Mesenchymal stem cells and their conditioned medium can enhance the repair of uterine defects in a rat model.

BACKGROUND: Our aim was to examine the roles of mesenchymal stem cell (MSC) transplantation in the repair of large uterine defects. METHODS: Uterine defects were created in both uterine horns of female rats by a punch instrument, and bone marrow-derived MSCs, MSC-conditioned medium (MSC-CM) or vehicle were injected into the myometrium around the defect. The rate of uterine defect repair was monitored on day 2 and 4 after operation. Cytokine array of MSC-CM was performed, followed by neutralizing antibody experiments to clarify the exact cytokine participating in the MSC-CM-enhanced wound repair. RESULTS: Transplantation of MSCs, but not myometrial cells, significantly enhanced uterine defect repair. The transplanted MSCs were detected in the uterine horn with no signs of rejection on day 4 after transplantation, when the MSC-transplanted uterine wound was nearly healed. Moreover, uterine defect repair was also accelerated by injection of MSC-CM, indicating the paracrine effects of MSCs on uterine wound healing. Cytokine array analysis further revealed that MSC-CM contained abundant cytokines and chemokines, among which high levels of interleukin-6 (IL-6) were found. Additionally, antibodies against IL-6 were shown to block MSC-CM-enhanced uterine defect repair. CONCLUSION: This study demonstrated that transplantation of MSCs could enhance uterine defect repair by paracrine effects involving IL-6, which are findings that may be applied to facilitate uterine wound healing in the removal of huge intramural masses.

Interleukin-18 deteriorates Fabry cardiomyopathy and contributes to the development of left ventricular hypertrophy in Fabry patients with GLA IVS4+919 G>A mutation.

RATIONALE: A high incidence of GLA IVS4+919 G>A mutation in patients with Fabry disease of the later-onset cardiac phenotype, has been reported in Taiwan. However, suitable biomarkers or potential therapeutic surrogates for Fabry cardiomyopathy (FC) in such patients under enzyme replacement treatment (ERT) remain unknown. OBJECTIVE: Using FC patients carrying IVS4+919 G>A mutation, we constructed an induced pluripotent stem cell (iPSC)-based disease model to investigate the pathogenetic biomarkers and potential therapeutic targets in ERT-treated FC. RESULTS AND METHODS: The iPSC-differentiated cardiomyocytes derived from FC-patients (FC-iPSC-CMs) carried IVS4+919 G>A mutation recapitulating FC characteristics, including low α-galactosidase A enzyme activity, cellular hypertrophy, and massive globotriaosylceramide accumulation. Microarray analysis revealed that interleukin-18 (IL-18), a pleiotropic cytokine involved in various myocardial diseases, was the most highly upregulated marker in FC-iPSC-CMs. Meanwhile, IL-18 levels were found to be significantly elevated in the culture media of FC-iPSC-CMs and patients' sera. Notably, the serum IL-18 levels were highly paralleled with the progression of left ventricular hypertrophy in Fabry patients receiving ERT. Finally, using FC-iPSC-CMs as in vitro FC model, neutralization of IL-18 with specific antibodies combined with ERT synergistically reduced the secretion of IL-18 and the progression of cardiomyocyte hypertrophy in FC-iPSC-CMs. CONCLUSION: Our data demonstrated that cardiac IL-18 and circulating IL-18 are involved in the pathogenesis of FC and LVH. IL-18 may be a novel marker for evaluating ERT efficacy, and targeting IL-18 might be a potential adjunctive therapy combined with ERT for the treatment of advanced cardiomyopathy in FC patients with IVS4+919 G>A mutation.

Thermosensitive chitosan-based hydrogels for sustained release of ferulic acid on corneal wound healing.

Oxidative damage to cornea can be induced by alkaline chemical burn which may cause vision loss or blindness. Recent studies showed that exogenous application of natural antioxidants may be a potential treatment for corneal wound healing. However, low ocular bioavailability and short residence time are the limiting factors of topically administered antioxidants. Ferulic acid (FA) is a natural phenolic compound and an excellent antioxidant. The study was aimed to investigate the effects of FA in corneal epithelial cells (CECs) under oxidative stress and evaluate the feasibility of use the thermosensitive chitosan-based hydrogel containing FA for corneal wound healing. The results demonstrated that post-treatment of FA on CECs could decrease the inflammation-level and apoptosis. In the rabbit corneal alkali burn model, post-treatment FA-loaded hydrogel may promote the corneal wound healing. The results of study suggest that FA-loaded hydrogel may have the potential applications in treating corneal alkali burn.

Poly(ADP-Ribose) Polymerase 1: Cellular Pluripotency, Reprogramming, and Tumorogenesis.

Poly(ADP-ribos)ylation (PARylation) is the catalytic function of the Poly(ADP-ribose) polymerases (Parps) family for post-translational modification in cellular process. Being a major member of Parps, Parp1 is a crucial nuclear factor with biological significance in modulating DNA repair, DNA replication, transcription, DNA methylation and chromatin remodeling through PARylation of downstream proteins. In addition, high expression level and activity of Parp1 are correlated with pluripotent status, reprogramming, and cancer. Furthermore, epigenetic modulation of Parp1 is explored for regulating wide variety of gene expression. Genetic and pharmaceutical disruption of Parp1 further confirmed the importance of Parp1 in cell growth, DNA repair, and reprogramming efficiency. Taken together, the proximity toward the understanding of the modulation of Parp1 including interaction and modification in different fields will provide new insight for future studies. In this review, the biological significance of Parp1 in transcription and the epigenetic modulation of Parp1 in pluripotent status, reprogramming process and cancer will be summarized.

CHD1L Regulated PARP1-Driven Pluripotency and Chromatin Remodeling During the Early-Stage Cell Reprogramming.

PARP1 and poly(ADP-ribosyl)ation (PARylation) have been shown to be essential for the initial steps of cellular reprogramming. However, the mechanism underlying PARP1/PARylation-regulated activation of pluripotency loci remains undetermined. Here, we demonstrate that CHD1L, a DNA helicase, possesses chromatin remodeling activity and interacts with PARP1/PARylation in regulating pluripotency during reprogramming. We found that this interaction is mediated through the interplay of the CHD1L macro-domain and the PAR moiety of PARylated-PARP1. Chromatin immunoprecipitation assays demonstrated the co-occupancy of CHD1L and PARP1 at Pou5f1, Nanog, and Esrrb pluripotency loci. Knockdown of CHD1L significantly blocked the binding activity of PARP1 at pluripotency loci and inhibited the efficiency of PARP1-driven reprogramming. Notably, we found that CHD1L-promoted reprogramming requires both a PARP1-interacting domain and DNA helicase activity, partly contributing to the chromatin-remodeling states of pluripotency loci. Taken together, these results identify CHD1L as a key chromatin remodeler involved in PARP1/PARylation-regulated early-stage reprogramming and pluripotency in stem cells.

A longitudinal cohort study of incidence rates of inguinal hernia repair in 0- to 6-year-old children.

BACKGROUND/PURPOSE: This study provides epidemiologic data on the incidence of inguinal hernia repair in preschool children using the Taiwan National Health Insurance Research Database. We believe that the data on hernia repair in said database provide a close approximation of the true incidence of inguinal hernia in young children. METHOD: A cohort of 1,073,891 deidentified individuals was randomly selected from an insured population of 23 million. Subjects born during the period 1997-2004 were followed from birth to 6 years. The chi-square test and logistic regression modeling were used for statistical analyses. RESULT: A total of 92,308 individuals were born during the study period. Of these individuals, 3881 underwent hernia repairs. The cumulative incidence of hernia repair in children aged 0 to 6 years was 4.20%/7 years. The boy/girl ratio was 4.27:1 and the unilateral/bilateral ratio was 3.77:1. The incidence of hernia repair among boys was highest during the first year of life, but then decreased with age. In contrast, the incidence among girls remained stable during the first 6 years of life. Boys younger than 1 year had more bilateral repairs than boys in other age groups (p<0.0001) and girls had significantly more bilateral repairs than boys (p<0.0001). Subjects with a history of preterm birth also had a higher incidence of hernia repair than subjects who were born at full term (odds ratio=2.34, p<0.0001). CONCLUSION: Yearly incidence of hernia repair was obtained from a nationwide database. Some of the observations have not been reported elsewhere.

A tumor necrosis factor-α inhibitor reduces the embryotoxic effects of endometriotic peritoneal fluid.

OBJECTIVE: To determine whether a tumor necrosis factor-α (TNF-α) inhibitor can reduce the embryotoxicity of the peritoneal fluid (PF) of women with endometriosis. DESIGN: Experimental clinical study. SETTING: University hospital. PATIENT(S): Twelve women with chocolate cysts and 12 control women without endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We collected the PF from patients with chocolate cysts (CH-PF) and patients without endometriosis (N-PF) during laparoscopic surgery. For the in vitro studies, development and apoptosis were evaluated in two-cell stage mouse embryos after incubation with CH-PF and N-PF, with or without a TNF-α inhibitor. RESULT(S): We found that CH-PF significantly decreased the rate of blastocyst development and increased the percentage of apoptotic cells in the embryos. Cytokine assays showed that the concentrations of several cytokines, including TNF-α, were higher in embryos incubated with CH-PF than in those incubated with N-PF. Furthermore, the treatment of embryos with TNF-α retarded development and induced apoptosis. Important, adalimumab, a TNF-α inhibitor, effectively abrogated the embryotoxicity that was induced by CH-PF. CONCLUSION(S): These data collectively highlight the crucial role of TNF-α in CH-PF-induced embryotoxicity and suggest that TNF-α inhibitors may be potential therapeutic agents for treating endometriosis-induced infertility.

Collagen IV significantly enhances migration and transplantation of embryonic stem cells: involvement of α2ß1 integrin-mediated actin remodeling.

Embryonic stem (ES) cell transplantation represents a potential means for the treatment of degenerative diseases and injuries. As appropriate distribution of transplanted ES cells in the host tissue is critical for successful transplantation, the exploration of efficient strategies to enhance ES cell migration is warranted. In this study we investigated ES cell migration under the influence of various extracellular matrix (ECM) proteins, which have been shown to stimulate cell migration in various cell models with unclear effects on ES cells. Using two mouse ES (mES) cell lines, ESC 26GJ9012-8-2 and ES-D3 GL, to generate embryoid bodies (EBs), we examined the migration of differentiating cells from EBs that were delivered onto culture surfaces coated with or without collagen I, collagen IV, Matrigel, fibronectin, and laminin. Among these ECM proteins, collagen IV exhibited maximal migration enhancing effect. mES cells expressed α2 and ß1 integrin subunits and the migration enhancing effect of collagen IV was prevented by RGD peptides as well as antibodies against α2 and ß1 integrins, indicating that the enhancing effect of collagen IV on cell migration was mediated by α2ß1 integrin. Furthermore, staining of actin cytoskeleton that links to integrins revealed well-developed stress fibers and long filopodia in mES cells cultured on collagen IV, and the actin-disrupting cytochalasin D abolished the collagen IV-enhanced cell migration. In addition, pretreatment of undifferentiated or differentiated mES cells with collagen IV resulted in improved engraftment and growth after transplantation into the subcutaneous tissue of nude mice. Finally, collagen IV pretreatment of osteogenically differentiated mES cells increased osteogenic differentiation-like tissue and decreased undifferentiation-like tissue in the grafts grown after transplantation. Our results demonstrated that collagen IV significantly enhanced the migration of differentiating ES cells through α2ß1 integrin-mediated actin remodeling and could promote ES cell transplantation efficiency, which may be imperative to stem cell therapy.

Non-classical estrogen receptors action on human dermal fibroblasts.

OBJECTIVE: To study the possible non-genomic effect of selective estrogen receptor modulators on human dermal fibroblasts (HDF). MATERIALS AND METHODS: WS1 cells were used to test the effect of raloxifene. The mRNA expressions of estrogen receptor (ER) α and ß and G protein-coupled ER 1(GRP30) were examined by reverse transcription polymerase chain reaction. Apoptosis was identified by TUNEL assay and FACS analysis. MAPK and PI3 K/Akt pathways were determined by immunoblotting analysis. RESULTS: Neither ERα nor ERß, but GPR30 was detected in WS1 cells. Raloxifene increased apoptosis, which was blocked by pertussis toxin, an inhibitor of G protein, or by LY294002. Phosphorylated p38 MAPK and Akt were also increased after raloxifene treatment. CONCLUSION: SERMs could induce apoptosis of HDF through G protein and PI3 K/Akt signaling, which may help understand the role of SERMs on the skin.

Roles of estrogen and progesterone in endometrial hemodynamics and vascular endothelial growth factor production.

BACKGROUND: The endometrium becomes receptive to the embryo after sequential actions of estrogen and progesterone. The purpose of this study was to examine the effects of estrogen and progesterone on endometrial hemodynamics and on secretion of vascular endothelial growth factor (VEGF) from endometrial epithelial cells (EEC). METHODS: Six early postmenopausal women taking sequential estrogen and progestin [days 1-11: estradiol valerate (estrogen) 2 mg daily; days 12-21: estradiol valerate 2 mg plus norethisterone acetate (progestin) 1 mg daily] were recruited. Three-dimensional power Doppler angiography (3D-PDA) was performed before hormone treatment (phase 0), on days 10-11 of hormone treatment (phase E), and on days 18-20 of hormone treatment (phase E + P). Ishikawa EEC were treated with or without 17-beta-estradiol and progesterone for 24 hours, followed by determination of VEGF concentrations in the supernatants. RESULTS: The endometrial volume was significantly increased in phase E and phase E + P as compared with that in phase 0. The vascularization index, flow index, and vascularization flow index in the subendometrial region, as measured by 3D-PDA, were significantly higher in phase E + P than in phase 0, but there were no significant differences in these indices between phase 0 and phase E. While treatment of EEC with 17-beta-estradiol had little enhancing effect on VEGF production, progesterone alone or in combination with 17-beta-estradiol significantly increased VEGF secretion from EEC. CONCLUSION: Our data suggested that progesterone could stimulate VEGF secretion from EEC and subsequently increase subendometrial vascularity and blood flow.

Induction of p38 mitogen-activated protein kinase-mediated apoptosis is involved in outgrowth of trophoblast cells on endometrial epithelial cells in a model of human trophoblast-endometrial interactions.

During embryo implantation in species with hemochorial placentation, such as the mouse and human, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Signs of apoptosis were demonstrated in luminal endometrial epithelial cells (EEC) adjacent to the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Because mitogen-activated protein kinases (MAPK) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, the possible involvement of MAPK during trophoblast-EEC interactions was studied. By coculturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction, we found that most spheroids rapidly attached to EEC monolayers and then progressively expanded, with marked dislodgment of EEC adjacent to the spreading trophoblast cells. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC after coculture. However, only SB203580 (a p38 MAPK inhibitor), but not PD98059 (an ERK inhibitor), inhibited trophoblast outgrowth on EEC monolayers through the suppression of p38 MAPK activation in EEC. Furthermore, trophoblast expansion caused prominent EEC apoptosis at the spheroid-EEC interface, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (which binds activated caspases) staining, and SB203580 significantly decreased the percentage of apoptotic cells. Our results, based on a model of human trophoblast-EEC interactions, establish that trophoblast cells cause activation of p38 MAPK in EEC and, consequently, induce apoptosis and displacement of EEC, a process that may facilitate implantation.

Distinct mechanisms account for beta-amyloid toxicity in PC12 and differentiated PC12 neuronal cells.

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.

Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers.

During implantation, complex embryo-endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo-endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25 x g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids were attached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200-1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst-EEC adhesion. Spheroid-EEC adhesion was inhibited by blocking Ca(2+) influx with extracellular Ca(2+) chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or a Ca(2+) channel blocker (verapamil) but not by interfering with Ca(2+) release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.