Marek Zygmunt

Angiogenesis and vasculogenesis in pregnancy

An adequate nutrient and substrate supply is essential for normal intrauterine development of the fetus. Disturbances in uterine blood supply are associated with higher perinatal morbidity and mortality caused by preterm delivery, pre-eclampsia or intrauterine growth restriction. Adaptation of the uterine vasculature to the rising needs of the fetus occurs through both vasodilation and development of new vessels. Angiogenesis is the process of neovascularization from pre-existing blood vessels in response to hypoxia or substrate demands of tissues. The endometrium, decidua and placenta are sources rich of angiogenic growth factors. In general, the angiogenic process is initiated by growth factors such as bFGF, VEGF, or placental growth factor (PlGF). Through a complex signal transduction machinery mediated by respective receptor-tyrosine kinases, an increase in the permeability of the maternal vessels is achieved to permit growth and invasion of endothelial cells. Their chemotactic migration, formation of a vessel lumen, and functional maturation of new capillaries complete the angiogenic process that involves the expression of specific adhesion receptors and extracellular matrix-degrading proteases. During vasculogenesis, endothelial progenitor cells--angioblasts--form a primitive vascular network. This process occurs mainly during fetal development, although recruitment of angioblasts from bone marrow and peripheral blood in response to ischemic insult have been described in adults. Our recent data indicate a novel function for human chorionic gonadotropin (hCG), a hormonal factor of trophoblastic origin in uterine adaptation to early pregnancy as well as in tumor invasion and underline the importance of hCG as an yet unrecognized angiogenic factor. Although there are striking similarities between, on the one hand, tumor invasion and tumor-induced vascularization and, on the other hand, trophoblast invasion and placental development, our understanding of the different molecular and functional aspects of these two different processes, in particular, the self-limitation of the trophoblastic invasion and vessels formation during gestation might allow the establishment of new therapeutic strategies for the treatment of both tumor and pregnancy related pathology.

Uterine blood flow--a determinant of fetal growth.

An adequate increase of uterine blood flow throughout gestation is essential for uterine, placental and fetal growth. Maternal cardiovascular adaptation has to provide the uterine perfusion that is necessary to meet the requirements of the developing and growing fetus by providing transport of nutrients and oxygen to the placenta and the fetus. Thus, uterine blood flow is inextricably linked to fetal growth and survival. Reductions of uterine blood flow can occur under acute or chronic conditions or in a combination of both. Chronic reductions of uterine blood flow can be observed in pregnancy-induced hypertension (PIH), diabetes mellitus in pregnancy and intrauterine growth restriction (IUGR). Chronic restrictions in uterine blood flow will elicit a placental and fetal response in the form of growth adaptation to the reduced supply of oxygen and nutrients to the conceptus. If compensatory growth restriction reaches its limits intrauterine fetal distress can ensue. Among the great number of experimental models of intrauterine growth restriction, those involving a generalized reduction in the uteroplacental blood supply are of significance to questions relating to human pregnancy. Despite physiological differences, particularly with regard to maternal metabolism and placentation, the occlusion model in the pregnant sheep is suitable for investigating questions about fetal and placental growth.

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro.

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-microm pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of alpha5 and alpha6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl cAMP (db cAMP) and the cAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell UEG-3) invasion. The collagenolytic activity of trophoblastic cells (EG-3) was increased by hCG stimulation. No changes were shown in the expression of alpha5 and alpha6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.

Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood

A significant number of hematopoietic stem/progenitor cells (HSPC) in human umbilical cord blood could serve as a reservoir for the placental vasculature, yet, their morphological and functional features are not completely understood. Here, we describe the characterization of purified CD133(+) progenitor cells from umbilical cord blood, a subset of CD34(+) hematopoietic progenitors that were grown in proliferation medium containing Flt3-ligand, thrombopoietin and stem cell factor. Following isolation and enrichment of the CD133(+) cells by immunomagnetic cell sorting, they remained non-adherent for up to 40 days in culture and expressed different pluripotency markers including Sox-1, Sox-2, FGF-4, Rex-1 and Oct-4.Oct-4 expression was confirmed by laser-assisted single cell picking with subsequent quantitative real-time RT-PCR. The expression of Oct-4 indicates a pluripotent phenotype of CD133(+) cells and appears to be of functional relevance: After three weeks in endothelial differentiation medium, suspended cells became adherent, developed an endothelial cell-like morphology, bound fluoresceine isothiocyanate-labeled Ulex europaeus agglutinin-1, took up acetylated Di-LDL, and expressed other endothelial markers such as PECAM-1 or VEGFR-2. Concomitantly, Oct-4 expression was significantly reduced. Moreover, following treatment with retinoic acid, CD133(+) cells exhibited neural morphology associated with the expression of beta-III-tubulin. CD133(+) cells were found to express the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, detected by RT-PCR and immunocytochemistry. The recombinant human chorionic gonadotropin induced proliferation of the CD133(+) cells in a dose-specific manner. Our results indicate that CD133(+) HSPC from umbilical cord blood may have a greater differentiation potential than previously recognized and give rise to proliferative endothelial cells participating in placental vasculogenesis.

The gonadotropins: Tissue-specific angiogenic factors?

The gonadotropins, whose members are human chorionic gonadotropin (hCG), lutenizing hormone (LH) and follicle-stimulating hormone (FSH) are a well characterized hormone family known to regulate reproductive functions in both females and males. Recent studies indicate that they can modulate the vascular system of reproductive organs. It was shown that gonadotropins not only influence the expression of vascular endothelial growth factor (VEGF) and both its receptors VEGFR-1 and -2, but also modulate other ubiquitously expressed angiogenic factors like the angiopoietins and their receptor Tie-2, basic fibroblast growth factor or placental-derived growth factor. Some recent data indicates a possible direct action of gonadotropins on endothelial cells. Thus, the gonadotropins act as tissue-specific angiogenic factors providing an optimal vascular supply during the menstrual cycle and early pregnancy in the female reproductive tract as well as in testis. In pathological conditions (e.g. preeclampsia, intrauterine growth restriction, ovarian hyperstimulation or endometriosis), these tightly regulated interactions between the gonadotropins and the ubiquitous angiogenic factors appear to be disturbed. The intent of this short manuscript is to review the current knowledge of the regulatory role of the gonadotropins in vasculo- and angiogenesis. We also review angiogenic actions of thyroid-stimulating hormone (TSH), a glycoprotein closely related to gonadotropins, which display strong gonodal actions.

Centralizing surgery for gynecologic oncology—A strategy assuring better quality treatment?

OBJECTIVEnThe objective of this study was to assess the association between the type of hospital and the previously reported shortcomings in surgical treatment for ovarian and endometrial carcinomas in Hesse, Germany.nnnMETHODSnThe types of hospitals)primary, secondary, tertiary and central care referral or university clinic) at which patients with endometrial and ovarian cancer were treated were correlates with the following variables: patients functional status, tumor stage (FIGO), the performance of lymphadenectomy and/or omentectomy, and the frequency of intraoperative and postoperative complications. Data came from the GQH project, which assessed all diagnostic, surgical, and postoperative gynecologic procedures undertaken in Hesse between 1997 and 2001.nnnRESULTSnIn 1119 cases of endometrial cancer significantly fewer (P < 0.001) lymphadenectomies were performed in primary care hospitals despite the fact that patients treated in primary care hospitals were younger and had a better functional status and lower tumor stage than patients treated in other types of hospitals. In ovarian cancer too, lymphadenectomy rates varied considerably with the type of hospital (P = 0.010) even when the analyses were restricted to patients whose functional status was good (ASA <III) and whose tumor stage was low (FIGO stage <III). However, the analyses still revealed striking shortcomings, even at tertiary care hospitals and central referral hospitals and university clinics where the lymphadenectomy rate ranged around 60%.nnnCONCLUSIONnThe type of hospital is an important factor in the quality of surgical treatment for endometrial and ovarian cancer. Restricting treatment to experienced specialist surgeons or hospitals offering high treatment standards seems necessary if treatment outcomes are to improve.

Invasion of Cytotrophoblastic (JEG‐3) Cells Is Up‐Regulated by Interleukin‐15 In Vitro

PROBLEM: Trophoblast invasion into the uterus is controlled by many factors. Some cytokines (interleukin [IL]‐1, IL‐6, and IL‐10) have been shown previously to play an important role in placentation. The human placenta is an important source of IL‐15, although the cellular source of IL‐15 in the placenta has not yet been specified. IL‐15 influences cell adhesion and migration by redistributing adhesion molecules in lymphocytes and has been shown to have effects on endothelial cells and in some human tumors.

Self-concept, body image, and use of unconventional therapies in patients with gynaecological malignancies in the state of complete remission and recurrence

OBJECTIVEnAre there differences in self-concept and body image in patients with cancer recurrence in comparison to patients with complete remission? What impact has cancer recurrence on use, users and non-users of unconventional cancer therapies?nnnPATIENTS AND METHODSnOne hundred and nine patients with no evidence of disease after gynaecological cancer and sixty-one patients with recurrent disease were analysed for self-concept with the Frankfurter Selbstkonzeptskalen and body image with the Frankfurter Körperkonzeptskalen. Use and motivation for unconventional therapies was assessed with a questionnaire.nnnRESULTSnWith respect to frequency of use and expected benefits of unconventional therapies no differences were observed between the groups. However, cancer recurrence was found to induce considerable changes of self-concept and body image, some indicating even positive changes due to cancer recurrence.nnnCONCLUSIONnIt may be beneficial to consider body therapy and psychotherapy as a mean to improve body image and self-esteem in cases with cancer recurrence.

Term breech and long-term morbidity — cesarean section versus vaginal breech delivery

STUDYnPerinatal morbidity and mortality of term fetuses have been discussed extensively both for vaginal breech delivery and cesarean section. However, information regarding long-term morbidity and psychomotoric development of these children are scarce.nnnDESIGNnData of 154 children delivered after breech presentation at our institution between 1988 and 1994 were analyzed using a specific, standardized questionnaire (Enzephalopathiefragebogen, Meyer-Probst) with emphasis on psychomotoric development and skills. Hyperkinetic disorders, social adaptation, emotional instability, and intelligence were evaluated as subcategories and compared to perinatal data.nnnRESULTSnpO(2) and base excess (BE) in the umbilical artery were lower in the vaginal group. pH, body weight and placental weight showed no difference between groups. Psychomotoric development and skills did not differ between children delivered vaginally or abdominally. Perinatal variables did not allow a prediction of long-term morbidity.nnnCONCLUSIONnRoute of delivery has negligible influence on the measured values in the umbilical artery and no influence on long-term morbidity of fetuses presenting breech.

Quantitative 3D micro-CT imaging of the human feto-placental vasculature in intrauterine growth restriction.

OBJECTIVEnPlacental vascular development matches fetal growth and development. Quantification of the feto-placental vasculature in placentas from pregnancies is complicated by intrauterine growth restriction (IUGR) revealed confounding results. Therefore, the feto-placental vascular volume in IUGR placentas was assessed by 3D micro-computed tomography (micro-CT).nnnMETHODS AND RESULTSnPlacental probes from IUGR (n=24) and healthy control placentas (n=40) were perfused in situ with Microfil or BaSO(4) and randomly chosen samples were scanned by micro-CT. Using 3D images, we quantitated the feto-placental vascular volume fraction (VVF). A subanalysis was performed at three different levels, reaching from the chorionic plate artery (level A), to intermediate arteries (level B) and capillary system (level C). Results were complemented by histology. The significance of differences in vascular volume measurements was tested with analysis of variance [ANOVA].nnnRESULTSnMicrofil perfused placentas showed a total vascular volume fraction of 20.5+/-0.9% in healthy controls. In contrast, the VVF decreased to 7.9+/-0.9% (p<0.001) in IUGR placentas. Significant differences were found between Microfil and BaSO(4) perfused placentas in the vascular volume fraction using micro-CT and histology. Micro-CT demonstrated localized concentric luminal encroachments in the intermediate arteries in placentas complicated by IUGR.nnnCONCLUSIONnMicro-CT imaging is feasible for quantitative analysis of the feto-placental vascular tree in healthy controls and pregnancies complicated by IUGR.