Transforming growth factor beta (TGF-β) has been implicated in anterior subcapsular cataract (ASC) development. In the first section of this thesis, an in-vitro rat lens model was used to determine the role of matrix metalloproteinases during TGF-β-induced ASC. In the second part, an in-vivo TGF-β transgenic and Smad3 knockout model was used to examine the role of Smad3 signaling pathway in TGF-β-induced ASC development. Lastly, an in-vitro rat lens epithelial explant culture model was used to investigate the potential role of heat shock proteins (Hsps) in TGF-β-induced epithelial-mesenchymal transition (EMT). Optical, morphological and molecular changes were analyzed in theses studies. Results from cultured rat lenses show a significant increase of back vertex distance variability (decrease of sharpness and focus) during ASC development. Inhibition of MMPs eliminated the TGF-β-induced plaque formation. Similarly, the overexpression of TGF-β1 in transgenic mouse lenses leads to ASC formation and a decrease in lens optical quality in comparison to wild-type lenses, while TGF-β1/Smad3-/- (null) lenses show diminished TGF-β-induced effects. The plaques formed in the TGF-β1/Smad3-/- lenses are substantially smaller than in the TGF-β1/Smad3+/+ lenses. The morphological and molecular changes of TGF-β2/FGF-2 treated rat lens epithelial explants are similar to those found in the TGF-β2 treated rat lenses and transgenic TGF-β1 mouse lenses. Heat shock treatment prior to TGF-β treatment significantly reduced the effects of EMT in rat LECs. In conclusion, MMP inhibition prevented TGF-β-induced ASC formation whereas heat shock treatment and the absence of Smad3 protein expression only reduced the severity of TGF-β-induced effects.

O objetivo do estudo foi analisar a cápsula e o epitéilo subcapsular cristaliniano (ESC) de pacientes submetidos à capsulotomia curvilínea contínua (CCC) utilizando o corante azul de tripano (AT) a 0,1%, através de microscopia óptica (MO), da técnica TUNEL, de imunohistoquímica e de microscopia eletrônica transmissão (MET). Realizamos um estudo prospectivo, controlado e randomizado utilizando 30 amostras de cápsulas e ESC obtidos de pacientes após CCC durante cirurgia de facectomia. Essas amostras foram divididas em dois grupos (15 espécimes cada) um utilizando o AT (grupo experimental) no ato cirúrgico e o outro sem o uso do corante (grupo controle). As cápsulas e o ESC destes grupos foram fixados e processados para análises estruturais posteriores com técnicas de MO de rotina, técnica TUNEL para detecção de morte celular por apoptose, imunohistoquímica para analisar a expressão da beclina-1 (um marcador de morte celular por autofagia), além de análise ultraestrutural por meio da MET. Foram realizadas análises morfométricas das imagens de microscopia após captura e digitalização, utilizando o programa Image Pró Plus (Cybernetics®, USA). Foram encontrados resultados positivos para a expressão de morte celular por apoptose e por autofagia no grupo submetido ao uso do AT, enquanto que no grupo controle os resultados foram negativos. As análises através da MET do ESC mostraram alterações em células coradas com o AT, incluindo ruptura mitocondrial, dilatação das cisternas do retículo endoplasmático, aumento da elétrondensidade citoplasmática e nuclear, e alteração no perfil nuclear. Os resultados estatísticos obtidos pelo teste de Mann-Whitney a partir da morfometria obtida de micrografias, demonstraram diferenças morfológicas significativas entre os grupos estudados, tanto nas dimensões dos maiores eixos nucleares, quanto na relação perímetro/área do núcleo celular (p=0,03). Em relação à espessura da cápsula, do epitélio subcapsular e do conjunto dessas estruturas obtidas a partir da MO de rotina não houve diferença estatisticamente significativa entre os grupos (p= 0,1). O AT provoca no ESC toxicidade celular com sinais indicativos de morte celular. Observamos nos aspectos morfológicos e moleculares morte celular tanto pelo mecanismo de apoptose quanto de autofagia. A partir destes achados podemos sugerir que a ação do AT, talvez possa ajudar a prevenir ou reduzir a opacificação da cápsula posterior do cristalino no período pós-operatório das facectomias / The purpose of this study was to evaluate the effect of trypan blue (TB) 0.1% staining on lens epithelial cells (LECs) and capsules of patients undergoing capsulorhexis using routine optical microscopy (OM), TUNEL technique, immunohistochemistry and transmission electron microscopy (TEM). In a prospective controlled and randomized study we evaluated 30 samples of capsules with LECs obtained after capsulorhexis during cataract surgery. Samples were randomly assigned to one of two groups (15 specimens each), one submitted to TB (experimental group) during the surgery and the other without the dye (control group). The capsule and the LECs of both groups were fixed and processed for later structural analysis with routine optical microscopy, immunohistochemistry for beclin-1 expression (a marker of cell death by autophagy), and the TUNEL technique to detect apoptosis, in addition to ultra-structural analysis by TEM. Morphometrical analysis were performed by using the Image Pro Plus software (Cybernetics®, USA). In the TB-stained group we have found positive results for the expression of cell death by autophagy and apoptosis while in the control group the results were negative. Analysis of LEC by TEM showed abnormalities in TB-stained cells including mitochondrial disruption, dilation of the endoplasmic reticulum cisterns, increased cytoplasmic and nuclear electron density and abnormalities in the nuclear profile. Statistical analysis using the Mann-Whitney test on morphometric data from micrographies showed significant morphologic differences between the two groups, both regarding longest nuclear axis difference and the ratio between the total nuclear perimeter and the cell area (p=0.03). No statistically significant difference was observed in capsule thickness, the LEC and the grouping of these two structures obtained from routine OM (p=0.1). Trypan blue is toxic to LECs, and cause abnormalities indicative of cell death. We observed molecular and morphologic aspects of cell death both by the mechanism of apoptosis and autophagy. Our findings lend support to the hypothesis that staining with 0.1% TB can help prevent or reduce the incidence of posterior capsule opacification following cataract surgery

L’Opacification Capsulaire Postérieure (OCP) est la fibrose de la capsule développée sur la lentille intraoculaire implantée (LIO) suite à la dé-différenciation de cellules épithéliales cristalliniennes (LECs) subissant une transition épithélio-mésenchymateuse (EMT). La littérature a montré que l'incidence de l’OCP est multifactorielle, dont l'âge ou la maladie du patient, la technique de chirurgie, le design et le matériau de la LIO. La comparaison des LIOs en acryliques hydrophiles et hydrophobes montre que les premières ont une OCP plus sévère, médiée par la transition EMT. En outre, il est également démontré que l'adhérence des LECs est favorisée sur des matériaux hydrophobes par rapport à ceux hydrophiles. Une stratégie biomimétique destinée à promouvoir l’adhérence des LECs sans dé-différenciation en vue de réduire le risque de développement de l’OCP est proposée. Dans cette étude, les peptides RGD, ainsi que les méthodes de greffage et de quantification sur un polymère acrylique hydrophile ont été étudiés. La surface fonctionnalisée des LIOs favorisant l'adhérence des LECs via les récepteurs de type intégrine peut être utilisée pour reconstituer la structure capsule-LEC-LIO en sandwich, ce qui est considéré dans la littérature comme un moyen de limiter la formation de l‘OCP. Les résultats montrent que le biomatériau innovant améliore l'adhérence des LEC, et présente également les propriétés optiques (transmission de la lumière , banc optique) similaires et mécaniques (force haptique de compression, force d'injection de la LIO) comparables à la matière de départ. En outre, par rapport au matériau hydrophobe IOL, ce biomatériau bioactif présente des capacités similaires vis à vis de l’adhérence des LECs, le maintien de la morphologie, et l'expression de biomarqueurs de l’EMT. Les essais in vitro suggèrent que ce biomatériau a le potentiel de réduire certains facteurs de risque de développement de l’OCP. / Posterior Capsular Opacification (PCO) is the capsule fibrosis developed onto the implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LEC) undergoing Epithelial-Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including patient’s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs show the former has more severe PCO after EMT transition. Additionally, the LEC adhesion is favored onto the hydrophobic materials compared to the hydrophilic ones. A biomimetic strategy to promote LEC adhesion without de-differentiation to reduce PCO development risk is proposed. RGD peptides, as well as their grafting and quantification methods on a hydrophilic acrylic polymer were investigated. The surface functionalized IOL promoting LEC adhesion via integrin receptors can be used to reconstitute the capsule-LEC-IOL sandwich structure, which is considered to prevent PCO formation in literature. The results show the innovative biomaterial improves LEC adhesion, and also exhibits similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties comparing to the starting material. In addition, comparing to the hydrophobic IOL material, this bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression. The in vitro assays suggest this biomaterial has the potential to reduce some risk factors of PCO development.

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