS This is the first study clearly showing the expression of NRP-1 in human decidua and trophoblast, suggesting an important role for the VEGF co-receptor NRP-1 besides the established receptor VEGFR2 at the embryo-maternal interface during embryonic implantation and placentation.

Expression of the vascular endothelial growth factor receptor neuropilin-1 in the human endometrium

Angiogenesis is a key process in the endometrium which undergoes dramatic changes during the menstrual cycle. Molecules such as vascular endothelial growth factor (VEGF), acting via two tyrosine kinase family receptors (VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1]), are potent modulators of angiogenesis and vascular remodelling in the endometrium. Recently, neuropilin-1 (NRP-1) was shown to be expressed in endothelial cells binding VEGF(165) and therewith enhancing the binding of VEGF(165) to VEGFR2. This suggests that NRP-1, in addition to the known VEGF receptors, may play an important role in VEGF-induced angiogenesis. In this study, the expression of NRP-1 in the cycling human endometrium has been investigated by reverse transcription (RT)-polymerase chain reaction (RT-PCR), semi-quantitative competitive RT-PCR (RT-cPCR) and immunohistochemical staining. NRP-1 was expressed in all 32 endometrium samples throughout the menstrual cycle. However, samples from the proliferative phase showed significantly higher expression levels of NRP-1 mRNA compared to samples from the secretory phase (t/c-ratio 2.13 vs. 0.84, p<0.05). Immunohistochemistry confirmed the results showing increased NRP-1 staining in vascular endothelium, glandular epithelium and stromal cells of the proliferative phase endometrium. This study demonstrates mRNA and protein expression of NRP-1 in human endometrium samples throughout the menstrual cycle. The enhanced expression of NRP-1 in the proliferative phase suggests that it may participate in hormonally regulated changes of endometrial angiogenesis, preparing the endometrium for the implantation of an embryo. NRP-1 expression might act as a co-factor for VEGF(165) enhancing the angiogenic stimulus.

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation.

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.

Genital schistosomiasis as a cause of female sterility and acute abdomen.

OBJECTIVE To report the case of a Nigerian patient who suffered from sterility and underwent abdominal laparotomy to remove an adnexal tumor. Histologic analysis revealed Schistosoma haematobium ova. DESIGN Case report. SETTING Multidisciplinary group practice and teaching hospital. PATIENTS One patient who underwent an abdominal laparotomy to remove an adnexal tumor. INTERVENTION(S) In vitro fertilization and adnexal tumor resection via laparotomy. MAIN OUTCOME MEASURE(S) Treatment of the adnexal tumor affecting the reproductive health. RESULT(S) Histologic analysis performed on the tissue removed at surgery revealed fibrous patches around aggregates of calcified Schistosoma haematobium eggs in the fallopian tube wall. CONCLUSION(S) Schistosomiasis needs to be considered as a differential diagnosis of female infertility and sterility.

Interleukin-1 system in the human fallopian tube-No spatial but a temporal regulation of mRNA and protein expression.

The human fallopian tube provides the environment for the first 5 days of embryonic development in vivo. The IL-1 system is involved in human embryo implantation. This study aimed to investigate IL-1beta, IL-1ra and IL-1R tI expression within the length of the human fallopian tube on mRNA- and protein-level in samples from proliferative versus secretory phase, postmenopause (PMP) samples and samples from intra- (IUP) and extrauterine pregnancies (EUP) to examine possible spatial and hormonal induced changes (fimbrial, ampullary and isthmic tube segments). On mRNA-level, IL-1beta was expressed in all samples except in PMP. IL-1R tI could be detected in all samples whereas IL-1ra was only expressed in secretory phase and the IUP sample. Immunohistochemically we could detect IL-1beta and IL-1R t1 protein in all proliferative and secretory phase samples with maximum intensity in secretory phase samples whereas IL-1ra was expressed in secretory phase samples only. Overall no spatial but temporal differences possibly due to hormonal changes could be observed suggesting a precise regulation of the IL-1 system, especially for IL-1ra and moreover a stable molecular architecture within the full length of the fallopian tube.

Molekulare Mechanismen der Embryoimplantation im Endometrium

ZusammenfassungDie menschliche Reproduktion und die Implantation des Embryos sind ein komplexer und dadurch störungsanfälliger Prozess. Ein genauer Zeitplan bestimmt die Synchronisation der Blastozystenreifung und die endometriale Vorbereitung auf die Einnistung. Eine Vertiefung unseres Wissens über die molekularen Abläufe während der physiologischen Präimplantationsentwicklung und Implantation sollte zu einer Verbesserung der In-vitro-Kulturbedingungen und damit ultimativ zu einer Steigerung der Erfolgsraten der In-vitro-Fertilisationsbehandlung führen. In dem vorliegenden Beitrag werden molekulare Mechanismen, wie hormonelle Faktoren, Zytokin- und Wachstumsfaktorensysteme, die Bedeutung von Metalloproteinasen und Adhäsionsmolekülen kurz beschrieben und in den Kontext aktueller Forschung gestellt. Die weitere Erforschung dieser Zusammenhänge wird verantwortlich dafür sein, dass in Zukunft ein besseres klinisches sowie therapeutisches Management im Hinblick auf Sterilität, Infertilität, jedoch auch Kontrazeption entwickelt werden kann.AbstractHuman Reproduction is a very complex and surprisingly inefficient process. The embryo development and the generation of a receptive endometrium needs an exact timing and synchronization. Understanding the factors involved in preimplantation embryo development and embryo-maternal interaction and implantation is crucial for improving success rates in reproductive medicine. The purpose of the present review is to describe briefly the function of adhesion- and hormonal factors, cytokines, growth factors and metalloproteinases. A better understanding of the molecular mechanisms of implantation will be responsible for the improvement of clinical and therapeutical management of sterility, infertility and contraception.

hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation.