Investigation of potential new targets for the diagnosis and/or the treatment of osteoarthritis

in Osteoarthritis and Cartilage (2013, April), 21(Supplement April 2013),

Purpose: Synovial inflammation plays a key role in the pathophysiology process of osteoarthritis (OA). We have previously compared the gene expression pattern of synovial cells isolated from inflammatory ... [more ▼]

Purpose: Synovial inflammation plays a key role in the pathophysiology process of osteoarthritis (OA). We have previously compared the gene expression pattern of synovial cells isolated from inflammatory (I) or normal/reactive (N/R) areas of a synovial membrane harvested from the same OA patient. We identified a large number of mediators belonging to key pathways involved in OA pathogenesis. The aim of this study was to validate different potential new targets for the diagnosis and/or the treatment OA. Methods: Synovial cells (SC) were isolated from synovial specimens obtained from OA patients undergoing knee replacement. The inflammatory status of the synovial membrane was characterized according to macroscopic criteria. The biopsies from N/R and I areas were cultured separately for a period of 7 days. Microarray gene expression profiling between N/R and I areas was performed. The biological relevance of up- and down-regulated genes was analyzed with Ingenuity Pathways Analysis. Western blot and immunohistochemistry confirmed the identified genes most differentially expressed in the key pathways. The production of the triggering receptor expressed on myeloid cells-1 (TREM1), the alarmin S100 calcium binding protein A9 (S100A9), the wingless-type MMTV integration site family, member 5A (Wnt-5A) and the stanniocalcin 1 (STC1) were evaluated by Western blot. S100A9, hyaluronan synthase-1 (HAS1) and STC1 expression and localization were investigated by immunohistochemistry. Results: 896 genes differentially expressed in N/R and I areas were identified. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling and angiogenesis. In the inflammatory gene pattern, TREM1 and S100A9 were strongly upregulated. We validated the production of these proteins in OA synovial biopsies by Western blot. TREM1 and S100A9 were increased in I compared to N/R synovial cells culture. S100A9 was observed in the perivascular area and in sublining cells in I synovial biopsies, but not in N/R biopsies. An increased staining was also observed in the intima lining layer of I when compared to N/R biopsies. The most upregulated anabolism enzyme in I synovial biopsies was HAS1. Using immunohistochemistry, we observed in I areas an increase of the HAS1-positive cells mainly in the intima lining. We also studied the protein production of Wnt-5A, the most upregulated intermediate of Wnt signaling pathway. The protein level was increased in I compared to N/R areas. Finally, in the angiogenesis pathway, one the most u-regulated gene was STC1. A significant increase of STC1 production was observed in I areas compared to N/R areas by Western blot. This result was also supported by the immunohistochemical analysis. In I area, the staining for STC1 was more intense in perivascular and sublining cells. Conclusions: Synovial membrane inflammation is a key target for OA treatments. In this work, we have identified proteins involved in the synovitis pathways like angiogenesis, cells infiltration and matrix remodeling. These proteins could be targeted by drugs and used as companion biomarkers for evaluating their efficacy. Although qualitative, our results could also yield to the identification of markers of the disease. This investigation has to be further pursued. [less ▲]

Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation

in Transplantation (2013), 95(3), 426-433

Background: Cryopreservation of cortex ovarian tissue before anti-cancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ... [more ▼]

Background: Cryopreservation of cortex ovarian tissue before anti-cancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ovarian graft causes massive follicle loss by apoptosis. VEGF111 is a recently described VEGF isoform that does not bind to the extracellular matrix, diffuse extensively and is resistant to proteolysis. These properties confer a significantly higher angiogenic potential to VEGF111 in comparison to the other VEGF isoforms. Methods: We evaluated the morphology of cryopreserved sheep ovarian cortex, grafted in the presence or absence of VEGF111. Ovarian cortex biopsies were embedded in type I collagen with or without VEGF111 addition before transplantation to SCID mice ovaries. Transplants were retrieved 3 days or 3 weeks later. Follicular density, vasculature network, haemoglobin content and cell proliferation were analysed. Results: Addition of VEGF111 increased density of functional capillaries (p=0.01) 3 days after grafting. By double immunostaining of Ki-67 and von Willebrand Factor (vWF) we demonstrated that proliferating endothelial cells were found in 83% of the VEGF111 group when compared to 33% in the control group (p=0.001). This angio-stimulation was associated with a significant enhancement of haemoglobin content (p=0.03). Three weeks after transplantation, the number of primary follicles was significantly higher in VEGF111 grafts (p=0.02). Conclusion: VEGF111 accelerates blood vessels recruitment, functional angiogenesis and improves the viability of ovarian cortex by limiting ischemia and ovarian cortex damage. [less ▲]

Expression et localisation spatio-temporelle de KISS1 et de son récepteur KISSR dans le placenta normal et pathologique.

Poster (2012, October)

Objectif : Etudier l’expression de KISS1 (métastatine) et de son récepteur KISS1R lors de la grossesse normale et pathologique. Matériels et méthodes : Nous avons étudié la localisation de KISS1 et KISS1R ... [more ▼]

Objectif : Etudier l’expression de KISS1 (métastatine) et de son récepteur KISS1R lors de la grossesse normale et pathologique. Matériels et méthodes : Nous avons étudié la localisation de KISS1 et KISS1R par immunohistochimie dans des placentas normaux (1 er et 3 ème trimestre). Par RT-PCR quantitative, nous avons évalué le niveau d’expression des ARNm dans les placentas et les lits placentaires correspondants. Les niveaux d’expression de ARNm ont été comparés entre les grossesses normales (GN, n=13) et les grossesses spathologiques Prééclampsiques -PE-, n=17 et retard de croissance intrautérine -RCIU-, n=9). Résultats : Au premier trimestre des GN, KISS1 est majoritairement localisé dans les syncitiotrophoblastes, alors que KISS1R est détecté dans le mesenchyme villositaire. Au cours du troisième trimestre, KISS1 est uniquement localisé dans le syncitiotrophoblaste au contact avec la décidue et dans le mésenchyme villositaire et KISS1R est détecté dans le trophoblaste extra-villeux ainsi que dans quelques cellules de la décidue. Les analyses par RT-PCR mettent en évidence une expression plus importante des ARNm de KISS1 (p<0,001) et de KISS1R (p=0.039) dans les placentas (GN,PE et RCIU) par rapport aux lits placentaires correspondants. Les niveaux d’expression de KISS1 et KISS1R ne sont pas, cependant, significativement modulés dans les grossesses pathologiques. Conclusions : Par immunohistochimie, nos résultats indiquent une expression spatiotemporelle différente pour KISS1 et KISS1R entre le 1 er et 3 ème trimestre des grossesses normales. Nous n’avons pas mis en évidence de modulation de l’expression des ARNm dans les grossesses pathologiques. [less ▲]

Differential expression of Vegfr-2 and its soluble form in preeclampsia.

in PLoS ONE (2012), 7(3), 33475

Background: Several studies have suggested that the main features of preeclampsia (PE) are consequences of endothelial dysfunction related to excess circulating anti-angiogenic factors, most notably ... [more ▼]

Background: Several studies have suggested that the main features of preeclampsia (PE) are consequences of endothelial dysfunction related to excess circulating anti-angiogenic factors, most notably, soluble sVEGFR-1 (also known as sFlt-1) and soluble endoglin (sEng), as well as to decreased PlGF. Recently, soluble VEGF type 2 receptor (sVEGFR-2) has emerged as a crucial regulator of lymphangiogenesis. To date, however, there is a paucity of information on the changes of VEGFR-2 that occur during the clinical onset of PE. Therefore, the aim of our study was to characterize the plasma levels of VEGFR-2 in PE patients and to perform VEGFR-2 immunolocalization in placenta. METHODOLOGY/PRINCIPAL FINDINGS: By ELISA, we observed that the VEGFR-2 plasma levels were reduced during PE compared with normal gestational age matched pregnancies, whereas the VEGFR-1 and Eng plasma levels were increased. The dramatic drop in the VEGFR-1 levels shortly after delivery confirmed its placental origin. In contrast, the plasma levels of Eng and VEGFR-2 decreased only moderately during the early postpartum period. An RT-PCR analysis showed that the relative levels of VEGFR-1, sVEGFR-1 and Eng mRNA were increased in the placentas of women with severe PE. The relative levels of VEGFR-2 mRNA as well as expressing cells, were similar in both groups. We also made the novel finding that a recently described alternatively spliced VEGFR-2 mRNA variant was present at lower relative levels in the preeclamptic placentas. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the plasma levels of anti-angiogenic factors, particularly VEGFR-1 and VEGFR-2, behave in different ways after delivery. The rapid decrease in plasma VEGFR-1 levels appears to be a consequence of the delivery of the placenta. The persistent circulating levels of VEGFR-2 suggest a maternal endothelial origin of this peptide. The decreased VEGFR-2 plasma levels in preeclamptic women may serve as a marker of endothelial dysfunction. [less ▲]

Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation

in Journal of Assisted Reproduction & Genetics (2012), 28(11), 1009

Hyperglycosylated human chorionic gonadotropin stimulates angiogenesis through TGF-beta receptor activation.

in FASEB Journal (2012), sous presse

Embryo implantation requires extensive angiogenesis at the maternal-fetal interface. Hyperglycosylated human chorionic gonadotropin (hCG-H), a trophoblast invasive signal produced by extravillous ... [more ▼]

Embryo implantation requires extensive angiogenesis at the maternal-fetal interface. Hyperglycosylated human chorionic gonadotropin (hCG-H), a trophoblast invasive signal produced by extravillous cytotrophoblasts and by choriocarcinoma, was evaluated for its angiogenic role. hCG-H was purified by HPLC from choriocarcinoma supernatant, and the glycosylation pattern was determined by 2D gel analysis. Angiogenesis models used were aortic ring assay with wild-type and LHCGR-knockout mice, endothelial and mural cell proliferation, and migration assays. The TGF-beta signaling pathway was studied by coimmunoprecipitation, competitive binding, TGF-beta reporter gene assays, and Smad immunoblotting. hCG-H displayed a potent angiogenic effect [3.2-fold increase of number of vessel intersections in wild-type aortic rings (11.406 to 36.964)]. hCG-H-induced angiostimulation was independent of the classic hCG signaling pathway since it persisted in LHCGR-knockout mice [4.73-fold increase of number of vessel intersections (10.826 to 51.288)]. Using TGF-beta signaling inhibitors, Tbeta-RII was identified as the hCG-H receptor responsible for its angiogenic switch. hCG-H exposure enhanced phosphorylation of Smad 2 in endothelial and mural cells and genomic activation of Smad-responsive elements. Interaction between hCG-H and Tbeta-RII was demonstrated by coimmunoprecipitation and binding competition with (125)I-TGF-beta. This new paracrine interaction between trophoblast and endothelial cells through the hCG-H and the TGF-beta receptor complex plays a key role in angiogenesis associated with placental development and tumorigenesis. [less ▲]

Abnormal vascular architecture at the placental-maternal interface in placenta increta

in American Journal of Obstetrics and Gynecology (2012), 207(3), 1881-9

Objective The objective of the study was to characterize the vascular architecture at the placental-maternal interface in pregnancies complicated by placenta increta and normal pregnancies. Study Design ... [more ▼]

Objective The objective of the study was to characterize the vascular architecture at the placental-maternal interface in pregnancies complicated by placenta increta and normal pregnancies. Study Design Vessel numbers and cross-section area density and spatial and area distributions in 13 placenta-increta placental beds were compared with 9 normal placental beds using computer-assisted image analysis of whole-slide CD31 immunolabeled sections. Results The total areas occupied by vessels in normal and placenta-increta placental beds were comparable, but vessels were significantly sparser and larger in the latter. Moreover, placenta-increta–vessel distributions (area and distance from the placental–myometrial junction) were more heterogeneous. Conclusion Size and spatial organization of the placenta-increta vascular architecture at the placental-maternal interface differed from normal and might partially explain the severe hemorrhage observed during placenta-increta deliveries. [less ▲]

Does isoforms 111 and 165 of vascular endothelial growth factor (VEGF111 and 165) improve transplanted ovarian tissue survival?

in Journal of Assisted Reproduction & Genetics (2011), 28

Transient reduction of placental angiogenesis in PAI-1 deficient mice

in Physiological Genomics (2011), 43(4), 188-98

Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and foetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently ... [more ▼]

Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and foetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at 10.5-11.5 day post-coitum (dpc). Knowing the key contribution of PAI-1 in the regulation of angiogenesis, we have now analyzed the consequence of PAI-1 deficiency on murine placentation. Morphological and quantitative computer-assisted image analysis revealed abnormal placental morphology in PAI-1 (-/-) mice at 10.5 and 12.5 dpc. At 10.5 dpc, the genetic ablation of PAI-1 resulted in a transient reduction of both maternal and foetal vascularizations in the placenta and increased trophoblast cell density. This was associated with a poorer development of the labyrinth and an extension of the decidua. A larger spongiotrophoblast layer appeared at 12.5 dpc in PAI-1 deficient mice. Placental morphology was normalized at 14,5 dpc. Microarray analyses performed on laser capture microdissected labyrinths revealed that 46 genes were differentially expressed at 10.5 dpc between the two genotypes. However, only 11 genes were still differently modulated at 14.5 dpc when normalization of placental morphology had take place. This transcriptomic profiling highlighted a dysregulation in the expression of placenta-related cathepsin family members. All together our data provide evidence for a transient impaired placental morphology in PAI-1-deficient mice which is then normalized leading to normal embryonic development. [less ▲]

Specific and extensive endometrial deregulation is present before conception in IVF/ICSI repeated implantation failures (IF) or recurrent miscarriages.

in Journal of Pathology (The) (2011), 225(4), 554-64

The objective was to examine if IVF/ICSI repeated implantation failures (IF) or recurrent miscarriages (RM) could be related to preconceptional endometrial deregulations. IF was defined as the absence of ... [more ▼]

The objective was to examine if IVF/ICSI repeated implantation failures (IF) or recurrent miscarriages (RM) could be related to preconceptional endometrial deregulations. IF was defined as the absence of pregnancy despite the transfer of at least ten IVF/ICSI good quality embryos, and RM as having at least three unexplained miscarriages. Fertile controls (FC) were women who had given birth at least once. Endometrial biopsy was performed in the mild luteal phase of a non-conceptual cycle (five women were selected in each group). Affymetrix chips (GeneChip Human Genome U133 Plus2.0 Array) were used for hybridization. Data were normalized by the gcRMA method, and raw p values adjusted by the Bonferroni procedure (1%). Differential expression of selected genes was analysed using real-time PCR. Gene networks and biological functions were explored using the Ingenuity Pathways Analysis software. Endometrial gene expression profiles at the time of uterine receptivity differ dramatically in the endometrium among FC, RM, and IF patients. Compared to FC, 2126 and 2477 genes are differentially expressed in IF and RM groups, respectively, and 2363 between IF and RM. In both conditions, differential gene expression referred mainly to DNA transcription and expression. Other main cellular functions deregulated in IF conditions correspond to cell morphology, cellular development, cell cycle, and cellular assembly, while in RM conditions, deregulated cellular functions relate to cell signalling (degradation of cyclic AMP and calcium metabolism) and cellular maintenance. In both conditions, there is an over-representation of deregulations related to the haematological system. In the IF condition, cell-mediated immune response and nervous system development and function are highly deregulated, while in RM patients, main deregulations are in organ and tissue development, humoral immune response, and muscular system development and function. Extensive endometrial deregulations are present before conception in patients who experienced IF or RM with both distinct and common deregulation. [less ▲]