Regulation of soluble vascular endothelial growth factor receptor 1 secretion from human endothelial cells by tissue inhibitor of metalloproteinase 1.

Vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-1) are key regulators in human ovarian angiogenesis. Produced by granulosa and ovarian theca interna cells, VEGF promotes blood vessel growth during follicular development and corpus luteum formation, whereas sVEGFR-1, which is secreted by endothelial cells, functions as an antagonist to VEGF activity by binding it. In order to gain further insights into the regulatory mechanisms of ovarian angiogenesis, the aim of the present study was to analyze the influence of tissue inhibitor of metalloproteinase 1 (TIMP-1), which is actively involved in the degradation and remodeling of the extracellular matrix, on sVEGFR-1 secretion of cultured human umbilical vein endothelial cells. sVEGFR-1 production was determined in the culture supernatant by Sandwich-ELISA. We showed that TIMP-1 produced by human granulosa cells and recombinant human TIMP-1 both significantly increased the production of sVEGFR-1 in endothelial cells. Also, the down-regulation of TIMP-1 expression by RNA interference resulted in a significant reduction of endothelial sVEGFR-1 secretion into the culture medium. Furthermore, TIMP-1 weakly inhibited proliferation of VEGF-stimulated endothelial cells. In conclusion, our results provide evidence that TIMP-1 increases the production of sVEGFR-1 in endothelial cells and thus may reduce VEGF bioavailability, leading to reduced blood vessel growth in the ovary.

Regulation of gene expression in endothelial cells: the role of human follicular fluid.

A precise regulation of angiogenesis is a prerequisite for an adequate maturation of ovarian follicles. Despite the production of vascular endothelial growth factor (VEGF) by granulosa cells in antral follicles, angiogenesis is restricted to the theca cell layer. The maturing follicle remains avascular before ovulation, implying regulatory mechanisms which prevent premature follicular vascularization. In order to investigate the role of follicular fluid and of granulosa cells in the regulation of endothelial gene expression, human umbilical vein endothelial cells (HUVECs) were incubated in vitro with media conditioned with human follicular fluid obtained from individual patients undergoing oocyte retrieval for in vitro fertilization procedures or with culture medium conditioned by human granulosa cells respectively. Using microarray technology, the gene expression pattern was compared between untreated monolayers of HUVECs and HUVECs treated either with follicular fluid or with granulosa cell conditioned media. We identified a total of 15 genes that were significantly up-regulated and 11 genes that were significantly down-regulated in endothelial cells treated with follicular fluid at least 2.5-fold in more than 70% of comparisons. Up-regulated genes involved in angiogenesis were the anti-angiogenic factors gro-beta (16.5-fold), angiopoietin-2 (3.9-fold), alpha-2-macroglobulin (24.3-fold) and the pro-angiogenic factors E-selectin (5.3-fold) and vascular cell adhesion molecule-1 (VCAM-1) (4.4-fold), whereas a significant down-regulation of the pro-angiogenic genes fibulin-5 (3.5-fold) and elastin (14.9-fold) could be observed. Culturing of HUVECs with conditioned medium from cultured human luteinized granulosa cells demonstrated a similar regulatory pattern of gene expression for fibulin-5, elastin, gro-beta, and E-selectin. The gene regulation in endothelial cells by follicular fluid could be confirmed by RT-PCR for gro-beta, angiopoietin-2, elastin, fibulin-5, and E-selectin. The present work reveals that compounds secreted by granulosa cells lead to the expression of anti-angiogenic factors on the transcript level in endothelial cells and thus could help to explain the temporal and spatial discrepancy between the high expression of VEGF and the restricted angiogenesis in the preovulatory follicle.

Cell-cell-communication during placental development and possible implications for trophoblast proliferation and differentiation.

Since direct cell-cell-communication plays a crucial role in the coordination of proliferation and differentiation processes during development we have focused on the expression patterns of gap junctions and their functional implication in the human placenta. The gap junction protein connexin40 (Cx40) is expressed in the proximal extravillous trophoblast of cell islands and columns. In accordance with these observations, isolated trophoblast cells from first and second trimester placentae and choriocarcinoma cells (Jeg-3) reveal Cx40 expression. This channel is not only characteristic of the trophoblast cells along the invasive pathway but also of endothelial cells. To elucidate the functional role of this channel for proliferation and invasion, the non-coupled Jeg-3 cells have been transfected with Cx26, Cx40 and Cx43, respectively. In contrast to Cx40, the Cx26 channel was more potent in reducing proliferation and inducing differentiation indicated by hCG-beta secretion. Using the nude mouse model to study invasion properties of choriocarcinoma cells, we demonstrated that malignant trophoblast cells were able to invade host vessels and to replace endothelial cells. Upregulation of endogeneous connexin genes in tumours grown in nude mice enforces further experimental strategies to investigate the importance of the different channels to fake the cell biological program of endothelial cells.

Connexin expression patterns in human trophoblast cells during placental development.

This study focuses on the gap junction expression pattern in trophoblast cells during human placental development in vivo and in vitro. Investigations of cell-cell communication properties within the subpopulations of trophoblast responsible for invasion, placental growth and feto-maternal transport seem of special interest because the intercellular channels are believed to coordinate proliferation and differentiation processes. From all gap junction connexins (Cx) investigated (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43), Cx40 was the only connexin clearly detected within the cytotrophoblast of human placenta, and was restricted to the extravillous trophoblast of cell islands and cell columns. Most intense staining was found in the juxtastromal area correlated to the proliferating extravillous trophoblast cells. Connexin protein expression was missing during trophoblast migration into the decidua but was re-expressed in trophoblast aggregates within the decidua. Cx40 expression decreased with progressing pregnancy and no connexins could be detected in villous or extravillous trophoblast of mature placentae. In parallel, isolated trophoblast cells of first and second trimester placentae revealed Cx40 expression and, in contrast to the situation in vivo, Cx43 was also found. In isolated cells of mature placentae, expression of both Cx40 and Cx43 transcripts was decreased to low levels and Cx40 immunoreactivity was absent. Cx43 protein, however, was still detectable in trophoblast cultures of term placentae. Our studies suggest that Cx40 is the characteristic channel for the proliferating cell population of cell islands and cell columns of first and second trimester placentae and isolated trophoblast and is probably involved in regulation and coordination of the invasive pathway.

Gap junction and tissue invasion: a comparison of tumorigenesis and pregnancy.

1. Trophoblast invasion during embryo implantation in some aspects resembles tumour cell invasion but, unlike tumour cells, trophoblast cells are able to differentiate and establish a placenta. Because direct cell-cell communication is believed to be involved in growth control and differentiation, we have investigated connexin (cx) gene expression during trophoblast development. 2. Pre-implantation embryos expressed cx43 as well as cx31 proteins from the 8-cell stage onwards. Following implantation, compartmentalization of both connexins occurred: cx31 expression was restricted to the invasive trophoblast cell population, whereas the embryo proper was characterized by cx43. Trophoblast differentiation was indicated by induction of cx26 in the labyrinth and cx43 in the spongiotrophoblast accompanied by a disappearance of cx31. Comparison with trophoblast cell lines revealed that rat trophoblast HRP-1 cells express connexin43, while malignant choriocarcinoma cells express cx31. Treatment with retinoic acid led to a disappearance of cx31 in the choriocarcinoma. Both cell lines reduced their invasion properties after retinoic acid treatment, but growth retardation was only observed in the malignant trophoblast. 3. It seems that the cx31 channel is needed for trophoblast cell populations to maintain the highly proliferative properties but does not alter their invasion properties.