GSK-3β is essential for physiological electric field-directed Golgi polarization and optimal electrotaxis

Endogenous electrical fields (EFs) at corneal and skin wounds send a powerful signal that directs cell migration during wound healing. This signal therefore may serve as a fundamental regulator directing cell polarization and migration. Very little is known of the intracellular and molecular mechanisms that mediate EF-induced cell polarization and migration. Here, we report that Chinese hamster ovary (CHO) cells show robust directional polarization and migration in a physiological EF (0.3–1 V/cm) in both dissociated cell culture and monolayer culture. An EF of 0.6 V/cm completely abolished cell migration into wounds in monolayer culture. An EF of higher strength (≥1 V/cm) is an overriding guidance cue for cell migration. Application of EF induced quick phosphorylation of glycogen synthase kinase 3β (GSK-3β) which reached a peak as early as 3 min in an EF. Inhibition of protein kinase C (PKC) significantly reduced EF-induced directedness of cell migration initially (in 1–2 h). Inhibition of GSK-3β completely abolished EF-induced GA polarization and significantly inhibited the directional cell migration, but at a later time (2–3 h in an EF). Those results suggest that GSK-3β is essential for physiological EF-induced Golgi apparatus (GA) polarization and optimal electrotactic cell migration

Disruption of the Basement Membrane after Corneal Débridement Animal Model

PURPOSE. To determine whether the native basement membrane left behind after manual débride-ment wounding is retained throughout healing in the Balb/c mouse. METHODS. Mouse corneas were subjected to either 1.5 mm (small) or limbus-to-limbus (large) epithelial débridement wounds and allowed to heal for times ranging from 12 hours to 3 days. For the larger wounds, care was taken to leave an approximately 0.5-mm zone of epithelial cells near the limbal border. Unwounded corneas served as control specimens. At each time point, confocal immunofluorescence microscopy was used to localize several proteins found in the basement membrane including laminin-5, entactin, and perlecan. In addition, ultrastructural studies were performed using transmission electron microscopy (TEM) to assess the basement membrane zone (BMZ) of the corneas at various times after injury. RESULTS. The smaller (1.5-mm) wounds healed within 24 hours, and the larger wounds healed at approximately 48 hours. Both wound sizes healed with little scarring or neovascularization. At all time points after 1.5-mm wounding, immunofluorescence confocal microscopy and TEM showed that both basement membrane proteins and the lamina densa were retained at the BMZ throughout healing. For the larger wounds, at time points after 24 hours, confocal microscopy showed patches along the denuded corneal stroma where there was a partial or complete loss of basement membrane markers at the BMZ. TEM confirmed that the lamina densa was partly or completely absent along the anterior surface of the exposed cornea at time points of more than 24 hours after the larger wounds. CONCLUSIONS. The denuded epithelial basement membrane was shown to be partially disassembled in response to manual débridement wounds when re-epithelialization took more than 24 hours. Regulated disassembly of the epithelial basement membrane probably plays a role in the healing of large-diameter débridement wounds. (Invest Ophthalmol Vis Sci. 2000;41:1045-1053 S tudies of corneal wound healing after manual débride-ment were first conducted more than 30 years ago and have been shown by numerous groups to leave the lamina densa of the basement membrane intact and in its native state after wounding. 1-5 Based on previous studies, 6,7 we know that the mouse cornea re-epithelializes after manual débridement between 20 to 24 hours in the 8-week-old Balb/c mouse after wounds removing no more than 40% of the epithelial surface (1.5 mm; small wounds). Re-epithelialization of small wounds is accompanied by increased expression of ␣6␤4 integrin, 8 a structural component of the hemidesmosomes and a signaling molecule known to regulate epithelial cell proliferation. 9 -11 Extracellular ligands for ␣6␤4 integrin are members of the laminin family of adhesive glycoproteins, primarily laminin-1 and -5. Laminin-5 is a component of the anchoring filaments of hemidesmosome adhesion complexes. It also has been shown recently to be secreted by migrating epithelial cells in the skin in response to blisters and deeper, more penetrating wounds 12 and by corneal epithelial cells in response to manual keratectomy wounds. 13 Laminin-1 is also a basement membrane component, 7 Unlike ␣6␤4, the functions of ␣9␤1 in epithelial cells are unclear. To determine whether alterations in the nature of the substrate, the basement membrane zone (BMZ), are associated with the increased expression of either ␣6␤4 or ␣9␤1 observed in response to corneal débridement wounding, we performed both small and large débridement wounds and evaluated tissues by immunohistochemistry for the localization of several different basement membrane proteins at a variety of different time points after wounding. We also evaluated the morphology of the corneal epithelial basement membrane directly by transmission electron microscopy (TEM)

Characteristics of Progenitor Cells Derived from Adult Ciliary Body in Mouse, Rat, and Human Eyes

PURPOSE. To isolate and characterize progenitor cells derived from adult mammalian ciliary body. METHODS. The authors isolated progenitor cells from the ciliary body of adult mice, rats, and human cadaver eyes and determined quantitative growth characteristics of groups of progenitor cells called neurosphere (NS) cells, including individual cell diameter, NS diameter, percentage of NS-forming cells, and cell number per eye in mouse, rat, and human eyes. The immunolabeling and ultrastructure of NS cells were investigated by confocal and transmission electron microscopy. RESULTS. Average diameters of individual cells and neurospheres after 1 week in culture were similar in mice, rats, and humans (cell diameters: 22 Ϯ 1.1, 21 Ϯ 0.3, 25 Ϯ 0.4 m; NS diameters: 139 Ϯ 22, 137 Ϯ 9, 141 Ϯ 11 m, respectively). Mean numbers of cells per NS were estimated to be 1183 in mice, 5360 in rats, and 685 in humans. Molecules that were identified by immunolabeling in NS cells included nestin, Chx-10, vimentin, GFAP, and Pax-6. Thy-1 was expressed in some NS cells. Ultrastructurally, NS cells displayed abundant rough endoplasmic reticulum and many cellular processes but no characteristics of mature retinal neurons or glia. CONCLUSIONS. Progenitor cells from adult mammalian ciliary body have significant, but limited, proliferation potential and express markers characteristic of other progenitor cells and seen during early retinal development. The ciliary body could be a source of cells for transplantation in experimental rodent eyes and for autotransplantation in human eyes. (Invest Ophthalmol Vis Sci

Electrical cues regulate the orientation and frequency of cell division and the rate of wound healing in vivo

Controlling cell division is fundamental. One environmental cue that exerts profound control over both the orientation and frequency of cell division in vivo is a naturally occurring, wound-induced electric field (EF). Wounds in rat corneas generate endogenous EFs in the plane of the epithelial sheet because the transcorneal potential difference (TCPD; +40 mV internally positive) collapses at the wound edge, but is maintained at normal levels at 0.5 mm back from the wound. We manipulated the endogenous EF this creates by using drugs with differing actions. The wound-induced EF controlled the orientation of cell division; most epithelial cells divided with a cleavage plane parallel to the wound edge and perpendicular to the EF vector. Increasing or decreasing the EF pharmacologically, respectively increased or decreased the extent of oriented cell division. In addition, cells closest to the wound edge, where the EF was highest, were oriented most strongly by the EF. Remarkably, an endogenous EF also enhanced the frequency of cell division. This also was regulated by enhancing or suppressing the EF pharmacologically. Because the endogenous EF also regulated the wound healing rate, it may act as one control of the interplay between cell migration and cell division during healing

β4 Integrin and Epidermal Growth Factor Coordinately Regulate Electric Field-mediated Directional Migration via Rac1

Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (β)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), β4 integrin-null human keratinocytes (β4−), or those in which β4 integrin was reexpressed (β4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of β4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding–defective β4 (β4+AD) or β4 with a truncated cytoplasmic tail (β4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the β4 integrin ligand–binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus

Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture

The authors have previously documented decreased epithelial basement membrane (BM) components and α(3)β(1) epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar–collagen gel at the air–liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 α5 chain, nidogen-1/entactin, integrin α(3)β(1), and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of α(3)β(1) integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized α(3)β(1) integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas