Advanced glycation end products (AGEs) have an important role in diabetic complications, with many responses mediated through AGE-receptors. The current study has investigated the binding and uptake of AGEs by retinal microvascular endothelium in an attempt to understand the nature of AGE-interaction with receptors on the cell surface. There has been special emphasis placed on the R1, R2, and R3 components of AGE-receptor complex (AGE-RC) and their localization to caveolin-rich membrane domains. Retinal microvascular endothelial cells (RMECs) were exposed to either AGE-modified BSA (AGE-BSA) or native BSA conjugated to colloidal gold (gAGE, gBSA) for various time periods, fixed, and processed for transmission electron microscopy (TEM). Localization of AGE-RC components in caveolae was investigated using confocal microscopy and ultrastructural immunogold labeling. Caveolae were extracted from RMECs using differential Triton X-100 solubility, and Western analysis was conducted to test for caveolae enrichment and the presence of AGE-RC complex components. Ligand blots determined 125I-AGE-BSA binding to caveolae-enriched extracts. Colloidal gold conjugates of AGE-BSA bound to caveolae and were internalized to be trafficked to lysosomal-like compartments. AGE-receptor complex components were significantly enriched within caveolae. The data suggest that AGEs interact with their receptors within caveolae. It is significant that the AGE-R complex localizes to these organelles, because this may have implications for AGE binding, internalization, signal transduction, and the modulation of AGE-receptor-mediated vascular cell dysfunction.
The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.
It has been demonstrated that inhibiting Notch signaling through γ-secretase inhibitor (GSI) treatment increases myogenesis, AKT/mTOR signaling, and muscle protein synthesis (MPS) in C2C12 myotubes. The purpose of this study was to determine if GSI-mediated effects on myogenesis and MPS are dependent on AKT/mTOR signaling. C2C12 cells were assessed for indices of myotube formation, anabolic signaling, and MPS following GSI treatment in combination with rapamycin and API-1, inhibitors of mTOR and AKT, respectively. GSI treatment increased several indices of myotube fusion and MPS in C2C12 myotubes. GSI-mediated effects on myotube formation and fusion were completely negated by treatment with rapamycin and API-1. Meanwhile, GSI treatment was able to rescue MPS in C2C12 myotubes exposed to rapamycin or rapamycin combined with API-1. Examination of protein expression revealed that GSI treatment was able to rescue pGSK3β Ser9 despite AKT inhibition by API-1. These findings demonstrate that GSI treatment is able to rescue MPS independent of AKT/mTOR signaling, possibly via GSK3β modulation.
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