Figure 1.
Representation of a chorionic villus at the implantation site.
Villous cytotrophoblasts (yellow) fuse to form the syncytiotrophoblast (green). The extravillous trophoblasts (red) proliferate to form multilayered columns of cells and then invade the decidua up to the upper third of the myometrium and the uterine arterioles. At the deciduo-muscular junction, EVCTs undergo final differentiation into multinucleated giant cells. Adapted from figure 1 of Tarrade et al. [12].
Figure 2.
Microarray and transcriptome analyses of rosiglitazone-treated EVCTs purified from first-trimester placentas.
SAM scatter plot of the observed d(l) versus the expected dE(l) relative difference between rosiglitazone-treated cells and their paired controls (n = 5), using a two-fold threshold. EVCTs were cultured for 24 h and then treated with rosiglitazone for 24 h. Gene profiles were compared with those of paired untreated control EVCTs from the same placenta. The solid blue line represents no difference between d(l) and dE(l), and the broken red lines show the delta limit of 0.93 from the solid blue line. A total of 175 probe sets outside the broken lines were significantly induced (red dots, n = 149) or suppressed (green dots, n = 26) in rosiglitazone-treated cells. The median estimated FDR was <5%.
Figure 3.
Heatmap of the 175 probe sets (117 unique genes) selected with the SAM procedure.
Each column represents an individual sample (5 controls and 5 samples treated with 1 µM rosiglitazone). Each row represents one probe. The gene expression level is depicted by the intensity of the green (low intensity) and red (high intensity) boxes. Probe and gene names are included in Figure S1.
Figure 4.
Networks of the 20 differentially expressed genes containing putative PPARγ response elements (PPREs) in their promoter.
The entire 117 unique gene list was uploaded into the Genomatix Pathway System (GePS 2.4.0). Only the 20 genes linked to PPARγ by a PPRE are represented. The function of each gene is that proposed by GePS software for receptors and co-factors, etc. (for further details see the Genomatix web site; http://www.genomatix.de). The pale salmon color represents genes expressed in human placenta (Unigene): 2 genes (UPK1A and GADD45A) are not mentioned as being expressed in human placenta on this web site, but GADD45A expression has recently been described in this tissue [100].
Figure 5.
Microarray and transcriptome analyses of rosiglitazone-treated EVCTs compared to paired controls: top 7 networks.
The 175 probe sets were loaded into Ingenuity Pathway Analysis software (IPA) and converted into gene networks. Genes shown in bold type are present in the input data list as upregulated (red) or downregulated (green). Genes involved in the network that are not included in the transcriptome results are shown in black. * genes with several probes present in the input data list.
Figure 6.
Relative RT-qPCR analysis of the effect of rosiglitazone on EVCT gene expression.
EVCTs were purified and treated with rosiglitazone in an identical manner as for microarray analyses. RNA expression estimated with relative RT-qPCR is shown A) for selected genes identified as differentially regulated by GeneChip analyses; and B) for the five known LOX isoforms. Five treated cultures (black bar) were normalized to control cultures (gray bar) from the same placenta. Results are expressed as the mean ± SEM of six (A) and five experiments (B) performed in duplicate. * p<0.05, ** p<0.01, *** p<0.001 versus untreated cultures; ns: not significant.
Figure 7.
Expression analyses of LOXs in early placental villi and EVCTs.
A) LOX, LOXL1 and LOXL2 RNA copy numbers per ng of total RNA in 48 h-cultured EVCTs were determined by absolute RT-qPCR. Each bar represents the mean ± SEM of five independent experiments performed in duplicate. *: p<0.05 for differences between isoforms; ns: not significant. B) Western blot analyses of LOX and LOXL1 proteins. Protein (20 µg) extracted with the T-PER reagent (Pierce) from 8- to 9-WA placental villous tissue (Vill), or with the M-PER reagent from 72 h-cultured primary EVCTs purified from placental tissue at the same term (EVCT, C), as well as 40 µg of protein from cell conditioned medium (CM) and the insoluble M-PER cell extract fraction (IF) taken up in Laemmli buffer were separated on 4–12% polyacrylamide Bis-Tris gels. Detection was achieved by using LOX and LOXL1 isoform-specific antibodies; ß -actin was used as a control.
Figure 8.
Immunohistological localization of LOX and LOXL1.
Experiments were carried out on PFA-fixed sections of 8-WA placental villi, using specific antibodies; non-specific rabbit IgG was used as a control. Immunostaining was performed with an universal streptavidin-peroxidase kit (Dako).
Figure 9.
Immunolocalization by confocal microscopy of LOX (Fig 9A) and LOXL1 (Fig 9B) proteins in rosiglitazone-treated and control EVCT primary cultures.
EVCTs were cultured for 72™. Anti-LOX and -LOXL1 rabbit antibodies (2.5 µg/mL) were used for immunostaining. Individual staining for CK7 (first column), topro-3 (second column) and LOXs (third column) is shown in grayscale and is merged in the last column; 9A third line: 2× magnification of LOX staining in control cells. LOX isoforms (FITC labeling in green) were detected as follows: LOX in the cytoplasm and around the cells, and LOXL1 mainly in the nucleus and nucleolus. CK7 (CY3 labeling) was used to identify the trophoblast cytoskeleton (in red) and Topro-3 to label the nuclei (in blue). Non-specific rabbit IgG was used as a negative control. The 1 µM rosiglitazone-treated cells (Rosi) were stained in the same conditions. Only the LOXL1 signal was enhanced in rosiglitazone-treated cells.
Figure 10.
Effect of LOX inhibition on EVCT invasion.
EVCTs were isolated from first-trimester placentas, cultured as described in Materials and Methods, and treated 24 h later with A) 100 µM or 200 µM BAPN for 48 h or B) 1 µM rosiglitazone or 100 µM BAPN, alone or combined, for 48 h. Cells were fixed and stained with DAPI (nuclei) and CK7 (cytotrophoblasts). CK7+ migrating cells, nuclei and pseudopodes were counted. The number of invasive EVCTs is expressed as a percentage of the total cell number. Each bar represents the mean ± SEM of four experiments performed in duplicate. Significant differences between treatments are marked as * p<0.05, ** p≤0.01, or *** p<0.001.