Diverse roles of Eph/ephrin signaling in the mouse lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.     

Effect of advanced glycation end products on lens epithelial cells in vitro

The extended exposure of proteins to reducing sugars leads to nonenzymatic glycation with the accumulation of advanced glycation end products (AGEs). Long-lived proteins, such as collagen and crystallins, are subjected to this modification, and are implicated as causal factors in several diseases including diabetic complications, cataracts, and arteriosclerosis. One means through which AGEs modulate cellular interactions is via binding to specific receptors. In the current study, the existence of AGEs in human anterior polar lens capsules of cataracts was confirmed using a combination of dot-immunoblot and fluorescent detection. Human lens epithelial cells (LECs) attached to anterior lens capsules expressed mRNA for the receptor for AGEs (RAGE). The interaction of LECs with AGEs using bovine lens epithelial explants demonstrated that AGEs induced mRNAs and proteins of fibronectin, collagen type I, aberrant extracellular matrix proteins, and alpha-SMA, a specific marker for myofibroblastic cells. These findings suggest that AGEs may alter cellular functions which induce mRNAs and proteins associated with fibrosis in LECs.     

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.     

Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions.     

Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats

A spontaneous focal polar anterior subcapsular lenticular opacity characterized by focal epithelial proliferation was found in Charles River Sprague-Dawley rats from various breeding facilities around the world (France, Japan, and the United States). The incidence of this change slightly increased with age up to a maximal incidence of 9.8% in 28- to 35-week-old male rats (French source). Over that period, there was little change in the size of the opacity; however some rats that were examined over longer periods (more than 2 years of age) developed secondary anterior cortical changes, and rarely, histologic findings of pigmentation and/or mineralization. The lenticular change was present throughout the life of the animals and had no sex predilection; mode of inheritance was not investigated. Due to its small size, this lens opacity is more easily identified by use of slit lamp biomicroscopy than by use of indirect ophthalmoscopy, and serial sections of the eye aid in locating it for histologic evaluation.     

The gamma S-crystallin gene is mutated in autosomal recessive cataract in mouse

We established a recessive cataract model from a spontaneous mutation in the KUNMING outbred mice. Lens opacity appears 11 days after birth. Slit lamp examination reveals that the opacity mainly localizes to the nuclear region of the lens. Histological analysis shows a severe degeneration of the epithelial cells underneath the anterior lens capsule, whereas those cells in the equatorial region display an excessive proliferation and migration. Within the cortical area underneath the posterior lens capsule, both vacuoles and morgagnian-like bodies are seen. Blue-stained spherical bodies are observed in the embryonic nucleus, forming a Y-like pattern. We mapped the disease locus and found a homozygous G to A nucleotide conversion at position 489 of Crygs in mutant mice, leading to a truncated gene product (Trp163Stop). This finding suggests that CRYGS is not only a lens structural protein, but is also likely to be involved in epithelial cell proliferation, apoptosis, and migration.     

Cataract Development in Atlantic Salmon (Salmo salar L) in Fresh Water

Irreversible bilateral cataracts were diagnosed by slit-lamp biomicroscopy in 178 of 200 farm-raised Atlantic salmon (Salmo salar L) fed a standard diet over a five-month period. Initial changes were anterior polar opacities, progressing to involve both the anterior and posterior cortex before changes in the lens nucleus were seen. The lens changes were recorded and given scores according to the severity of the cataracts. At each of 3 samplings, after 2, 4 and 5 months, 200 fish were measured, weighed and examined by slit-lamp biomicroscopy. At all 3 samplings, there was a significant correlation between body length and both cataract incidence and cataract severity. There was also a significant correlation between body weight and cataract incidence and severity for the 2 last samplings. There was a significant correlation between K-factor as a measure of the shape of the fish, and both cataract incidence and severity, at all 3 samplings. Evaluation of specific growth rate in the periods between the examinations showed that the rapidly-growing fish were most susceptible to cataract formation. After cataract developed, however, the growth rate slowed. Follow-up examination of severely affected fish 3 months after transfer to sea water showed a normal cortical zone in the periphery of the lens in 24 out of 28 fish.     

Osmotic cataract causes reduced vision in wild Atlantic salmon postsmolts

Osmotic cataracts were diagnosed in all of 191 Atlantic salmon Salmo salar L. postsmolts caught during 8 trawl hauls on the western side of the V?ringsplateau, Norwegian Sea, in June 2001. The changes varied from a hazy opacity in the anterior part of the lens to cataracts affecting the whole lens. Severely affected lenses appeared swollen and large vacuoles were visible in the opaque areas. Large vacuoles in otherwise clear lenses were diagnosed in 1 of 4 adult salmon examined. Histologically, widened sutures, vacuolation of lens epithelium and cortex, and proteinaceous lakes subjacent to the epithelium were the most frequent changes, while extensive cortical necroses and epithelial proliferation were seen in a few cases. UV-absorbance of the aqueous humor was determined and levels compared to plasma levels and also to levels in farmed Atlantic salmon of the same developmental stage. Wild salmon generally showed higher levels of protective factors than farmed fish. The osmotic type of cataract diagnosed leads to poor vision and is a potential cause of reduced survival in postsmolts. The cause of the cataracts could not be determined, but defective osmoregulation is suspected.     

Focus on lens connexins

The lens is an avascular organ composed of an anterior epithelial cell layer and fiber cells that form the bulk of the organ. The lens expresses connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50). Epithelial Cx50 has critical roles in cell proliferation and differentiation, likely involving growth factor-dependent signaling pathways. Both Cx46 and Cx50 are crucial for lens transparency; mutations in their genes have been linked to congenital and age-related cataracts. Congenital cataract-associated connexin mutants can affect protein trafficking, stability and/or function, and the functional effects may differ between gap junction channels and hemichannels. Dominantly inherited cataracts may result from effects of the connexin mutant on its wild type isotype, the other co-expressed wild type connexin and/or its interaction with other cellular components.     

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Vitreoretinal influences on lens function and cataract

The lens is composed of a thin metabolically active outer layer, consisting of epithelial and superficial fibre cells. Lying within this outer shell are terminally differentiated, metabolically inactive fibre cells, which are divided into an outer cortex and central nucleus. Mature fibre cells contain a very high protein concentration, which is important for the transparency and refractive power of the lens. These proteins are protected from oxidation by reducing substances, like glutathione, and by the low-oxygen environment around the lens. Glutathione reaches the mature fibre cells by diffusing from the metabolically active cells at the lens surface. With age, the cytoplasm of the nucleus becomes stiffer, reducing the rate of diffusion and making nuclear proteins more susceptible to oxidation. Low pO(2) is maintained at the posterior surface of the lens by the physical and physiological properties of the vitreous body, the gel filling the space between the lens and the retina. Destruction or degeneration of the vitreous body increases exposure of the lens to oxygen from the retina. Oxygen reaches the lens nucleus, increasing protein oxidation and aggregation and leading to nuclear cataract. We suggest that maintaining low pO(2) around the lens should prevent the formation of nuclear cataracts.     

Lens-Specific Expression of PDGF-A Alters Lens Growth and Development

The vertebrate lens provides anin vivomodel 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 byin situhybridization. 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-α receptor (PDGF-αR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-αR 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 developmentin vivo,we generated transgenic mice that express human PDGF-A in the lens under the control of the αA-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 β-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cellsin vivo.Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.     

Platelet-derived growth factor D, tissue-specific expression in the eye, and a key role in control of lens epithelial cell proliferation

Platelet-derived growth factor D (PDGF-D), also known as Iris-expressed growth factor, is a member of the PDGF/vascular endothelial growth factor family. The expression of PDGF-D in the eye is tissue-specific. In the anterior segment, it is localized to iris and ciliary body, whereas in the retina, PDGF-D is restricted to the outer plexiform layer. PDGF-D is present in aqueous humor but is not detectable in mature lens or in mouse lens-derived alphaTN4-1 cells. However, it is expressed in rabbit lens-derived N/N1003A cells. N/N1003A cell-conditioned medium stimulates proliferation in rat lens explants, and this is blocked by immunodepletion of PDGF-D. Immunopurified PDGF-D also stimulates cell proliferation in rat lens explants and in NIH 3T3 cells. In organ culture of rat eye anterior segments, anti-PDGF-D strongly inhibits lens epithelial cell proliferation. This finding suggests a major in vivo role for PDGF-D in the mechanisms of coordinated growth of eye tissues. Intervention in the PDGF-D pathway in the eye, perhaps by antibody or blocking peptide, could be useful in the treatment of certain cataracts, including post-operative secondary cataract.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.