Traducción y validación del cuestionario Visual Function and Corneal Health Status (V-FUCHS) al idioma castellano / Translation and validation of the Visual Function and Corneal Health Status (V-FUCHS) questionnaire into Spanish language

Objetivo: Traducir y validar al castellano el cuestionario V-FUCHS en una población de pacientes que padecen distrofia endotelial de Fuchs (DEF).MétodosEl V-FUCHS consta de 15 preguntas, que valoran aspectos visuales de la calidad de vida en los pacientes con DEF, las cuales se pueden agrupar en 7 que valoran el factor «dificultad visual» y 8 que valoran el factor «deslumbramiento». Para la traducción y adaptación se siguieron las normas estandarizadas, destacando, una traducción, una retro-traducción y una aplicación en pacientes con DEF.ResultadosEn una primera fase se llegó al consenso de la traducción al castellano del V-FUCHS. Posteriormente, se incluyeron 25 pacientes para realizar la fase pre-test con el objetivo de valorar la aplicabilidad y la viabilidad del test. La puntuación de las mismas obtuvo un valor mínimo de −0,88 y un valor máximo de +2,44, según la escala probabilística de Rasch. El valor medio obtenido de las preguntas que conforman el factor dificultad visual fue de 0,61 (±0,71) y la media del factor deslumbramiento (Glare) fue de 0,41 (±0,51).ConclusiónLa validación del cuestionario V-FUCHS, tras su traducción y adaptación al castellano, demostró ser una herramienta útil para la valoración de la calidad visual de los pacientes con DEF. Los pacientes con un estado más avanzado de la enfermedad presentaron una mayor severidad en el resultado de la prueba. Asimismo, el factor deslumbramiento se correlaciona mejor con el aumento paquimétrico que con la agudeza visual del paciente. (AU)

Purpose: To translate and validate the V-FUCHS questionnaire into Spanish in a population of patients with Fuchs endothelial dystrophy (DEF).MethodsThe V-FUCHS consists of 15 short, easily understandable questions that assess visual aspects of quality of life in patients with DEF, which can be gathered into a group of seven that assess the “Visual Difficulty” factor and another group of eight that assess the “Glare Factor”. For the translation and cultural adaptation, the standardized norms for this process were followed, among other phases, a translation, a back-translation and an application in patients with DEF.ResultsIn the first phase, consensus was reached on the Spanish translation of the V-FUCHS. Subsequently, 25 patients were included to carry out the pre-test phase with the aim of assessing the applicability and feasibility of the test. The score obtained a minimum value of −0.88 and a maximum value of +2.44, according to the Rasch probabilistic scale. The mean value obtained from the Visual Difficulty factor was 0.61 (±0.71), while the mean for the Glare factor was 0.41 (±0.51).ConclusionThe validation of the V-FUCHS questionnaire, after its translation and adaptation into Spanish, proved to be a useful tool for assessing the visual quality of patients with DEF. Patients with a more advanced stage of the disease presented a greater severity in the test result. Likewise, the Glare factor (Glare) correlates better with the pachymetric increase than with the visual acuity of the patient. (AU)

Translation and validation of the Visual Function and Corneal Health Status (V-FUCHS) questionnaire into Spanish language.

PURPOSE: To translate and validate the V-FUCHS questionnaire into Spanish in a population of patients with Fuchs endothelial dystrophy (DEF). METHODS: The V-FUCHS consists of 15 short, easily understandable questions that assess visual aspects of quality of life in patients with DEF, which can be gathered into a group of seven that assess the "Visual Difficulty" factor and another group of eight that assess the "Glare Factor". For the translation and cultural adaptation, the standardized norms for this process were followed, among other phases, a translation, a back-translation and an application in patients with DEF. RESULTS: In the first phase, consensus was reached on the Spanish translation of the V-FUCHS. Subsequently, 25 patients were included to carry out the pre-test phase with the aim of assessing the applicability and feasibility of the test. The score obtained a minimum value of -0.88 and a maximum value of +2.44, according to the Rasch probabilistic scale. The mean value obtained from the Visual Difficulty factor was 0.61 (±0.71), while the mean for the Glare Factor was 0.41 (±0.51). CONCLUSION: The validation of the V-FUCHS questionnaire, after its translation and adaptation into Spanish, proved to be a useful tool for assessing the visual quality of patients with DEF. Patients with a more advanced stage of the disease presented a greater severity in the test result. Likewise, the Glare Factor (Glare) correlates better with the pachymetric increase than with the visual acuity of the patient.

Pterygium surgery with lyophilized versus cryopreserved amniotic membrane graft.

PURPOSE: To evaluate surgical outcomes (recurrence rate, aesthetics and symptoms) of pterygium surgery with two different amniotic membrane preservation approaches - lyophilized (LAM) and cryopreserved (CAM). METHODS: Primary pterygium patients were randomized to either LAM or CAM surgery. Demographic data, ocular surface disease index (OSDI), aesthetic grading (1 to 4), recurrences and complications were recorded over a 6-month follow-up period. RESULTS: Twenty-nine patients were recruited. Recurrence at month 6 was detected in 11 cases (37.9%) and was more prevalent with CAM grafts, without reaching statistical significance (P=0.196). Aesthetic outcome grading showed no differences between LAM and CAM at month 6 (P=0.124). Aesthetic results were mostly unsatisfactory (grade 3 and 4) without statistical differences between groups (P=0.514). Baseline OSDI was similar in both groups (P=0.888), and it significantly decreased by the last follow-up visit (P<0.001) for both the LAM and CAM groups. This decrease did not significantly differ between amniotic membrane preservation approach surgery groups (P=0.714). CONCLUSION: LAM might be considered a legitimate alternative to CAM, showing no inferiority in outcomes, since clinical and aesthetic outcomes were similar for both groups.

Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix.

BACKGROUND: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure. METHODS: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue. RESULTS: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue. CONCLUSIONS: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients.

Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Determination of the Culture Time Point to Induce Corneal Epithelial Differentiation in Induced Pluripotent Stem Cells.

BACKGROUND: Limbal stem cells (LSC) are progenitor cells in the ocular surface that renew the corneal epithelium. Limbal stem cell deficiency usually induces blindness through the loss of corneal transparency, and bilateral cases do not an accurate treatment because of the lack of an autologous source of stem cells. METHODS: Induced pluripotent stem cells (iPSC) are promising for use in cell therapy because of their autologous origin and the capability to differentiate into corneal epithelial cells. However, there are not standardized protocols to achieve a complete corneal epithelial differentiation. We examined the expression of several markers in a human episomal iPSC line after an induction period from embryoid bodies. RESULTS: Progenitor LSC and corneal epithelial differentiation markers, some extracellular matrix protein adhesion molecules, and wingless signaling pathway were studied. Overall, LSC progenitor and corneal epithelium differentiation markers increased after maintaining cell culture in specific conditions for 14 days, whereas pluripotency markers decreased. CONCLUSIONS: Our approach indicated that the optimal time point to initiate iPSC differentiation into LSC and corneal phenotypes, with the use of specific medium, is from 14 days after initial embryoid bodies treatment induction.

Pathological findings in the lens capsules and intraocular lens in chronic pseudophakic endophthalmitis: an electron microscopy study.

PURPOSE: The aim of this study was to describe the pathological findings in lens capsules and intraocular lens (IOL) studied by scanning and/or transmission electron microscopy (SEM and TEM, respectively) in a series of four eyes with chronic pseudophakic endophthalmitis (CPE). PATIENTS AND METHODS: We performed a retrospective study of four patients presenting CPE in whom surgical treatment with pars plana vitrectomy, capsulectomy with extraction of the IOL, and intravitreous antibiotic therapy was thereafter performed. The extracted IOL and the capsular remains were studied by SEM and/or TEM and microbiologic analysis of aqueous humour and vitreous aspirate was also carried out in all the cases. RESULTS: The presence of microorganisms was observed in the material analysed in all the cases studied. The use of TEM identified bacterial contamination by Staphylococcus spp and mixed contamination with microorganisms presenting a bacillar morphology suggestive of infection by Propionibacterium acnes in addition to the presence of cocci in the capsular remains. In another two cases, SEM localized colonies of Staphylococcus spp on the surface of the IOL in one case and mixed bacterial colonization with cocci plus filamentous bacteria in the other. The presence of macrophages associated with bacteria was observed in the capsular remains. CONCLUSIONS: Microorganisms were found in the IOL or the capsular material in the four cases studied, thereby explaining the refractoriness and severity of infection. The possible presence of polymicrobial infections, especially in the cases with filamentous bacteria, also explains the recurrence of infection.

[Efficacy of sodium carboxymethylcellulose in the treatment of dry eye syndrome]. / Eficacia de la carboximetilcelulosa sódica para el tratamiento del síndrome del ojo seco.

AIM: To assess the efficacy of sodium carboxymethylcellulose in the treatment of dry eye. MATERIAL AND METHODS: We carried out a prospective, randomized, masked-observer, control/problem group, single-center clinical assay during a period of 12 months in 19 patients that presented mild or moderate forms of dry eye. Patients were clinically evaluated each 3 months and treated with a 0.5% isotonic solution of sodium carboxymethylcellulose (CMC) or balanced salt solution. Subjective symptoms, functional tests and conjunctival impression cytology were performed according preexistent schedule study visits. To compare data between groups chi squared (chi2) analysis was applied. RESULTS: We observed a significant (p<0.05) decrease in the frequency of subjective symptoms and a significant (p<0.05) improvement of tearfilm interface stability after CMC treatment. There was a tendency to improve the degree of corneal surface wettability and the tearfilm integrity with higher percentage improvements in the CMC group compared to controls. Improved baseline values in at least one of the objective functional tests carried out (p<0.05) was also observed in an elevated percentage of patients in the CMC group (83.3%) compared with controls (34%). Furthermore, we observed a tendency to diminish the frequency of associated subjective symptoms after treatment. Conjunctival impression cytology did not provide significant differences related with therapeutic response. CONCLUSIONS: The results show a significant beneficial effect of CMC to improve clinical parameters in mild and moderate forms of dry eye.

Eficacia de la carboximetilcelulosa sódica para el tratamiento del síndrome del ojo seco / Efficacy of sodium carboxymethylcellulose in the treatment of dry eye syndrome

Objetivo: Evaluar la eficacia de la carboximetilcelulosasódica para el tratamiento del Síndrome deOjo Seco (SOS).Material y métodos: Se realizó un ensayo clínicoprospectivo randomizado unicéntrico y enmascaradode tipo grupo problema/control con 19 pacientesque padecían un SOS leve o moderado, durante unperíodo de 12 meses. Los pacientes fueron clínicamenteevaluados cada 3 meses y tratados con unasolución isotónica de carboximetilcelulosa sódica(CMC) al 0,5% o BSS. La toma de los síntomassubjetivos, las pruebas objetivas de funcionalidadclínica, y la citología de impresión conjuntival fueronrealizadas según el protocolo preestablecido.Para la comparación de los datos entre los grupos seutilizó un análisis estadístico mediante prueba dechi cuadrado (X2).Resultados: Se ha observado una disminución significativa(p<0,05) en la frecuencia de la sintomatologíasubjetiva asociada a una mejoría significativa(p<0,05) en la estabilidad de la interfase de la películalagrimal tras el tratamiento con CMC. Hubouna tendencia a la mejoría del grado de humectación de la superficie corneal y de la integridad de lapelícula lagrimal, con un porcentaje superior demejorías en el grupo problema. Se constató que unmayor porcentaje de pacientes en el grupo problema(83,3%) con relación al grupo control (34%),mejoraron por lo menos en una de las pruebas funcionalesde evaluación (p<0,05). Asimismo, seobservó una tendencia a la disminución de la frecuenciade síntomas subjetivos concomitantes trasel tratamiento con CMC. La citología de impresión(CI) no ha permitido establecer diferencias significativascon relación a la respuesta clínica al tratamiento.Conclusiones: Se pudo constatar un efecto significativamentefavorable de la CMC en la mejoría delos parámetros clínicos del SOS leve y moderado

Aim: To assess the efficacy of sodium carboxymethylcellulose in the treatment of dry eye. Material and methods: We carried out a prospective, randomized, masked-observer, control/problem group, single-center clinical assay during a period of 12 months in 19 patients that presented mild or moderate forms of dry eye. Patients were clinically evaluated each 3 months and treated with a 0.5% isotonic solution of sodium carboxymethylcellulose (CMC) or balanced salt solution. Subjective symptoms, functional tests and conjunctival impression cytology were performed according preexistent schedule study visits. To compare data between groups chi squared (X2) analysis was applied. Results:We observed a significant (p<0.05) decrease in the frequency of subjective symptoms and a significant (p<0.05) improvement of tearfilm interface stability after CMC treatment. There was a tendency to improve the degree of corneal surface wettability and the tearfilm integrity with higher percentage improvements in the CMC group compared to controls. Improved baseline values in at least one of the objective functional tests carried out (p<0.05) was also observed in an elevated percentage of patients in the CMC group (83.3%) compared with controls (34%). Furthermore, we observed a tendency to diminish the frequency of associated subjective symptoms after treatment. Conjunctival impression cytology did not provide significant differences related with therapeutic response. Conclusions: The results show a significant beneficial effect of CMC to improve clinical parameters in mild and moderate forms of dry eye

[Utility of ultrasonography in toxoplasmic uveitis]. / Utilidad de la ecografía en las uveítis por toxoplasmosis.

PURPOSE: To analyze the main indications and the most common ultrasonographic features observed in uveitis due to toxoplasmosis. MATERIAL AND METHODS: We carried out a retrospective, observational and descriptive study performed in 97 exams corresponding to 89 patients with uveitis during 7 consecutive years (1994-2000) using B-ultrasonography evaluation. RESULTS: The main ultrasonographic indication in toxoplasmosis was vitreous opacity. We observed that the most frequent findings were: a) intravitreous punctiform echoes, b) thickening of posterior hyaloid, c) partial or total posterior vitreous detachment and d) focal retinochoroidal thickening. This last finding should be highlighted due to its significant correlation (p<0.01) with toxoplasmosis. CONCLUSIONS: Our results suggest that ultrasonography plays an important role in the diagnosis and clinical follow-up of toxoplasmic uveitis.

Cloning and characterization of human syndecan-3.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.

Integrating signals from T-cell receptor and serum by T cells enhance translation of tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF-alpha in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF-alpha due to serum could be inhibited by the phosphatidylinositol (PI) 3-K inhibitors, wortmannin and LY294002, suggesting that PI 3-K is involved in the translational control of TNF-alpha by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF-alpha secretion and this was up-regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3-Kalpha increased the production of TNF-alpha in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen-activated protein kinase (MAPK), potentially downstream of PI 3-kinase, PD98059 and SB203580. Differently from with PI 3-K inhibitors, the accumulation of TNF-alpha mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3-K is an important step in controlling TNF-alpha protein synthesis in response to growth factors.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells.

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

[The use of collagen in the cicatrization of wounds]. / El uso del colágeno en la cicatrización de las heridas.

The authors review the use of collagen in the cicatrization of wounds, analyzing what this process consists of and what its regeneration and reparation phases are. The authors also summarize some fundamental biological aspects collagen has, their functions in hemostasia and in cicatrization; they develop the use of heterologous collagen in the cicatrization process. Expressive illustrations and a selection of bibliographical references accompany this article.

Wortmannin inhibits translation of tumor necrosis factor-alpha in superantigen-activated T cells.

The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-alpha expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-alpha as detected by cytofluorimetry and immunocytochemistry were CD4(+) T lymphocytes. The expression of TNF-alpha by CD4(+) T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-alpha at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC(50) = 1. 4 microM) significantly inhibited the expression of TNF-alpha but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-alpha showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-alpha translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-alpha but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-alpha. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-alpha production in T cells.

Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells.

PURPOSE: To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR). METHODS: Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated. RESULTS: Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time. CONCLUSIONS: Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.

Syndecan-2 induces filopodia by active cdc42Hs.

The syndecans, a family of transmembrane heparan sulfate proteoglycans, are ubiquitous molecules whose intracellular function is still unknown. To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycan in fibroblasts, we performed transfection studies in COS-1 and Swiss 3T3 cells. Endogenous syndecan-2 colocalized with F-actin in cortical structures. Overexpression of full-length syndecan-2 induced the formation of long filopodia-like structures. These changes correlated with a rearrangement of the actin cytoskeleton, which strongly colocalized with syndecan-2. Overexpression of syndecan-2 lacking the extracellular domain increased the number of microspikes on the cell surface but failed to induce filopodia. Addition of heparin blocked the effect of full-length syndecan-2, suggesting that heparan sulfate chains in the extracellular domain are necessary to induce filopodia. Coexpression of cdc42Hs negative-dominant N17 blocked syndecan-2-induced filopodia and cdc42Hs positive-dominant V12 had a synergic effect. This indicates that active cdc42Hs is necessary for syndecan-2 induction of filopodia. These results provide a link between syndecan-2, actin cytoskeleton, and cdc42Hs.