Recovery of Corneal Endothelial Cells from Periphery after Injury

Background

Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.

Aim

To investigate the recovery process of corneal endothelial cells (CECs) from corneal endothelial injury.

Methods

Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group). Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.

Results

Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.

Conclusions

CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.     

Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.     

NGF-induced moesin phosphorylation is mediated by the PI3K,Rac1 and Akt and required for neurite formation in PC12 cells

Moesin is a member of ERM family proteins which act as the cross-linkers between plasma membrane and actin-cytoskeleton and is activated by phosphorylation at Thr-558. In neurons, suppression of radixin and moesin alters the growth cone morphology. However, the significance of phosphorylation of ERM proteins in neuronal cells has not been fully investigated. In this study, we studied the signaling pathways responsible for moesin phosphorylation and its functional importance in NGF-treated PC12 cells. NGF rapidly induced the phosphorylation of moesin at Thr-558 in PC12 cells which was dependent on PI3K and Rac1. We found that Akt interacted and phosphorylated with moesin both in vitro and in vivo. Inhibition of PI3K and Rac1 abolished the NGF-induced Akt activation, indicating that Akt is at the downstream of PI3K and Rac1. To examine the functional role of phosphorylated ERM proteins, a dominant negative mutant form of moesin (T558A) was introduced into PC12 cells. The mutant significantly reduced the frequency of cells with neurites following NGF treatment. Our results indicate that PI3K, Rac1 and Akt-dependent phosphorylation of moesin is required for the NGF-induced neurite formation in differentiating PC12 cells.     

Activation of RhoA and FAK induces ERK-mediated osteopontin expression in mechanical force-subjected periodontal ligament fibroblasts

The precise mechanism by which Rho kinase translates the mechanical signals into OPN up-regulation in force-exposed fibroblasts has not been elucidated. Human periodontal ligament fibroblasts (hPLFs) were exposed to mechanical force by centrifuging the culture plates at a magnitude of 50 g/cm² for 60 min. At various times of the force application, they were processed for analyzing cell viability, trypan blue exclusion, and OPN expression at protein and RNA levels. Cellular mechanism(s) of the force-induced OPN up-regulation was also examined using various kinase inhibitors or antisense oligonucleotides specific to mechanosensitive factors. Centrifugal force up-regulated OPN expression and induced a rapid and transient increase in the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and Elk1. Pharmacological blockade of RhoA/Rho-associated coiled coil-containing kinase (ROCK) signaling markedly reduced force-induced FAK and ERK1/2 phosphorylation. Transfecting hPLFs with FAK antisense oligonucleotide diminished ERK1/2 activation and force-induced OPN expression. Further, ERK inhibitor inhibited significantly OPN expression, Elk1 phosphorylation, and activator protein-1 (AP-1)-DNA binding activation, but not FAK phosphorylation, in the force-applied cells. These results demonstrate that FAK signaling plays critical roles in force-induced OPN expression in hPLFs through interaction with Rho/ROCK as upstream effectors and ERK-Elk1/ERK-c-Fos as downstream effectors.     

Establishment of a resource population of SLA haplotype-defined Korean native pigs

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.     

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.     

Strategies for oral delivery of macromolecule drugs

Advances in biotechnology, gene manipulation, and protein engineering for macromolecule drugs, such as insulin, parathyroid hormone (PTH), calcitonin, human growth hormone, erythropoietin (EPO), and peptide YY (PYY) allow commercial production in large scale for diverse therapeutic uses. Other macromolecules, such as mucopolysaccharide heparin, have expanded markets through improvements in their pharmacokinetic and pharmacological effects. However, most products are available only as injectable forms and are limited to patients with no alternative therapeutic choices. Orally available macromolecule formulations are still unmet needs for improving patient compliance and expanding administration paradigms and indications. Oral delivery technologies including carrier systems, absorption enhancers, protease inhibitors, and modification by conjugating transporter or receptor recognition molecules have been developed and some are undergoing clinical studies. In this review, we discuss major obstacles for oral absorption of macromolecule drugs and summarize recent strategies to overcome the huddles related to enhancing intestinal permeation.