Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells

In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.     

Cell sources, regulatory factors and gene expression in the regeneration of the crystalline lens and retina in vertebrate animals

Over the past century extensive experimental materials have been accumulated concerning cell sources of lens and retina regeneration, successive transformations of the cells, regulatory factors, and gene expression during restitution of these eye structures. The use of nuclear and cytoplasmic markers provided convincing evidence that the removed lens is restituted from the dorsal iris cells in vivo or from embryonic cells of the pigment epithelium and retina in vitro. The removed or destroyed retina is restituted as a result of transdifferentiation of the pigment epithelium cells in amphibians, fish, birds, and mammals during embryogenesis, in larvae of some anuran amphibians, and in adult newts. Cell precursors of rods are a cell source of retina regeneration in adult fish. A subpopulation of randomly distributed cells, which are a cell source of rod formation during the normal development of the eye was found in the external nuclear layer with the use of electron microscopy and nuclear and cytoplasmic markers. These cells are not only a source of regeneration of rods, but also of cones and cells of the internal nuclear layer after destruction of the corresponding retina layers. There is a peripheral growth area in the retina of vertebrates, where multi- and unipolar cells are localized, which provide for the retina growth during ontogenesis. A paradox of retina regeneration consists in that these little differentiated cells are not a source of complete restitution of the removed or destroyed retina. They make only a small contribution to its regeneration corresponding to the growth potential of cells of this eye region, while restitution of the retina proceeds only at the expense of cells of another type of differentiation. A factor controlling the differentiated state of the cell was found in the dorsal iris during studies of lens regeneration. Removal of this factor in the early stages of cell transformations leads to the initiation of lens regeneration. The factor is not specific and was identified in many cells of vertebrates, including the pigment epithelium and limb tissues, which, as is known, may be fully restituted. Studies of gene expression during lens and retina regeneration are now at the initial stage. The greatest advances were achieved on the model of transdifferentiation of the pigment epithelium cells of chick embryos into lentoids. Expression of genes MMP115 and pP344 was established in the pigment epithelium cells, which characterize the pigmented phenotype of the initial cells. Expression of the alpha-, beta-, and delta-crystallin genes was found in the lentoids, which characterize the phenotype of regenerating structures. The gene activity appears to be switched at an intermediate stage during cell dedifferentiation. Expression of the gamma-crystallin genes during lens regeneration in adult newts is initiated after completion of dedifferentiation and cell proliferation in the dorsal iris. The genes specifically expressed in the dorsal and ventral iris and in the retina rudiment have been identified by the method of gene subtraction. Expression of homeobox-containing genes from the family of PAX genes was found during lens regeneration in adult newts and retina regeneration in adult fish. The role of growth factors (FGF) as morphogenetic factors was proved, which are involved in a yet unknown way of altering the differentiation pathway of the initial cells during formation of the neuroepithelium rudiment in chick embryos, adult newts, and fish.     

Properties of lens epithelial cells in albino rabbits--morphological alteration and its influence

We studied changes of epithelial cells in injured lens, after extracapsular cataract extraction (ECCE) in culture solution and in vivo changes in epithelial cells after phacoemulsification and aspiration (PEA) using light and electron microscopy. The epithelial cells at the injured site of the lens were transformed into spindle cells indicating healing and contraction of the lesion. In the culture solution, epithelial cells which had been seen only on the side of the anterior capsule extended and proliferated over the equatorial zone to the posterior capsule covering the whole posterior capsule 6 weeks after an ECCE. Spindle cells were seen in the region with folds in the posterior capsule. In vivo, formation of Soemmering's ring was observed in the equatorial zone after PEA suggesting regeneration of the lens in the electron microscopic aspect. At the edge of the anterior capsule, appearance of spindle cells formation of folds in the anterior and posterior capsules at this site as well as formation of membrane similar to the lens capsule could be seen, and this phenomenon resembled the healing process of the injured lens. Observation of the spindle cells at the cut edge of the anterior capsule showed cells which were extending psudopodia into collagen and they appeared to be engaged in ameboid movement. We considered that this ameboid movement would trigger off contraction of the wound and formation of folds at the cut edge of the anterior capsule.     

In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies

PURPOSE: To evaluate different in-vitro cell culture models for their suitability to study drug transport through cell monolayers. METHODS: Bovine turbinate cells (BT; ATCC CRL 1390), human nasal septum tumor cells (RPMI, 2650; ATCC CCL 30), and primary cell cultures of human nasal epithelium were characterized morphologically and histochemically by their lectin binding properties. The development of tight junctions in culture was monitored by actin staining and transepithelial electrical resistance measurements. RESULTS: The binding pattern of thin-sections of excised human nasal respiratory epithelium was characterized using a pannel of fluorescently-labelled lectins. Mucus in goblet cells was stained by PNA, WGA and SBA, demonstrating the presence of terminal N-acetylglucosamine, N-acetylgalactosamine and galactose residues respectively in the mucus of human nasal cells. Ciliated cells revealed binding sites for N-acetylglucosamine, stained by WGA, whereas Con A, characteristic for mannose moieties, labelled the apical cytoplasm of epithelial cells. Binding sites for DBA were not present in this tissue. Comparing three different cell culture models: BT, RPMI 2650, and human nasal cells in primary culture using three lectins (PNA, WGA, Con A) as well as intracellular actin staining and transepithelial electrical resistance measurements we found, that only human nasal epithelial cells in primary culture showed differentiated epithelial cells, ciliated nasal cells and mucus producing goblet cells, which developed confluent cell monolayers with tight junctions. CONCLUSIONS: Of the in-vitro cell culture models studied, only human nasal cells in primary culture appears to be suitable for drug transport studies.     

Role of small GTP-binding proteins in lovastatin-induced cataracts

PURPOSE: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent. METHODS: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays. RESULTS: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes. CONCLUSIONS: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.     

Activation of the Jak-STAT-signaling pathway in embryonic lens cells

Previous studies showed that lens epithelial cells proliferate rapidly in the embryo and that a lens mitogen, most likely derived from the blood, is present in the anterior chamber of the embryonic eye (Hyatt, G. A., and Beebe, D. C., Development 117, 701-709, 1993). Messenger RNAs for several growth factor receptors have been identified in embryonic lens epithelial cells. We tested several growth factors that are ligands for these receptors for their ability to maintain lens cell proliferation. Embryo serum, PDGF, GM-CSF, and G-CSF maintained lens cell proliferation, but NGF, VEGF, and HGF did not. This and a previous study (Potts, J. D., Harocopos, G. J., and Beebe, D. C., Curr. Eye Res. 12, 759-763, 1993) detected members of the Janus kinase family (Jaks) in the developing lens. Because Jaks are central players in the Jak-STAT-signaling pathway, we identified STAT proteins in the lens and tested whether they were phosphorylated in response to mitogens. STAT1 and STAT3, but not STAT 5 were detected in chicken embryo lens epithelial cells. Only STAT3 was found in terminally differentiated lens fiber cells. STAT1 and STAT3 were phosphorylated in lens cells analyzed immediately after removal from the embryo and when lens epithelial explants were treated with embryo serum, PDGF, or GM-CSF, but not with NGF. Chicken embryo vitreous humor or IGF-1, factors that stimulate lens cell differentiation, but not proliferation, did not cause STAT phosphorylation. When lens epithelial cells were cultured for 4 h in unsupplemented medium, STAT1 and STAT3 declined to nearly undetectable levels. Treatment with PDGF or embryo serum for an additional 15 min restored STAT1 and -3 levels. This recovery was blocked by cycloheximide, but not actinomycin D, suggesting that STAT levels are regulated at the level of translation. STAT levels were maintained in epithelial explants by lens mitogens, but not by factors that stimulated lens fiber differentiation. Both factors that stimulated lens cell proliferation and those that caused fiber differentiation protected cultured lens epithelial cells from apoptosis. These data suggest that the factor(s) responsible for lens cell proliferation in vivo activates the Jak-STAT-signaling pathway. They also indicate that growth factors maintain STAT protein levels in lens epithelial cells by promoting the translation of STAT mRNA, an aspect of STAT regulation that has not been described previously. Signaling by most of the growth factors and cytokines known to activate the Jak-STAT pathway has been disrupted in mice by mutation or targeted deletion. Consideration of the phenotypes of these mice suggests that the factor responsible for lens cell proliferation in vivo may be a growth factor or cytokine that has not yet been described.     

Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels

Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in beta 1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for beta 1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the beta 1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The alpha 5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and alpha 3 continues to be expressed at about the same level as in the epithelium. alpha 6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for alpha 5 beta 1. RGD peptides do not inhibit the transformation but antibodies to beta 1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the beta 1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial-mesenchymal transition.     

Responsibilities of regulatory agencies

If the eye lens of the adult newt, Notophthalmus viridescens, is removed, a new lens will regenerate and only from the dorsal, not the ventral, iris. The source, pigmented epithelial cells, would normally no longer divide, but upon lentectomy they do re-enter the cell cycle and form lens. The cause for this capability is unknown, but the mitogenic Fibroblast Growth Factors and their receptors may be involved. We have demonstrated that FGF receptors are present and operative in lens regeneration, since receptor-directed mitotoxins inhibit regeneration; heterogeneity and differential density in FGF-binding and receptor localization in iris sectors is also present. We propose that the spatial distribution of FGF receptors, especially the amphibian homolog of FGFR-3, is important in initiation of regeneration of eye lens.     

Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats

BACKGROUND: Tubulointerstitial fibrosis is the final common pathway to end-stage renal failure. The present study investigated the potential role of tubular epithelial cells (TEC) in progressive fibrosis in the rat remnant kidney model. METHODS: Rats underwent 5/6 nephrectomy or a sham operation (control), and groups of six animals were killed at weeks 1, 3, 5, 9, 13, 17 and 21. RESULTS: Immunohistochemistry staining and in situ hybridization at week 3 after nephrectomy demonstrated de novo expression of alpha-smooth muscle actin (alpha-SMA)--a marker of smooth muscle cells and myofibroblasts--by TEC that was invariably associated with disruption of the tubular basement membrane (TBM). This phenotypic evidence of tubular epithelial-myofibroblast transdifferentiation was supported by ultrastructural studies identifying the presence of characteristic actin microfilaments and dense bodies within TEC with a transformed morphology. In the late stage of this apparent tubular epithelial-myofibroblast transdifferentiation, TEC lost apical-basal polarity and tight junctions, became elongated, detached from the TBM, separated from neighboring cells and appeared to migrate into the peritubular interstitium through the damaged basement membrane. Indeed, focal peritubular accumulation of alpha-SMA+ myofibroblasts and local tubulointerstitial fibrosis was closely associated with alpha-SMA+ tubules, suggesting a tubular epithelial origin for some of these cells. Quantitative analysis found a significant correlation between the number of alpha-SMA+ TEC and the accumulation of interstitial alpha-SMA+ myofibroblasts and the severity of tubulointerstitial fibrosis (both P < 0.001). CONCLUSIONS: This study provides phenotypic and morphological evidence to support the hypothesis that TEC are pro-fibrogenitor cells capable of tubular epithelial-myofibroblast transdifferentiation in progressive renal fibrosis. In addition, we postulate that disruption of the TBM, which facilitates epithelial cell contact with the interstitial matrix, promotes this process of transdifferentiation.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

We observed the three dimensional structure of cellular aggregates formed from chick retinal cells in a floating culture system for 2 months. The aggregated cells partially mimicked the structure of the retina and showed differentiation of photoreceptor cells and Müller cells with numerous synapses. Immunohistochemical studies showed the number of anti-rhodopsin positive cells increasing over time. In the long-term culture, increasing anti-crystalline positive cells appeared late in the culture, indicative of differentiation of lens epithelial cells. Nerve, epidermal, and basic fibroblast growth factors, and co-culture with retinal pigment epithelial cells stimulated to some degree the growth of dendrites in retinal cellular aggregates. Epidermal growth factor, in particular, promoted the production of rhodopsin in photoreceptor cells. Retinal cellular aggregates in a floating culture system could be used to examine the effect of various factors on differentiation of the neuroretina.     

Structural changes in lenses of mice lacking the gap junction protein connexin43

PURPOSE: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization. METHODS: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections. RESULTS: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions. CONCLUSIONS: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.     

Antibiotic susceptibility among aerobic gram-negative bacilli in intensive care units in 5 European countries. French and Portuguese ICU Study Groups

BACKGROUND: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. METHODS: In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. RESULTS: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy. CONCLUSION: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.     

Feline ocular epithelial response to growth factors in vitro

OBJECTIVE: To examine the proliferative abilities of growth factors known to participate in wound healing on feline lens, iris pigment, ciliary, and retinal pigment epithelium cultured in vitro. ANIMALS: 8 clinically normal cats. PROCEDURE: Iris pigment, lens, ciliary, and retinal pigment epithelia of normal eyes of cats were isolated and cultured. Morphologic characteristics of primary cell cultures were studied by light and electron microscopy. Subcultures of epithelial cells were exposed to media supplemented with 0.5% fetal bovine serum plus various combinations of insulin and/or growth factors, including transforming growth factor-alpha, epidermal growth factor, acidic fibroblast growth factor, and basic fibroblast growth factor. Growth promoting effects were evaluated by counting with an electronic cell counter. RESULTS: Cells retained many of the morphologic characteristics of in vivo cells. Cell proliferation assays indicated that transforming growth factor-alpha stimulated lens and ciliary epithelial cell growth, and epidermal growth factor enhanced lens and iris pigment epithelial cell growth. Acidic fibroblast growth factor had proliferative effects on lens, iris pigment, and ciliary epithelium. Basic fibroblast growth factor was the most potent stimulator of all mitogens used, and caused substantial proliferation in all cell types. Insulin alone stimulated lens and ciliary epithelial proliferation but, combined with other growth factors, had a synergistic effect with those causing cell proliferation, except acidic fibroblast growth factor with iris pigment epithelium. CONCLUSION: Morphologic studies support the argument that pigment-producing cells are involved in feline ocular sarcoma. Growth factor studies indicated that ciliary epithelium has the most profound proliferative effect of all growth factors used. These data may help guide future studies in determining the cell of origin for feline ocular sarcoma.     

The conjunctiva in corneal epithelial wound healing

BACKGROUND/AIMS: During the healing of corneal epithelial wounds with limbal involvement, conjunctival epithelium often migrates across the denuded limbus to cover the corneal surface. It is believed that, over a period of time, conjunctival epithelium covering the cornea assumes characteristics of corneal epithelium by a process referred to as conjunctival transdifferentiation. The purpose of this study was to examine, clinically, the fate of conjunctival epithelial cells covering the cornea and to assess the healing of corneal epithelial wounds when the conjunctival epithelium was removed or actively prevented from crossing the limbus and extending onto the cornea. METHODS: 10 patients with conjunctivalisation of the cornea were followed for an average of 7.5 months. Five patients in this group had their conjunctival epithelium removed from the corneal surface and allowed to heal from the remaining intact corneal epithelium. In another four patients with corneal epithelial defects, the conjunctival epithelium was actively prevented from crossing the limbus by mechanically scraping it off. RESULTS: The area of cornea covered by conjunctival epithelium appeared thin, irregular, attracted new vessels and was prone to recurrent erosions. Conjunctivalisation of the visual axis affected vision. Removal of conjunctival epithelium from the cornea allowed cells of corneal epithelial phenotype to cover the denuded area with alleviation of symptoms and improvement of vision. It was also established that migration of conjunctival epithelium onto corneal surface could be anticipated by close monitoring of the healing of corneal epithelial wounds, and prevented by scraping off conjunctival epithelium before it reached the limbus. CONCLUSION: This study shows that there is little clinical evidence to support the concept that conjunctival transdifferentiation per se, occurs in humans. "Replacement" of conjunctival epithelium by corneal epithelial cells may be an important mechanism by which conjunctival "transdifferentiation" may occur. In patients with partial stem cell deficiency this approach can be a useful and effective alternative to partial limbal transplantation, as is currently practised.     

Lens epithelium: a primary target of UVB irradiation

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.     

The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.     

Lens-specific expression of PDGF-A alters lens growth and development

The vertebrate lens provides an in vivo model to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development by in situ hybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-alpha receptor (PDGF-alphaR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-alphaR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens development in vivo, we generated transgenic mice that express human PDGF-A in the lens under the control of the alphaA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific beta-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cells in vivo. Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Expression of the beta 4 integrin subunit in the mouse heart during embryonic development: retinoic acid advances beta 4 expression

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H2O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between approximately 10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction.