Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Biomaterials used for tissue engineering of barrier-forming cell monolayers in the eye.

Cell monolayers that form a barrier between two structures play an important role for the maintenance of tissue functionality. In the anterior portion of the eye, the corneal endothelium forms a barrier that controls fluid exchange between the aqueous humor of the anterior chamber and the corneal stroma. This monolayer is central in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). FECD is a common corneal disease, in which corneal endothelial cells deposit extracellular matrix that increases the thickness of its basal membrane (Descemet's membrane), and forms excrescences (guttae). With time, there is a decrease in endothelial cell density that generates vision loss. Transplantation of a monolayer of healthy corneal endothelial cells on a Descemet membrane substitute could become an interesting alternative for the treatment of this pathology. In the back of the eye, the retinal pigment epithelium (RPE) forms the blood-retinal barrier, controlling fluid exchange between the choriocapillaris and the photoreceptors of the outer retina. In the retinal disease dry age-related macular degeneration (dry AMD), deposits (drusen) form between the RPE and its basal membrane (Bruch's membrane). These deposits hinder fluid exchange, resulting in progressive RPE cell death, which in turn generates photoreceptor cell death, and vision loss. Transplantation of a RPE monolayer on a Bruch's membrane/choroidal stromal substitute to replace the RPE before photoreceptor cell death could become a treatment alternative for this eye disease. This review will present the different biomaterials that are proposed for the engineering of a monolayer of corneal endothelium for the treatment of FECD, and a RPE monolayer for the treatment of dry AMD.

The Presence of Guttae in Fuchs Endothelial Corneal Dystrophy Explants Correlates With Cellular Markers of Disease Progression.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by an accelerated depletion of corneal endothelial cells. There is growing evidence that mitochondrial exhaustion is central in the pathology. Indeed, endothelial cells loss in FECD forces the remaining cells to increase their mitochondrial activity, leading to mitochondrial exhaustion. This generates oxidation, mitochondrial damage, and apoptosis, fueling a vicious cycle of cells' depletion. This depletion ultimately causes corneal edema and irreversible loss of transparency and vision. Concurrently to endothelial cells loss, the formation of extracellular mass called guttae on the Descemet's membrane, is a hallmark of FECD. The pathology origins at the center of the cornea and progress outward, like the appearance of guttae. Methods: Using corneal endothelial explants from patients with late-stage FECD at the time of their corneal transplantation, we correlated mitochondrial markers (mitochondrial mass, potential, and calcium) and the level of oxidative stress and apoptotic cells, with the area taken by guttae. The different markers have been analyzed using fluorescent-specific probes and microscopic analysis. Results: We observed a positive correlation between the presence of guttae and the level of mitochondrial calcium and apoptotic cells. We found a negative correlation between the presence of guttae and the level of mitochondrial mass, membrane potential, and oxidative stress. Conclusions: Taken together, these results show that the presence of guttae is correlated with negative outcome in the mitochondrial health, oxidative status, and survival of nearby endothelial cells. This study provides insight on FECD etiology that could lead to treatment targeting mitochondrial stress and guttae.

Rescuing cellular function in Fuchs endothelial corneal dystrophy by healthy exogenous mitochondrial internalization.

Fuchs endothelial corneal dystrophy (FECD) is characterized by an accelerated loss of corneal endothelial cells. Since the function of these cells is to maintain the cornea in a state of deturgescence necessary for its transparency, the depletion of corneal endothelial cells ultimately causes corneal edema and irreversible loss of vision. Evidence is accumulating regarding the central involvement of mitochondria in FECD. As we have previously shown, when endothelial cells die and are not replaced, the mitochondria of surviving cells must provide more energy to compensate, leading to a phenomenon we have called mitochondrial burnout. This burnout causes cell death, thus exacerbating an irreversible vicious circle responsible for FECD progression. Corneal transplantation, for which the transplant supply is insufficient, is the only curative alternative for FECD. It thus becomes imperative to find other avenues of treatment. In this article, we tested whether incorporating healthy mitochondria into FECD cells would improve pathological molecular markers of the disease. Using corneal endothelium explants from FECD patients, we demonstrated that incorporation of exogenous mitochondria into FECD cells by co-incubation reduces oxidative stress, increases mitochondrial membrane potential, and reduces mitophagy. In addition, internalization of exogenous mitochondria significantly reduces apoptosis (57% in FECD vs 12% in FECD with internalized mitochondria). Taken together, these results suggest that the internalization of exogenous mitochondria reverses the vicious circle involved in FECD, thus revealing a much-needed novel treatment alternative for FECD.

Isolation efficiency of collagenase and EDTA for the culture of corneal endothelial cells.

Purpose: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplification of functional corneal endothelial cells (CECs). The goal of this study was to compare two common isolation methods, collagenase A and EDTA (EDTA), and determine whether they influence cell viability, morphology, and barrier function. Methods: Human eye bank research-grade corneas were used to isolate and cultivate CECs. All donors were more than 40 years old. Two Descemet membranes from the same donor were used separately to compare the collagenase A and EDTA cell isolation methods. The number of isolated cells, cell viability, morphology, and barrier functionality were compared. Results: A higher isolation efficiency of viable CECs and a higher circularity index (endothelial morphology) were obtained using collagenase A. Passage 3 cells presented similar barrier functionalities regardless of the isolation method. Conclusions: This study showed that isolation of CECs using collagenase A yields higher isolation efficiency than EDTA, delaying the loss of endothelial morphology for early passage cells.

TGF-ß-Mediated Modulation of Cell-Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells.

Purpose: Transforming growth factor-beta (TGF-ß) is known to influence many cell functions. In the corneal endothelium, TGF-ß1 exerts contextual effects, promoting endothelial-mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-ß isoforms participate in the formation of corneal endothelial intercellular junctions. Methods: Corneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-ß isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-ß2 were also studied. The beneficial effect of TGF-ß2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor. Results: The results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-ß isoforms were individually added during the maturation phase, and TGF-ß2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-ß2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points. Conclusions: These results suggest that TGF-ß2 can strengthen cell-cell and cell-substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells.

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Hydrodynamic forces influence the gene transcription of mechanosensitive intercellular junction associated genes in corneal endothelial cells.

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium. Native and tissue-engineered (TE) corneal endothelium were cultured in a corneal bioreactor for 7 days with 16 mmHg IOP and 5 µl/ml of medium flow. RNA was harvested, and gene expression was quantified. Cells that were used to reconstruct the TE corneal endothelia were also seeded on plastic to characterize their morphology by calculating their circularity index. For native endothelia, hydrodynamic forces increased gene expression of GJA1 (connexin 43), CDH2 (N-cadherin), TJP1 (ZO-1), ITGAV (integrin subunit αv), ITGB5 (integrin subunit ß5) and CTNND1 (p120-ctn) by 1.68 ± 0.40, 1.10 ± 0.27, 3.80 ± 0.56, 1.82 ± 0.33, 1.32 ± 0.21 and 3.04 ± 0.63, respectively. For TE corneal endothelium, this fold change was 1.72 ± 0.31, 1.58 ± 0.41, 6.18 ± 1.03, 1.80 ± 0.71, 1.77 ± 0.55, 2.42 ± 0.71. Furthermore, gene transcription fold changes (hydrodynamic/control) increased linearly with TE corneal endothelium cells population morphology with r = 0.83 for TJP1 (ZO-1) and r = 0.58 for CTNND1 (p120-ctn). In fact, the more elongated the cells populations were, the greater hydrodynamic conditions increased the transcription of TJP1 (ZO-1) and CTNND1 (p120-ctn). These results suggest that hydrodynamic forces contribute to the maintenance of tight and adherens junctions of native corneal endothelial cells, as well as to the formation of tight and adherens junctions of corneal endothelial cells that are in the process of forming a functional endothelial barrier.

Matrix metalloproteinases and their inhibitors in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is characterized by a progressive loss of corneal endothelial cells (CECs) and an abnormal accumulation of extracellular matrix in Descemet's membrane leading to increased thickness and formation of excrescences called guttae. Extracellular matrix homeostasis is modulated by an equilibrium between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). This study aimed to investigate MMPs and TIMPs profile in FECD, taking into account cell morphology. Populations of FECD and healthy CECs were cultured and their conditioned media collected for analysis. The presence of proteases in the conditioned media was studied using a semi-quantitative proteome profiler array, and MMPs levels were assessed using quantitative assays (ELISA and quantitative antibody array). MMP activity was determined by zymography and fluorometry. The expression pattern of the membrane type 1-MMP (MT1-MMP, also known as MMP-14) was examined by immunofluorescence on ex vivo FECD and healthy explants of CECs attached to Descemet's membrane. Finally, MMPs and TIMPs protein expression was compared to gene expression obtained from previously collected data. FECD and healthy CEC populations generated cultures of endothelial, intermediate, and fibroblastic-like morphology. Various MMPs (MMP-1, -2, -3, -7, -8, -9, -10, and -12) and TIMPs (TIMP-1 to -4) were detected in both FECD and healthy CECs culture supernatants. Quantitative assays revealed a decrease in MMP-2 and MMP-10 among FECD samples. Both these MMPs can degrade the main extracellular matrix components forming guttae (fibronectin, laminin, collagen IV). Moreover, MMPs/TIMPs ratio was also decreased among FECD cell populations. Activity assays showed greater MMPs/Pro-MMPs proportions for MMP-2 and MMP-10 in FECD cell populations, although overall activities were similar. Moreover, the analysis according to cell morphology revealed among healthy CECs, both increased (MMP-3 and -13) and decreased (MMP-1, -9, -10, and -12) MMPs proteins along with increased MMPs activity (MMP-2, -3, -9, and -10) in the fibroblastic-like subgroup when compared to the endothelial subgroup. However, FECD CECs did not show similar behaviors between the different morphology subgroups. Immunostaining of MT1-MMP on ex vivo FECD and healthy explants revealed a redistribution of MT1-MMP around guttae in FECD explants. At the transcriptional level, no statistically significant differences were detected, but cultured FECD cells had a 12.2-fold increase in MMP1 and a 4.7-fold increase in TIMP3. These results collectively indicate different, and perhaps pathological, MMPs and TIMPs profile in FECD CECs compared to healthy CECs. This is an important finding suggesting the implication of MMPs and TIMPs in FECD pathophysiology.

In Vitro Expansion of Corneal Endothelial Cells for Transplantation.

The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.

Chronology of cellular events related to mitochondrial burnout leading to cell death in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a degenerative eye disease characterized by corneal endothelial cell (CEC) death and the formation of guttae, an abnormal thickening of CEC's basement membrane. At the tissue level, an oxidative stress causing mitochondrial damage and CEC death have been described to explain FECD pathogenesis. At the cellular level, our group has previously observed significant variability in the mitochondrial mass of FECD CECs. This led us to hypothesize that mitochondrial mass variability might play a key role in the chronology of events eventually leading to CEC death in FECD. We thus used different fluorescent markers to assess mitochondrial health and functionality as a function of mitochondrial mass in FECD corneal endothelial explants, namely, intra-mitochondrial calcium, mitochondrial membrane potential, oxidation level and apoptosis. This has led us to describe for the first time a sequence of events leading to what we referred to as a mitochondrial burnout, and which goes as follow. FECD CECs initially compensate for endothelial cell losses by incorporating mitochondrial calcium to help generating more ATP, but this leads to increased oxidation. CECs then resist the sustained need for more ATP by increasing their mitochondrial mass, mitochondrial calcium and mitochondrial membrane potential. At this stage, CECs reach their maximum capacity and start to cope with irreversible oxidative damage, which leads to mitochondrial burnout. This burnout is accompanied by a dissipation of the membrane potential and a release of mitochondrial calcium, which in turn leads to cell death by apoptosis.

Characterization of a tissue-engineered choroid.

The choroid of the eye is a vascularized and pigmented connective tissue lying between the retina and the sclera. Increasing evidence demonstrates that, beyond supplying nutrients to the outer retina, the different choroidal cells contribute to the retina's homeostasis, especially by paracrine signaling. However, the precise role of each cell type is currently unclear. Here, we developed a choroidal substitute using the self-assembly approach of tissue engineering. Retinal pigment epithelial (RPE) cells, as well as choroidal stromal fibroblasts, vascular endothelial cells and melanocytes, were isolated from human eye bank donor eyes. Fibroblasts were cultured in a medium containing serum and ascorbic acid. After six weeks, cells formed sheets of extracellular matrix (ECM), which were stacked to produce a tissue-engineered choroidal stroma (TECS). These stromal substitutes were then characterized and compared to the native choroid. Their ECM composition (collagens and proteoglycans) and biomechanical properties (ultimate tensile strength, strain and elasticity) were similar. Furthermore, RPE cells, human umbilical vein endothelial cells and choroidal melanocytes successfully repopulated the stromas. Physiological structures were established, such as a confluent monolayer of RPE cells, vascular-like structures and a pigmentation of the stroma. Our TECS thus recaptured the biophysical environment of the native choroid, and can serve as study models to understand the normal interactions between the RPE and choroidal cells, as well as their reciprocal exchanges with the ECM. This will consequently pave the way to derive accurate insight in the pathophysiological mechanisms of diseases affecting the choroid. STATEMENT OF SIGNIFICANCE: The choroid is traditionally known for supplying blood to the avascular outer retina. There has been a renewed attention directed towards the choroid partly due to its implication in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Since AMD involves the dysfunction of the choroid/retinal pigment epithelium (RPE) complex, a three-dimensional (3D) model of RPE comprising the choroid layer is warranted. We used human choroidal cells to engineer a choroidal substitute. Our approach takes advantage of the ability of cells to recreate their own environment, without exogenous materials. Our model could help to better understand the role of each choroidal cell type as well as to advance the development of new therapeutics for AMD.

Physiological pressure enhances the formation of tight junctions in engineered and native corneal endothelium.

Cells and tissues are influenced by environmental conditions. In vivo, the corneal endothelium is subjected to hydrostatic intraocular pressure (IOP) and to the hydrokinetic pressure of the moving aqueous humor in the anterior chamber. In this paper, we used a corneal bioreactor to recreate the IOP condition and investigated the effect of the in vivo hydrodynamic environment of corneal endothelial cells on the formation of tight junctions. Native ex vivo corneas and engineered corneal endothelia subjected to pressure showed an increase in ZO-1 expression at the cell periphery. Pressure also improved the corneal transparency of engineered and native corneas. Corneal thickness was accordingly reduced from 926 ±â€¯70 µm to 651 ±â€¯70 µm for the engineered corneal endothelium and from 847 ±â€¯27 µm to 571 ±â€¯23 µm for the native endothelium. These results suggest that the hydrodynamic pressure of the anterior chamber is important for the cell junction integrity of the corneal endothelium.

Function-Related Protein Expression in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Models.

Fuchs endothelial corneal dystrophy (FECD) is a corneal pathology that affects the endothelial cell's ability to maintain deturgescence, resulting in a progressive loss of corneal transparency. In this study, we investigated the expression of function-related proteins in corneal endothelial cells using FECD or healthy corneal endothelial cells, either in a cell culture two-dimensional model or in an engineered corneal endothelium three-dimensional tissue model. No statistically significant difference in gene regulation was observed for the function-related families ATP1, SLC4, SLC16, AQP, TJP, and CDH between the FECD and the healthy cell models. Similarly, no difference in barrier integrity (transendothelial electrical resistance measurements and permeability assays) was observed in vitro between FECD and healthy cultured cells. Protein expression of the key function-related families was decreased for Na+/K+-ATPase α1 subunit, monocarboxylate transporters 1 and 4 in native ex vivo end-stage FECD specimens, whereas it returned to levels comparable to that of healthy tissues in the engineered FECD model. These results indicate that cell expansion and tissue engineering culture conditions can generate a corneal endothelium from pathologic FECD cells, with levels of function-related proteins similar to that of healthy tissues. Overall, these results explain why it is possible to reform a functional endothelium using corneal endothelial cells isolated from nonfunctional FECD pathologic specimens.

TGF-ß1 promotes cell barrier function upon maturation of corneal endothelial cells.

Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor ß (TGF-ß) could be a key factor in this phenomenon; however, TGF-ß is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-ß1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-ß1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-ß1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-ß1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.

Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model.

Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1-3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and ß1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor ß-induced protein (TGFßIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin ß5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and ß1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFßIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

Alternatives to eye bank native tissue for corneal stromal replacement.

Corneal blindness is a major cause of blindness in the world and corneal transplantation is the only widely accepted treatment to restore sight in these eyes. However, it is becoming increasingly difficult for eye banks to meet the increasing demand for transplantable tissue, which is in part due to population aging. Donor tissue shortage is therefore a growing concern globally and there is a need for alternatives to human donor corneas. Biosynthetic corneal substitutes offer several significant advantages over native corneas: Large-scale production offers a powerful potential solution to the severe shortage of human donor corneas worldwide; Good manufacturing practices ensure sterility and quality control; Acellular corneal substitutes circumvent immune rejection induced by allogeneic cells; Optical and biomechanical properties of the implants can be adapted to the clinical need; and finally these corneal substitutes could benefit from new advances in biomaterials science, such as surface coating, functionalization and nanoparticles. This review highlights critical contributions from laboratories working on corneal stromal substitutes. It focuses on synthetic inert prostheses (keratoprostheses), acellular scaffolds with and without enhancement of endogenous regeneration, and cell-based replacements. Accent is put on the physical properties and biocompatibility of these biomaterials, on the functional and clinical outcome once transplanted in vivo in animal or human eyes, as well as on the main challenges of corneal stromal replacement. Regulatory and economic aspects are also discussed. All of these perspectives combined highlight the founding principles of the clinical application of corneal stromal replacement, a concept that has now become reality.

Restoration of Mitochondrial Integrity, Telomere Length, and Sensitivity to Oxidation by In Vitro Culture of Fuchs' Endothelial Corneal Dystrophy Cells.

PURPOSE: Fuchs' endothelial corneal dystrophy (FECD), a degenerative disease of the corneal endothelium that leads to vision loss, is a leading cause of corneal transplantation. The cause of this disease is still unknown, but the implication of oxidative stress is strongly suggested. In this study, we analyzed the impact of FECD on mitochondrial DNA (mtDNA) integrity and telomere length, both of which are affected by the oxidative status of the cell. METHODS: We compared the levels of total mtDNA, mtDNA common deletion (4977 bp), and relative telomere length in the corneal endothelial cells of fresh Descemet's membrane-endothelium explants and cultured cells from healthy and late stage FECD subjects. Oxidant-antioxidant gene expression and sensitivity to ultraviolet A (UVA)- and H2O2-induced cell death were assessed in cultured cells. RESULTS: Our results revealed increased mtDNA levels and telomere shortening in FECD explants. We also found that cell culture restores a normal phenotype in terms of mtDNA levels, telomere length, oxidant-antioxidant gene expression balance, and sensitivity to oxidative stress-induced cell death in the FECD cells compared with the healthy cells. CONCLUSIONS: Taken together, these results bring new evidence of the implication of oxidative stress in FECD. They also show that FECD does not evenly affect the integrity of corneal endothelial cells and that cell culture can rehabilitate the molecular phenotypes related to oxidative stress by selecting the more functional FECD cells.

In Vivo Functionality of a Corneal Endothelium Transplanted by Cell-Injection Therapy in a Feline Model.

PURPOSE: To evaluate the functionality of a corneal endothelium reconstituted by injection of corneal endothelial cells (CEC) in the anterior chamber of a feline model. METHODS: We operated the right eyes of 16 animals. Eight underwent central endothelial scraping and injection with 2 × 10(5) (n = 4) or 1 × 10(6) (n = 4) feline CEC supplemented with Y-27632 and labeled with 3,3'-Dioctadecyl-5,5'-Di(4-Sulfophenyl)Oxacarbocyanine (SP-DiOC18[3] or DiOC). After total endothelial scraping, two eyes were injected with 1 × 10(6) labeled CEC and Y-27632. The central (n = 3) or entire (n = 3) endothelium was scraped in six eyes followed by Y-27632 injection without CEC. Subjects were positioned eyes down for 3 hours. Outcomes included graft transparency, pachymetry, CEC morphometry, histology, electron microscopy, and function and wound healing-related protein immunostaining. RESULTS: Postoperatively, corneas grafted with 2 × 10(5) CEC and centrally scraped controls displayed the best transparency and pachymetry. Corneas grafted with 1 × 10(6) CEC yielded intermediate results. Entirely scraped controls remained hazy and thick. Histopathology revealed a confluent endothelial monolayer expressing sodium-potassium adenosine triphosphatase (Na(+)/K(+)-ATPase) and zonula occludens-1 (ZO-1) in corneas grafted with 2 × 10(5) CEC and centrally scraped controls, a nonuniform endothelial multilayer without expression of functional proteins in centrally scraped corneas grafted with 1 × 10(6) CEC, and a nonfunctional fibrotic endothelium in entirely scraped grafts and controls. Expression of DiOC in grafts was scarce. CONCLUSIONS: Injected CEC contributed little to the incompletely functional endothelium of grafted corneas. Y-27632 injection without CEC following scraping reconstituted the healthiest endothelium. Further studies investigating the therapeutic effect of Y-27632 alone are needed to validate these conclusions.