Retinal Epithelium Research Articles

Electrical signaling in control of ocular cell behaviors

Epithelia of the cornea, lens and retina contain a vast array of ion channels and pumps. Together they produce a polarized flow of ions in and out of cells, as well as across the epithelia. These naturally occurring ion fluxes are essential to the hydration and metabolism of the ocular tissues, especially for the avascular cornea and lens. The directional transport of ions generates electric fields and currents in those tissues. Applied electric fields affect migration, division and proliferation of ocular cells which are important in homeostasis and healing of the ocular tissues. Abnormalities in any of those aspects may underlie many ocular diseases, for example chronic corneal ulcers, posterior capsule opacity after cataract surgery, and retinopathies. Electric field-inducing cellular responses, termed electrical signaling here, therefore may be an unexpected yet powerful mechanism in regulating ocular cell behavior. Both endogenous electric fields and applied electric fields could be exploited to regulate ocular cells. We aim to briefly describe the physiology of the naturally occurring electrical activities in the corneal, lens, and retinal epithelia, to provide experimental evidence of the effects of electric fields on ocular cell behaviors, and to suggest possible clinical implications.

Effects of different wave-length lights on proliferation and secretion of growth factors in human retinal pigment epithelium cells

Background The study of myopia development is always the hotspot worldwide. Recently,scientist found that some growth factor secreted by retinal nerve epithelium cells and retinal pigment epithelium(RPE)cells are associated with the development of myopia. Whenever, the absorption of RPE cells to different wave-length lights is different. ObjectiveThis study was to investigate the effects of different wave-lengths lights on the proliferation of human RPE cells, and explore the influence of different wave-lengths lights on RPE cells secreting hepatocyte growth factor(HGF) ,basic fibroblast growth factor(bFGF) and transforming growth factor-β(TGF-β).Methods The fourth to fifth passages of human embryonic RPE cells were exposed to blue light( λ =480 nm),red light( λ =775 nm) and white light. The cells of control group were harvested in normal condition. The proliferation and growth of RPE cells were assayed by MTT,and the ultrastructure of cells was examined under the transmission electron microscopy at 48 hours after light exposure of RPE cells. Enzyme linked immunosorbent assay(ELISA) was adopted to determine the concentrations of HGF,bFGF and TGF-β in the culture medium in 12,24,48,72 hours. The expression of HGF mRNA in RPE cells was detected by RT-PCR. This study was approved by Ethic committee of Fudan University. Results The A490 values of the cells exposed to blue light,red light,white light and white light were 0. 0218±0. 0014 ;0. 0353±0. 0025 ;0. 0371 ±0. 0024 and 0. 0445 +0. 0046 respectively with the significant difference among 4 groups ( F =12. 579, P＜0.05 ), and A490 value in blue light group, red light group were significantly lower than that of the control group ( t =2.043 ; t =2.024, P＜0.05 ). ELISA showed that the concentrations of HGF and TGF-β in culture medium were evidently elevated as the prolongation of light exposure in various light exposure groups in 72 hours(HGF) and 48 hours(TGF-β) compared with 12 hours with a predominating rise in the control group. The statistically significant differences were found in the concentrations of HGF and TGF-β between control group and blue light group or red light group in the( all P＜0. 05 ). The bFGF level was decreased with the time increase of various light exposure with the significant differences in 72 hours compared with 12 hours( P＜0.05 ). RT-PCR revealed the considerable difference about expression of HGF mRNA in RPE cells among these four groups( P＜0. 05 ), and the lest expression in HGF mRNA was in the blue light group compared with control group( t =3. 972,P＜0.05 ). Thinning of the chromatin, decreasing of organelle and loss of cellular membrane were seen in the blue light group, but no obvious change of ultrastructure of human embryo RPE cells was found in the ret and white light groups. Conclusions The irradiation of different wave-length light can effect the growth and proliferation and secretion of HGF,bFGF and TGFβ in human RPE cells in vitro,implying myopia formation is associated to exposure of different wave-length light. Key words: Myopia; Retinal pigment epithelial cell; Hepatocyte growth factor; Basic fibroblast growth factor; Transforming growth factor-β ; Light exposure

Intravitreal Ranibizumab for retrofoveolar neovascular Age Related Macular Degeneration, in Pseudo vitelliform and/or Drusenoid pigment epithelium detachment shape

AbstractPurpose To evaluate the functional, anatomical, vascular flow, effects of intravitreal Ranibizumab injections for retrofoveolar neovascular Pseudo vitelliform and/or Drusenoid pigment epithelium detachment (PED) Age Related Macular Degeneration (AMD), at 3 years 1/2 evolution.Methods 48 eyes of 40 patients, 15 men, 25 women, with neovascular Pseudo vitelliform(35 eyes) and/or Drusenoid PED (13eyes) AMD. Patients received intravitreous Ranibizumab, 3 times, every 4 weeks in an inductive treatment, the next injections (IVT), depending on the follow‐up results, were done by series of 3 IVT. First and 2 months’ interval follow‐up exam included ETDRS visual acuity (VA), complete ophthalmic examination, fluorescein and infracyanine (ICG) angiography, and optical coherence tomography (OCT).VA and OCT were done before each IVT.Results VA improved in 52% cases. Angiographic leakage disappeared in 78% cases, reduced about 75% in 40% cases. At ICG ,vascular flow, vessel’s diameter were 2/3 time less in 45% cases, low‐flow in 83% cases .Diffuse edema was normalized in 60% cases , pigment epithelial detachment(PED) was less dense in most of cases, by OCT. No ocular or systemic side effects observed.Better contrast Inductive treatment was sufficient in 24 cases, needed 2 IVT Series in 40% cases, failed in 1 case. In few cases, retinal and pigment epithelium atrophy was induced, Drusenoid PED disappeared.Discussion is open.Conclusion The results, with generally improved visual function, lack of fluorescein leakage, low neovascularisation’s flow in ICG, reduction of exudation on OCT, suggest Series of 3 Ranibizumab IVT Protocol seems effective, in those usually poor resort considered cases.

Microglia/Macrophages Migrate through Retinal Epithelium Barrier by a Transcellular Route in Diabetic Retinopathy: Role of PKCζ in the Goto Kakizaki Rat Model

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

The use of optical coherence tomography to aid in diagnosing solar maculopathy

Background Solar maculopathy is most commonly diagnosed after the direct viewing of solar eclipses. The damage to the retina is located in the outer segments of photoreceptors and in the retinal pigmentary epithelium. It may be acceptable to use optical coherence tomography (OCT) to identify retinal damage caused by the sun. Case report Here we describe a case of a patient who reported with a mild decrease in best-corrected visual acuities. Dilated examination found macular changes that were present in both eyes resembling solar maculopathy. OCT was utilized to aid in the diagnosis of solar maculopathy. Conclusions With the large number of individuals who spend their time in the sun, a practitioner should consider the possible repercussions of this activity. We are all aware of the damage that the sun can cause to the skin and, as in the case of cataracts, its effects on the eye. In cases in which macular changes are noted we should consider the possibility of solar maculopathy. As is in the case presented here, OCT may be an effective means of assessing the maculopathy.

Prise en charge chirurgicale des hémorragies sous-rétiniennes secondaires à une vasculopathie polypoïdale

We report the case of a 34-year-old black woman with acute and severe unilateral loss of sight related to idiopathic polypoidal choroidal vasculopathy responsible for a sub macular haemorrhage (1/10 on the Monoyer scale). The patient underwent a pars plana vitrectomy associated with a sub retinal administration of tissue plasminogen activator (100 μg) and a pneumatic displacement by gas (C2F6) with facedown positioning for 5 days. There were no intraoperative complications and the clot was lysed and totally displaced from the macula. There was no recurrence of the disease and the retinal epithelium detachment decreased progressively. The final visual acuity was 7/10. This case report illustrates the capacity and efficacy of this surgical procedure in the management of sub macular haemorrhage related to polypoidal choroidal vasculopathy. It provides effective displacement of the clot, limiting retinal damage induced by sub macular haemorrhage. Furthermore, it allows early treatment of the polypoidal aneurysm by laser or dynamic phototherapy and increases final visual acuity. Randomised studies are expected to determine the indication for this surgical procedure in the management of polypoidal choroidal vasculopathy and the possible association of laser, dynamic phototherapy, or anti-VEGF treatments.

Self-organizing optic-cup morphogenesis in three-dimensional culture

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

BackgroundThe physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences.ResultsSmall molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment.ConclusionsThe results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

A frameshift mutation in SANS results in atypical Usher syndrome

A frameshift mutation in <i>SANS</i> results in atypical Usher syndrome

Quantifiable Biomarkers of Normal Aging in the Japanese Medaka Fish (Oryzias latipes)

BackgroundSmall laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research.Principal FindingsThe median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain.SignificanceThe results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.

Distribution of CB2 cannabinoid receptor in adult rat retina

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT-PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT-PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina.

Natural evolution from macular retinoschisis to full-thickness macular hole in highly myopic eyes

to evaluate the morphological changes that occurred during the development of a full-thickness macular hole (FTMH) from macular retinoschisis (MRS) in highly myopic eyes by optical coherence tomography (OCT). the clinical characteristics and OCT images of five eyes of five patients with myopic MRS who developed FTMH during the follow-up period were evaluated. the natural evolution from MRS to FTMH was classified into two patterns by OCT findings. In FTMH formation pattern 1, a focal area of the external retinal layer was elevated and followed by the development of a small outer lamellar macular hole (OLMH) and retinal detachment (RD). The OLMH and RD were then enlarged horizontally and elevated vertically until the OLMH was attached to the overlying retinal layer. A FTMH finally developed when the roof of RD opened. In FTMH formation pattern 2, the opening of the roof of MRS or cystoid space caused an inner lamellar macular hole (ILMH). The MRS was then gradually resolved except the residual MRS beneath the ILMH, the ILMH would finally proceed into a FTMH as long as the remained external retinal layer beneath the ILMH continued splitting posteriorly until it reached retinal epithelium pigment. our longitudinal study revealed two patterns of FTMH formation in highly myopic eyes with MRS. This finding might be useful in further understanding the pathogenesis of macular hole formation in high myopia.

Alopecia and ocular alterations: a role for Minoxidil?

Research into ocular alterations in alopecia patients is limited and tends to overlook hypertrichotic agents medication. It was very recently reported that retinal changes in the form of degenerative changes, pigmentary clumping, and abnormal vascular changes are significantly more prevalent in alopecia areata (AA) patients than in the sex- and age-matched controls (1). Symptomless punctate lens opacities were significantly higher in patients than in control patients. Also, fundus alterations were significantly higher in AA patients than in the controls (2). Ophthalmological examinations showed significantly higher percentages of lens changes and alterations in the retinal epithelium in AA patients than in the controls (3). However, there is currently no proposed mechanism to explain this observation. Ocular findings are not rare conditions in other types of alopecia. In fact, a review of the literature will reveal a considerable number of similar reports. It would be difficult to establish a precise figure on what percent of AA patients or other types of alopecia patients apply topical Minoxidil. However, if it proved to be a significant figure, it would logically account for at least part of the high number of cases with retinal changes in patients using this agent. As there is scant data in the literature regarding possible angiogenic properties of Minoxidil—a potent hypertrichotic agent and one of the most commonly used medications in alopecia—the relation of an anti-hair loss product to retinal changes is less well-documented and has received little attention. The problem with Minoxidil is that it lacks tissue selectivity (4).

Abstract 4206: Utilizing a Drosophila high throughput screening technology for cancer drug discovery

Abstract MEDROS has developed unique high-throughput 96-well drug screening methods to model human diseases in the context of a whole organism, the fruit fly Drosophila melanogaster. Our approach is designed to address disease complexity and whole animal drug pharmacology, aspects poorly addressed by in vitro or cell screening methods. Here we focus on cancer, a complex disease that will benefit from the powerful tools available in Drosophila. We have designed a cancer fly model where the human oncogenes Ras and Src, centrally important in a variety of major human cancers, are targeted together to the retinal epithelium. We utilize inducible systems to calibrate their activation, generating reliably metastatic tumors that progress in a manner that is readily screened. In one iteration, these ‘tumors’ lead to organismal lethality that we have developed into a robust high-throughput screen. The kinase inhibitor Sorafenib rescued this phenotype, allowing flies to survive to adulthood and providing important validation of our screening platform. In a second iteration we permit Ras and Src to more mildly transform the eye epithelium to generate a ‘rough’ or ‘tumorous’ eye. We demonstrate that these phenotypes provide a second, more sensitive assay of Ras/Src oncogenic activity; again, Sorafenib rescued these cancer-like phenotypes. This work has important implications regarding novel drug screening methods for cancer and provides useful insights about the metastatic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4206.

Gardner Syndrome

Gardner syndrome is a variant of familial adenomatous polyposis (FAP) and results in the manifestation of numerous external and internal symptoms including gastrointestinal polyps, osteomas, tumors, and epidermoid cysts. As such, it is highly recommended that physicians conduct full body examinations to catch the key clinical features of the disease when it is suspected. Stemming from a mutation in the adenomatous polyposis coli (APC) gene, Gardner syndrome shares genetic correlations with the FAP phenotype; as a result, it becomes all the more crucial for physicians to be able to discern Gardner syndrome from other differential diagnoses such as Turcot syndrome, FAP, and other attenuated forms of familial polyposis. Fortunately, Gardner syndrome has characteristic polyps in the colon, osteomas, and also exhibits abnormalities in the retinal epithelium that discern it from others. Surgery is the most effective method of management for Gardner syndrome; restorative proctocolectomy with ileal pouch anal anastomosis with mucosectomy is the top choice for colonic malignancies, and skin manifestations can be treated through a variety of excisions and therapy depending on location, size, and number of malignancies. Currently, there are no specific screening recommendations for Gardner syndrome, but testing following general screening recommendations for extra-colonic malignancies, genetic counseling, and endoscopy are encouraged.

Mutations in N-cadherin and a Stardust homolog, Nagie oko, affect cell-cycle exit in zebrafish retina

It has been reported that the loss of apicobasal cell polarity and the disruption of adherens junctions induce hyperplasia in the mouse developing brain. However, it is not fully understood whether hyperplasia is caused by an enhanced cell proliferation, an inhibited neurogenesis, or both. In this study, we found that the ratio of the number of proliferating progenitor cells to the total number of retinal cells increases in the neurogenic stages in zebrafish n-cadherin ( ncad) and nagie oko ( nok) mutants, in which the apicobasal cell polarity and adherens junctions in the retinal epithelium are disrupted. The cell-cycle progression was not altered in the ncad and nok mutants. Rather, the ratio of the number of cells undergoing neurogenic cell division to the total number of cells undergoing mitosis decreased in the ncad and nok mutant retinas, suggesting that the switching from proliferative cell division to neurogenic cell division was compromised in these mutant retinas. These findings suggest that the inhibition of neurogenesis is a primary defect that causes hyperplasia in the ncad and nok mutant retinas. The Hedgehog-protein kinase A signaling pathway and the Notch signaling pathway regulate retinal neurogenesis in zebrafish. We found that both signaling pathways are involved in the generation of neurogenic defects in the ncad and nok mutant retinas. Taken together, these findings suggest that apicobasal cell polarity and epithelial integrity are essential for retinal neurogenesis in zebrafish.

Topographic and age-related changes of the retinal epithelium and Bruch’s membrane of rhesus monkeys

To examine structural differences in the retinal pigmented epithelium (RPE) and Bruch's membrane of rhesus monkeys (Macaca mulatta) as a function of topography and age. The retinas of two old (24 and 26 years old) and two young (1 and 6 years old) female monkeys were examined by light fluorescence and electron microscopy at the macula, equator, and ora serrata. All monkeys lacked fluorescence and lipofuscin granules in the RPE at the ora serrata where photoreceptors are absent. The equator and macula showed intense fluorescence and many lipofuscin granules in the RPE of the old but not the young monkeys. At the ora, the RPE contained many dense round melanin granules throughout the cell. At the equator and macula, melanin granules were more apical, less frequent, and often elongated. Mitochondria were clustered at the basal side of the RPE cell near infolds of the plasma membrane. Both mitochondria and infolds tended to increase toward the macula. In all regions, the basal lamina of the RPE did not penetrate the extracellular space adjacent to infolds. The elastin layer of Bruch's membrane was wide at the ora and equator and thinner at the macula. In the old monkeys, drusen were found at all retinal regions between the basal lamina and the internal collagen layer of Bruch's membrane. The drusen were often membrane-bound with a basal lamina and contained material resembling structures in the RPE. Lack of fluorescence and lipofuscin in the RPE at the ora serrata, where photoreceptors are absent, confirms that RPE fluorescence occurs only where outer segments are phagocytized. Mitochondrial clustering indicates that the basal side of the RPE cell uses the most energy and this becomes maximal at the macula. The presence of age-related degenerative changes and drusen at all retinal locations in the older monkeys, even at the ora where RPE lipofuscin was absent, indicates that these processes are not dependent on local lipofuscin accumulation. Therefore lipofuscin toxicity may not be the sole cause of age-related RPE degeneration.

Macular Hole Following Intravitreal Ranibizumab Injections for Choroidal Neovascularization

Purpose: To report a case of macular hole following intravitreal ranibizumab injection in choroidal neovascularization associated with age-related macular degeneration. Case summary: A 73-year-old man was referred for a sudden decline in vision in his right eye. The best corrected visual acuity was 0.15 in the right eye and 0.9 in the left eye. There was a choroidal neovascularization associated with subretinal hemorrhage in the right eye. Optical coherence tomogram (OCT) showed subretinal hemorrhage and pigment retinal epithelium detachment in the right eye. The patient received intravitreal ranibizumab injection. Four weeks after the initial treatment, the best corrected visual acuity of the right eye was improved to 0.3. The patient received a second intravitreal ranibizumab injection. Four weeks after the second injection, the patient presented with further decreased vision in his right eye, with a best corrected visual acuity of 0.02. Although the fundus examination was indistinct, OCT confirmed the presence of a full thickness macular hole. The patient received a third intravitreal ranibizumab injection. Four weeks after receiving the third injection, the patient underwent pars plana vitrectomy with internal limiting membrane peeling and fluid-gas exchange. Four months later, the macula hole was closed completely and visual acuity was 0.1. Conclusions: Although it is rare, intravitreal ranibizumab injection in the treatment of choroidal neovascularization secondary to age-related macular degeneration may cause complicated by a macular hole.

Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5--the lens pit stage--there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates.

The ubiquitin proteasome pathway ‐ Repair or degradation of damaged proteins

Abstract Purpose Accumulation of methylglyoxal (MGO), a highly reactive side‐product of glycolysis can modify proteins. Increased levels of MGO in cells have been implicated in diabetic vascular complications. In physiological conditions, proteolytic systems and chaperones together ensure maintenance of protein quality control. We hypothesize that MGO impairs the function of UPS and on molecular chaperones. Methods Rats with moderate type 2 diabetes (GK) and retinal epithelium pigment cell line were used. Protein oxidation was assessed by formation of carbonyl groups. Production of intracellular ROS was assessed in frozen sections of diabetic retinas by DHE incorporation. 20S proteasome activities were assessed by fluorogenic peptides. Ubiquitin (Ub) conjugation activity was determined by the ability of retinal extracts to conjugate 125I‐Ub to endogenous substrates. Ub conjugates, Hsp90, Hsc70, Hsp40 and CHIP levels were assessed by WB. Cell viability was determined by MTT while proliferation was assessed by BrdU‐incorporation. Results Data show that accumulation of endogenous Ub conjugates in the presence of MGO is associated with an increased ability of retinal extracts to conjugate 125I‐Ub to endogenous substrates. Moreover, MGO significantly decreases the 20S ptoteasome activity. Data further show that MGO decreases the levels of the molecular chaperones Hsp90 and Hsc70 and promotes aggregation of Hsp40 and CHIP. Moreover, these aggregates revealed immunoreactivity against Ub. Consistently, these effects are associated with increased cell protein oxidation, decreased cell proliferation and viability. Conclusion In diabetes, accumulation of MGO may impair the UPP and the protein quality control, leading to accumulation of obsolete proteins and cell injury.