METHODS: Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph.
RESULTS: 15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.
CONCLUSION: We conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.
Methods: BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages.
Results: Corneal stem cell markers β1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct.
Conclusions: In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.
METHOD AND MATERIAL: Fresh, vaginally delivered placentae from ten normotensive pregnant women and nine women with pre-eclampsia were carefully dissected and 4 gm each of amnion, chorion laeve, placental plate chorion, fetal placenta (fetal surface of the placenta) and maternal placenta (surface of the placenta attached to the uterine wall) were obtained. These tissues were then thoroughly washed in a 0.5 M phosphate buffer, pH 7.5, at room temperature and then individually homogenized for one minute in 4 ml of the same buffer. After centrifugation the supernatant was removed. The pellet was re-suspended in buffer, re-homogenized and then centrifuged. The supernatant was removed and the procedure was repeated once again and the three supernatants of each tissue were pooled. Endothelin-1 was estimated by RIA. All results are presented as mean+/-SEM. Statistical analysis was performed using students 't' test for unpaired samples and a 'p' value of <0.05 was considered significant.
RESULTS: In tissues from normotensive pregnant women, no significant differences were evident in endothelin-1 concentrations in the chorion laeve, fetal placenta and maternal placenta but were significantly higher than those in the amnion and placental plate chorion (p<0.01). In tissues from pre-eclamptic women, no significant differences were evident between endothelin-1 concentrations in the chorion laeve, placental plate chorion and fetal placenta. Mean endothelin-1 concentration in the amnion and maternal placenta were significantly lower than those in chorion laeve, placental plate chorion and fetal placenta (p<0.01). Endothelin-1 concentrations were significantly higher in the amnion, chorion laeve, placental plate chorion and fetal placenta from women with pre-eclampsia when compared to tissues from normotensive pregnant women (p<0.01).
CONCLUSIONS: Endothelin-1 levels were significantly higher in the placental tissues from women with pre-eclampsia. Endothelin-1, being a powerful vasoconstrictor, could cause significant vasoconstriction in the placental vasculature, and alterations in endothelin-1 levels in placental vasculature may therefore have a role in the pathogenesis of pre-eclampsia.