RESUMEN
Background: This study investigated the expression and localization of cathepsins B, D, and G in relationship to the embryonic stem cell (ESC)-like population we have previously identified in microcystic lymphatic malformation (mLM). Methods and Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in cervicofacial mLM tissue samples from 11 patients. Immunofluorescence staining of two representative mLM samples showed localization of cathepsins B and D to the OCT4+ and the c-MYC+ cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+/tryptase+ cells within the stroma. Transcript expression of cathepsins B, D, and G was confirmed using reverse transcription quantitative polymerase chain reaction (RT-qPCR; n = 5). Western blotting (n = 3) performed on the mLM tissue samples revealed protein expression of cathepsins B and D, which were demonstrated to be enzymatically active using enzymatic activity assays. Conclusion: This study demonstrated expression of cathepsins B and D by the ESC-like cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+ phenotypic mast cells within the stroma of mLM.
Asunto(s)
Catepsina B , Catepsina D/genética , Catepsina G/genética , Anomalías Linfáticas , Western Blotting , Catepsina B/genética , Células Madre Embrionarias , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The endometrium lines a women's uterus becoming receptive, and allowing embryo implantation to occur, for just a few days during the post-ovulatory mid-secretory phase of each menstrual cycle. We investigated whether concentrations of proposed receptivity biomarkers (VEGF, IL8, FGF2, CSF3 sFlt-1, sGP130 and PlGF) secreted by the endometrium into the uterine cavity and forming the microenvironment for embryo implantation is altered among a population of age-matched women with unexplained (idiopathic) infertility compared to fertile women during the receptive mid-secretory phase (nâ¯=â¯16 fertile, 18 infertile) and the prior pre-receptive early secretory phase (nâ¯=â¯19 fertile, 18 infertile) of their cycle. In the mid-secretory cohort significantly elevated concentrations of five biomarkers; PlGF (pâ¯=â¯0.001), IL8 (pâ¯=â¯0.004), sGP130 (pâ¯=â¯0.009), sFlt-1 (pâ¯=â¯0.021), and CSF3 (pâ¯=â¯0.029) was present in uterine fluid of infertile women during the mid-secretory phase, but only CSF3 was significantly elevated in the pre-receptive early secretory phase (pâ¯=â¯0.006). In vitro studies of glycosylated and non-glycosylated forms of CSF3 at representative fertile (20â¯ng/mL) and infertile (70â¯ng/mL) effects on endometrium and embryo behaviour were performed. Non-glycosylated CSF3 at fertile concentrations significantly (pâ¯<â¯0.001) elevated endometrial epithelial cell proliferation however chronic treatment or elevated (infertile) concentrations of CSF3 in glycosylated form abrogated the positive effects. Both forms of CSF3 increased trophoblast cell invasion (pâ¯<â¯0.001) regardless of concentration. Mouse embryo outgrowth was significantly (pâ¯<â¯0.01) increased at fertile but not at infertile concentrations. The study confirmed potential utility of five biomarkers of endometrial receptivity for future application in the mid-secretory phase while highlighting CSF3 is elevated in the earlier pre-receptive phase. Our data provides evidence that CSF3 acts on both human endometrium and embryo in a manner that is concentration and glycosylation dependent.