Autologous Platelet-Rich Plasma in Clitoral Reconstructive Surgery After Female Genital Mutilation/Cutting: A Pilot Case Study.

BACKGROUND: Postoperative healing after clitoral reconstruction (CR) for female genital mutilation/cutting can be long and painful due to prolonged clitoral re-epithelialization time (up to 3 months). Autologous platelet-rich plasma (A-PRP) might reduce postoperative clitoral epithelialization time and pain. OBJECTIVES: The authors assessed postoperative clitoral re-epithelialization time and pain after intraoperative clitoral administration of A-PRP. METHODS: Five consecutive women underwent CR (Foldès technique) followed by the administration of A-PRP Regen Lab SA (Le Mont-sur-Lausanne, Switzerland) plasma and glue, injected inside and applied above the re-exposed clitoris, respectively. We recorded surgical complications, postoperative clitoral pain (visual analogue scale), painkiller intake, time to complete re-epithelialization, and the experienced subjective changes in sexual response and perception of their own body image referred by women. RESULTS: Sexual distress/dysfunction as well as the desire to be physically and symbolically "repaired" were the reasons behind women's requests for surgery. None of the women suffered from chronic vulvar or non-vulvar pain. All women achieved complete clitoral epithelialization by day 80, 3 women between day 54 and 70, and only 1 woman was still taking 1 g of paracetamol twice a day at 2 months postoperative. She had stopped it before the 3-month control. There were no short- or long-term complications. All women described easier access and stimulation of their clitoris as well as improved sexual arousal, lubrication, and pleasure and claimed to be satisfied with their restored body image. CONCLUSIONS: A-PRP could expedite postoperative clitoral epithelialization and reduce postoperative pain after CR after female genital mutilation/cutting.

Autologous Platelet-Rich Plasma for Clitoral Reconstruction: A Case Study.

Clitoral reconstruction after female genital mutilation/cutting (FGM/C) is associated with significant post-operative pain and months-long recovery. Autologous platelet-rich plasma (A-PRP) reduces the time of healing and pain in orthopedic and burn patients and could also do so in clitoral reconstruction. In the present case, a 35-year-old Guinean woman who had undergone FGM/C Type IIb presented to our clinic for clitoral reconstruction. Her request was motivated by low sexual satisfaction and body image. We surgically reconstructed the clitoris using the Foldès method and applied plasma and glue of A-PRP. The patient was highly satisfied with the procedure. Two months post-operatively, her pain had ceased entirely and re-epithelialization was complete. We conclude that A-PRP may improve pain and healing after clitoral reconstruction. Extensive studies investigating long-term outcomes are needed.

Amphetamine-Type Stimulant Dependence and Association with Concurrent Use of Cocaine, Alcohol, and Cannabis: A Cross-Sectional Study.

INTRODUCTION: Amphetamine-type stimulants (ATSs) are the second most commonly used class of illegal substances in Europe. Although concurrent substance use has been subject to research, little is known about associations between concurrent use of cocaine, alcohol, or cannabis and ATS dependence. We expect that the concurrent use of any of the substance, especially cannabis and cocaine, is associated with ATS dependence. METHODS: Cross-sectional data were gathered within the European ATTUNE study in 2018/2019. Participants (N = 721) were asked about their consumption patterns and social, psychological, and economic situation. Multivariate logistic regressions were carried out for associations between ATS dependence and use combinations of frequent cocaine, alcohol, or cannabis, with the reference group of no frequent concurrent use (model 1). Model 2 calculated associations for ATS dependence with lifetime methamphetamine use for respective use combinations. RESULTS: The study population was on average 28.9 years old (SD = 7.7), with the majority being male (63.5%). In model 1, the adjusted odds ratio (aOR) for frequent alcohol use was 0.70 (confidence interval [CI] 0.41-1.20). Similar results were shown for model 2 (aOR 0.82, CI 0.42-1.62). Frequent cannabis use significantly reduced the chance for ATS dependence by 50% in adjusted model 1 (aOR 0.50, CI 0.28-0.89) and by 62% in model 2 (aOR 0.38, CI 0.18-0.82). For frequent cocaine use, models 1 and 2 report an aOR at 1.37 (CI 0.58-3.25) and 2.39 (CI 0.77-7.43), although not statistically significant. Frequent users of all 3 substances had a significant 3-fold chance for ATS dependence (model 1: aOR 2.98, CI 1.16-7.63; model 2: aOR 2.95, CI 1.02-8.58). DISCUSSION: Against initial hypotheses, frequent concurrent use of alcohol or cannabis generally decreased chances for ATS dependence. An explanation could be the study population, which consists of many irregular users of ATS, who mainly consume alcohol or cannabis. Cocaine generally increased chances, although results were not significant. The frequent use of all 3 substances together with ATS in the last year was significantly associated with dependence, thus reporting important information for treatment services. Further research is needed for disentangling causal relationships underlying these associations and for pinpointing consequences for relapse prevention and retention success.

NF-κB and Angiogenesis Inhibitors from the Aerial Parts of Chresta martii.

The ethyl acetate extract of the aerial parts of Chresta martii showed significant in vitro NF-κB inhibition. Bioactivity-guided isolation was undertaken using HPLC microfractionation to localize the active compounds. Different zones of the HPLC chromatogram were linked to NF-κB inhibition. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and UHPLC-TOF-HRMS were used for the early identification of some of the compounds present in the extract and to get a complete phytochemical overview. The isolation of the compounds was performed by high-speed counter-current chromatography and further semipreparative HPLC. Using this approach, 14 compounds were isolated, two of them being new sesquiterpene lactones. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, ECD, NMR, and HRMS. All isolated compounds were evaluated for their inhibitory activity of NF-κB and angiogenesis, and compound 2 showed promising NF-κB inhibition activity with an IC50 of 0.7 µM. The isolated compounds 1, 2, 5, 7, and 8 caused a significant reduction in angiogenesis when evaluated by an original 3D in vitro angiogenesis assay.

A Bivalent Role of Genistein in Sprouting Angiogenesis.

The effects of genistein on angiogenesis remain poorly understood. Some studies claim an antiangiogenic effect and others claim a pro-angiogenic one. Thus, the aim of this study was to determine if genistein may exhibit bivalent angiogenic effects. To address this question, genistein angiogenic modulatory effects were examined using an in vitro 3D angiogenesis model using human umbilical vein endothelial cells. In this model, a bivalent effect of genistein was demonstrated on sprouting angiogenesis, with angiogenic stimulation at low concentrations (0.001 - 1 µM) and inhibition at higher ones (25 - 100 µM). Enhancement of the endothelial tube formation correlated with an increase in human umbilical vein endothelial cell metabolic activity and proliferation. Inhibition of angiogenesis correlated with a decreased metabolic activity, proliferation, and migration. Moreover, high concentrations of genistein influenced human umbilical vein endothelial cell morphology. Expression of genes involved in the angiogenic process in response to genistein was measured to study the mechanism of action. Secretome profiling revealed that angiogenic regulators were modulated with genistein treatment. These results suggested a bivalent effect of genistein on human umbilical vein endothelial cell growth and angiogenesis, and further investigations on the benefit of genistein for cancer chemoprevention, cancer treatment, or pro-angiogenic therapies have to be carefully considered.

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

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-ß signaling pathway was studied by coimmunoprecipitation, competitive binding, TGF-ß 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-ß signaling inhibitors, Tß-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 Tß-RII was demonstrated by coimmunoprecipitation and binding competition with (125)I-TGF-ß. This new paracrine interaction between trophoblast and endothelial cells through the hCG-H and the TGF-ß receptor complex plays a key role in angiogenesis associated with placental development and tumorigenesis.

Platelet-Rich Plasma Promotes the Expansion of Human Myoblasts and Favors the In Vitro Generation of Human Muscle Reserve Cells in a Deeper State of Quiescence.

Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.

Evidence for cross-talk between the LH receptor and LH during implantation in mice.

The present study investigated the first interaction that occurs between the blastocyst and endometrium during implantation. Given the ethical objections to studying implantation in humans, a mouse model was used to study the dialogue between luteinising hormone (LH) and luteinising hormone receptor (LHCGR). Several studies performed on LHCGR-knockout mice have generated controversy regarding the importance of the dialogue between LH and LHCGR during implantation. There has been no demonstration of a bioactive LH-like signal produced by the murine blastocyst. The first aim of the present study was to examine and quantify, using radioimmunoassay, the generation of a bioactive LH signal by the murine blastocyst. We went on to examine and quantify endometrial Lhcgr expression to validate the mouse model. Expression of LHCGR in mouse uteri was demonstrated using immunohistochemistry and western blot analysis. To quantify the expression of Lh in the mouse blastocyst and Lhcgr in the endometrium, reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative (q) RT-PCR were performed. The results demonstrate that Lhcgr expression in BALB/c mouse endometrial epithelium is increased at the time of implantation and indicate that LHCGR may contribute to the implantation process. In support of this hypothesis, we identified a bioactive LH signal at the time of murine blastocyst implantation.

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

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.

Swiss Medical Devices for Autologous Regenerative Medicine: From Innovation to Clinical Validation.

Regenerative medicine, based on the use of autologous tissues and embryonic, stem or differentiated cells, is gaining growing interest. However, their preparation, in a manner compliant with good practices and health regulations, is a technical challenge. The aim of this manuscript is to present the design of reliable CE marked medical devices for the preparation of standardized platelet-rich plasma (PRP) and other autologous biologics intended for therapeutic uses. There are numerous PRP isolation processes. Depending on the methodology used, PRP composition varies greatly in terms of platelet concentration, platelet quality, and level of contamination with red and white blood cells. This variability in PRP composition might affect the clinical outcomes. The devices presented here are based on a specific technology, patented all over the world, that allows the precise separation of blood components as a function of their density using thixotropic separator gels in closed systems. This allows the preparation, in an automated manner, of leukocyte poor PRP with a standardized composition. Production of different forms of PRP is a clinical asset to suit various therapeutic needs. Therefore, we are offering solutions to prepare PRP either in liquid or gel form, and PRP combined with hyaluronic acid. These biologics have been successfully used in many different therapeutic domains, resulting in more than 150 published clinical studies. We also developed the CuteCell technology platform for cell culture expansion for further autologous cell therapies. This technology enables the safe and rapid in vitro expansion of cells intended for therapeutic use in good manufacturing practices (GMP) and autologous conditions, using blood-derived products as culture media supplementation. We summarize in this article our 20 years' experience of research and development for the design of PRP devices and, more recently, for PRP combined with hyaluronic acid.

Use of Autologous Platelet Rich Plasma (A-PRP) for Postpartum Perineal Repair Failure: A Case Report.

Perineal wound dehiscence is an uncommon but important postpartum complication. In many cases, it leads to extreme pain and urinary and defecation problems. For up to several weeks, it can interfere with the mother's daily activity, affecting psychosexual health and body image. The best way to manage perineal wound breakdown (resuturing vs. spontaneous closure) after childbirth remains controversial. A-PRP is the autologous human plasma containing an increased platelet concentration, rich in growth factors, and mediators with hemostatic, anti-inflammatory, and antimicrobial properties. It accelerates the natural healing process. Even though A-PRP is widely used in orthopedics and dermatology, its use in gynecological injuries is limited. We describe here a case of a woman with postpartum perineal dehiscence treated with A-PRP with positive outcomes.

Soluble forms of VEGF receptor-1 and -2 promote vascular maturation via mural cell recruitment.

Two soluble forms of vascular endothelial growth factor (VEGF) receptors, sVEGFR-1 and sVEGFR-2, are physiologically released and overproduced in some pathologies. They are known to act as anti-VEGF agents. Here we report that these soluble receptors contribute to vessel maturation by mediating a dialogue between endothelial cells (ECs) and mural cells that leads to blood vessel stabilization. Through a multidisciplinary approach, we provide evidence that these soluble VEGF receptors promote mural cell migration through a paracrine mechanism involving interplay in ECs between VEGF/VEGFR-2 and sphingosine-1-phosphate type-1 (S1P)/S1P1 pathways that leads to endothelial nitric oxyde synthase (eNOS) activation. This new paradigm is supported by the finding that sVEGFR-1 and -2 perform the following actions: 1) induce an eNOS-dependent outgrowth of a mural cell network in an ex vivo model of angiogenesis, 2) increase the mural cell coverage of neovessels in vitro and in vivo, 3) promote mural cell migration toward ECs, and 4) stimulate endothelial S1P1 overproduction and eNOS activation that promote the migration and the recruitment of neighboring mural cells. These findings provide new insights into mechanisms regulating physiological and pathological angiogenesis and vessel stabilization.

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion.

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient's own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

Angiogenesis Is Differentially Modulated by Platelet-Derived Products.

Platelet-derived preparations are being used in clinic for their role in tissue repair and regenerative processes. The release of platelet-derived products such as autologous growth factors, cytokines and chemokines can trigger therapeutic angiogenesis. In this in vitro study, we evaluated and compared the ability of three platelet-derived preparations: platelet-rich-plasma (PRP), PRP-hyaluronic acid (PRP-HA) and platelet lysates (PL) at various concentrations (5-40%) to modulate human umbilical vein endothelial cells (HUVEC) biological effects on metabolism, viability, senescence, angiogenic factors secretion and angiogenic capacities in 2D (endothelial tube formation assay or EFTA) and in 3D (fibrin bead assay or FBA). HUVEC exocytosis was stimulated with PRP and PRP-HA. Cell viability was strongly increased by PRP and PRP-HA but mildly by PL. The three preparations inhibit HUVEC tube formation on Matrigel, while PRP enhanced the complexity of the network. In the fibrin bead assay (FBA), PRP and PRP-HA stimulated all steps of the angiogenic process resulting in massive sprouting of a branched microvessel network, while PL showed a weaker angiogenic response. Secretome profiling revealed modulation of 26 human angiogenic proteins upon treatment with the platelet derived preparations. These in vitro experiments suggest that PRP and PRP-HA are effective biological therapeutic tools when sustained therapeutic angiogenesis is needed.

Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of "Endothelial Tube Formation Assay" and "Fibrin Bead Assay".

Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells. Among the different methods applied to study angiogenesis, the most commonly used is the "Endothelial Tube Formation Assay" (ETFA). In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. An alternative approach to ETFA is the "Fibrin Bead Assay" (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. We previously developed the "Angiogenesis Analyzer" for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition. We detailed these two algorithms and used the new AA version to compare both methods (i.e. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.

Platelet Transforming Growth Factor-ß1 Induces Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6.

The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor ß1 (TGF-ß1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-ß1 antibody or a TGF-ß1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-ß1 ß1 in the priming phase of liver regeneration.

[Defective placental implantation and its effects on maternal endothelial function]. / Les défauts d'implantation placentaire et leurs conséquences sur la fonction endothé1iale maternelle.

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, edema and proteinuria, resolves spontaneously on placental delivery. Its pathogenesis is thought to involve placental hypoxia, which leads to maternal vascular dysfunction through increased placental release of anti-angiogenic factors such as the soluble form of VEGF receptor-1 (VEGFR1). VEGFR1 binds VEGF and PIGF, which are also produced by villous trophoblastic cells. In the absence of VEGF and PIGF in the maternal circulation, endothelial dysfunction occurs in several vascular territories (liver, kidneys, brain, heart, lungs, etc.). In experimental models, sVEGFR1 not only has an anti-angiogenic action but also augments endothelial expression of NO synthase through intracellular transduction. When NO production is increased, pericytes and perivascular smooth muscle cells are recruited and their adhesion to endothelial cells is strongly stimulated. This can hinder both trophoblast invasion and increase uteroplacental flow during preeclampsia.

Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion.

Nowadays autologous fibroblast application for skin repair presents an important clinical interest. In most cases, in vitro skin cell culture is mandatory. However, cell expansion using xenogeneic or allogenic culture media presents some disadvantages, such as the risk of infection transmission or slow cell expansion. In this study, we investigated an autologous culture system to expand human skin fibroblast cells in vitro with the patient's own platelet-rich plasma (PRP). Human dermal fibroblasts were isolated from patients undergoing abdominoplasty, and blood was collected to prepare nonactivated PRP using the CuteCell™ PRP medical device. Cultures were followed up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Fibroblasts cultured in medium supplemented with PRP showed dose-dependently significantly higher proliferation rates (up to 7.7 times with 20% of PRP) and initiated a faster migration in the in vitro wound healing assay compared with FBS, while chromosomal stability was maintained. At high concentrations, PRP changed fibroblast morphology, inducing cytoskeleton rearrangement and an increase of alpha-smooth muscle actin and vimentin expression. Our findings show that autologous PRP is an efficient and cost-effective supplement for fibroblast culture, and should be considered as a safe alternative to xenogeneic/allogenic blood derivatives for in vitro cell expansion. Impact Statement Autologous dermal fibroblast graft is an important therapy in skin defect repair, but in vitro skin cell culture is mandatory in most cases. However, cell expansion using xenogeneic/allogenic culture media presents some disadvantages, such as the risk of infection transmission. We demonstrated that an autologous culture system with the patient's own platelet-rich plasma is an efficient, cost-effective, and safe supplement for fibroblast culture. As it respects the good manufacturing practices and regulatory agencies standards, it should be considered as a potent alternative and substitute to xenogeneic or allogenic blood derivatives for the validation of future clinical protocols using in vitro cell expansion.

Hypoxia is responsible for soluble vascular endothelial growth factor receptor-1 (VEGFR-1) but not for soluble endoglin induction in villous trophoblast.

BACKGROUND: Pre-eclampsia is a pregnancy disorder characterized by a maternal endothelial cell dysfunction associated with low levels of circulating placental growth factor (PlGF) and increased levels of total vascular endothelial growth factor (VEGF), soluble VEGF receptor-1 (sVEGFR-1), and soluble endoglin, a transforming growth factor beta1 and 3 coreceptor. Here, we tested the hypothesis that these altered levels of angiogenic cytokines and of the anti-angiogenic soluble forms of cytokine receptors could be the consequence of hypoxia. METHODS: Normal human umbilical vein endothelial cells, immortalized first trimester extravillous trophoblast cells (HTR8/SVneo) and first trimester placental villi explants (8-14 weeks) were used for culture under normoxia (20% O(2)) or hypoxia (1% O(2)). Culture media were collected for the measurement of cytokines by enzyme-linked immunosorbent assay. Total RNA was extracted for RT-PCR analysis. RESULTS: Under hypoxia, villous trophoblast expressed higher levels of VEGF, VEGFR-1, sVEGFR-1 and VEGFR-2 mRNAs (P < 0.001), and secreted more VEGF and sVEGFR-1 proteins (P < 0.05). In contrast, PlGF mRNA and protein were decreased in 1% O(2) (P < 0.001), whereas endoglin (Eng) was not modulated. Additionally, sVEGFR-1 directly abolished VEGF/PlGF-induced angiogenesis in the rat aortic ring assay. CONCLUSIONS: Our results support the hypotheses that, in pre-eclampsia, (i) overproduction of VEGF family factors by pre-eclamptic placenta is a consequence of induced hypoxia; (ii) overproduction of sVEGFR-1 by hypoxic villous trophoblast accounts for maternal free VEGF depletion; (iii) low circulating level of free PlGF is not only related to sVEGFR-1 overproduction, but also to hypoxia-induced mRNA down-regulation; (iv) Eng is not modulated by hypoxia.

Earlier onset of tumoral angiogenesis in matrix metalloproteinase-19-deficient mice.

Among matrix metalloproteinases (MMP), MMP-19 displays unique structural features and tissue distribution. In contrast to most MMPs, MMP-19 is expressed in normal human epidermis and down-regulated during malignant transformation and dedifferentiation. The contribution of MMP-19 during tumor angiogenesis is presently unknown. In an attempt to give new insights into MMP-19 in vivo functions, angiogenic response of mutant mice lacking MMP-19 was analyzed after transplantation of murine malignant PDVA keratinocytes and after injection of Matrigel supplemented with basic fibroblast growth factor. In situ hybridization and immunohistochemical analysis revealed that MMP-19 is produced by host mesenchymal cells but not by endothelial capillary cells or CD11b-positive inflammatory cells. Based on a new computer-assisted method of quantification, we provide evidence that host MMP-19 deficiency was associated with an increased early angiogenic response. In addition, increased tumor invasion was observed in MMP-19-/- mice. We conclude that, in contrast to most MMPs that promote tumor progression, MMP-19 is a negative regulator of early steps of tumor angiogenesis and invasion. These data highlight the requirement to understand the individual functions of each MMP to improve anticancer strategies.