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 membraneendothelium explants and cultured cells from healthy and late stage FECD subjects. Oxidantantioxidant 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

Effets du stress oxydatif induit par les rayons UVA et endogène sur la cornée humaine : étude des délétions de l'ADN mitochondrial, des modifications dans la matrice extracellulaire cornéenne et de la dystrophie cornéenne endothéliale de Fuchs

Le stress oxydatif peut provenir de sources exogènes comme les UVA ou de sources endogènes comme la chaîne respiratoire (OXPHOS). L'oxydation des composants cellulaires a été associée avec la dégénération, des phénotypes de vieillissement et des pertes de fonctionnalités des tissus. Les UVA sont les plus efficaces des rayons UV à induire de l’oxydation, tel que démontré par la formation de dommages oxydatifs à l'ADN et par l'apparition de délétions mitochondriales qui en résultent. La délétion mitochondriale de 4977 pb (ADNmtCD4977), la plus commune, et celle de 3895 pb (ADNmt3895) sont deux délétions reliées au photovieillissement cutané et à l'exposition au stress oxydant. Le phénomène de vieillissement dans la peau est bien documenté et se traduit par une dégradation de la matrice extracellulaire, une perte d'élasticité et la formation de rides. Toutefois, peu d'études portent sur la cornée humaine alors qu'elle est un tissu exposé directement aux rayonnements UV au même titre que la peau. Nous avons donc tenté mieux comprendre l'effet de l'oxydation exogène et endogène sur cette structure. L'analyse de la localisation des délétions ADNmtCD4977 et ADNmtCD4977 dans l'oeil humain a permis de révéler qu'elles se concentrent principalement dans le stroma cornéen et s'accumule avec l'âge. Le stroma cornéen est la couche cellulaire qui confère la transparence et la rigidité à la cornée humaine. Ces résultats nous ont suggéré une implication des UVA dans le photovieillissement de la cornée. Nous avons donc entrepris de vérifier les changements liés à l'exposition aux UVA dans le stroma cornéen puisque les UVA sont connus pour causer des altérations à la matrice extracellulaire (ECM) au niveau cutané. Nous avons donc créé un modèle de photovieillisement par une exposition chronique aux UVA sur des kératocytes avec lesquels nous avons fait sécréter une ECM. Nos résultats nous ont démontré qu'une exposition chronique aux UVA cause des altérations à l'ECM cornéen semblable à des phénotypes de photvieillissement. En effet, nous avons dénoté des changements transcriptomiques et protéomiques pour certains collagènes et protéoglycans. Une atteinte aux collagènes par le vieillissement cornéen se traduit entre autres par une rigidification, une opacification et un changement dans son pouvoir réfractif qui mène à une perte de la vision. Par ailleurs, notre avons également investigué l'implication du stress oxydatif dans la dystrophie cornéenne endothéliale de Fuchs (FECD), une maladie dégénérative de l'endothélium cornéen, qui mène à une perte de vision et est une cause principale de greffe cornéenne. L'étiologie de la maladie est encore inconnue, mais le stress oxydatif est soupçonné de jouer un rôle important dans la pathogenèse. Nos résultats ont amené de nouvelles évidences de l'implication de l'oxydation dans la maladie par l'augmentation de la quantité d'ADN mitochondrial et un raccourcissement des télomères dans des explants de cornées pathologiques. Nos résultats nous ont également démontré que la mise en culture de cellules FECD permettait la sélection de cellules fonctionnelles et comparables à des cellules saines en termes de quantité d'ADN mitochondrial et de son intégrité, de sensibilité à l'oxydation et de longueur télomérique. Les résultats obtenus soutiennent ainsi la possibilité d'employer les cellules FECD fonctionnelles sélectionnées pour utilisation en génie tissulaire afin de créer des cornées autologues pour pallier aux manques de greffes cornéennes. Enfin, nos résultats apportent de nouvelles évidences quant à l'implication du stress oxydatif dans le photovieillissement cornéen et dans l'étiologie de la FECD.Oxidative stress can arise from exogenous sources like UVA or endogenous source like the respiratory chain (OXPHOS). Oxidation of cellular components is associated with degenerative disease, aging phenotypes and loss of tissue functions. UVA is the most efficient components of UV light to induce oxidation, like it have been shown by the formation of oxidative damage on DNA and the appearance of resulting mitochondrial deletions. The mitochondrial deletion of 4977 bp (mtDNACD4977), the most common, and the 3895 bp (mtDNA3895) are both connected to skin photoaging and exposition to oxidative stress. Skin photoaging is well documented and is portrayed by a degradation of the extracellular matrix, a loss of elasticity and formation of wrinkles. However, few studies have been done on the human cornea even if this structure is directly exposed to UV light like the skin. Thus, we have tried to understand the effect of exogenous and endogenous oxidative stress on this eye structure. Analysis of mtDNACD4977 and mtDNA3895 in the human eye has shown that these deletions are localized in the corneal stroma and accumulate with age. The corneal stroma is the cellular layer conferring transparency and rigidity to the human cornea. Our results have suggested that UVA is implicated in the photoaging of the cornea. Thus, we checked for changes linked to UVA exposure in the corneal stroma since UVA are known to cause alteration to the extracellular matrix (ECM). Thereby, we created a photoaging model by exposing keratocytes to chronic UVA doses and then making them secrete an ECM. Our results have shown that chronic UVA exposure causes changes similar to photoaging phenotypes. We noted changes in the transcriptomic and proteomic expression of collagen and proteoglycans. Alteration to collagens composition by aging leads to corneal rigidity, cloudening and a loss of refractive power of the cornea, which can result in a loss of vision. On the other hand, we also investigated the implication of oxidative stress in the Fuch’s Endothelial Corneal Dystrophy, a degenerative disease of the corneal endothelium, which leads to a vision loss and is the leading cause of corneal transplantation. The etiology of the disease is not well known, but oxidative stress is suspected of playing an important role in the pathogenesis. Our results have shown new evidences of oxidative stress in the pathology by displaying an increase in mitochondrial DNA and a telomeric shortening in cells from corneal explants. Our results have also shown that using cell culture on FECD cells can allow the selection of functional cells that can compare to healthy cells terms of mitochondrial amount and integrity, sensitivity to oxidation and telomere length. Our results support the fact that functional FECD cells could be used to create autologous cornea by tissue engineering to solve the lack of corneal graft. Thus, our findings bring new evidences to the implication of oxidative stress in corneal photoaging and in the etiology of FECD

Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced

DDB2, XPC and p53 follow same induction/translocation pattern after UV exposure in NHEK and HCEC.

<p>Quantification and distribution of DDB2, XPC and p53 after UV exposure in NHEK and HCEC was assessed by Western immunoblot. (A) Representative image of Western immunoblots for DDB2 and XPC analysed from chromatin bound (CB) and for DDB2, XPC and p53 from nuclear soluble (NS) protein extracts of NHEK and HCEC cultures harvested before (-) and at different time points following a 400 J/m² of UVB irradiation. (B) Quantitative analysis of Western blots shows a rapid increase of DDB2 on the CB fraction and a (E) rapid decrease in NS post-UV irradiation (unirradiated control vs 0h post-UV) in both NHEK and HCEC. This is followed by a partial or complete restoration of DDB2 level to the unirradiated control level in NS and CB fractions, respectively. (C) and (F) For XPC, the protein level varies to a lesser extent post-irradiation and was considered non-significant in both NHEK and HCEC. No significant difference in DDB2 and XPC levels modulation post-irradiation is observed between NHEK and HCEC. (G) For p53, the protein levels increase drastically 1h post-irradiation for at least 4h. Each western is quantified independently and is not compared with each other. The protein levels of DDB2, XPC or p53 post-irradiation are calculated as the percentage of variation in relation to the unirradiated level for each protein. A value of 1 is attributed to the NoUV for each protein and the post-irradiation levels are related to the NoUV. Data information: Data presented as means ± SEM. No significant difference was raised; Student’s <b><i>t</i></b>-test. Experiment was repeated on 5 NHEK (N = 5) and 4 HCEC (N = 4) in duplicate (n = 2).</p

NER damage recognition proteins DDB2 and XPC are longer-lived in HCEC than NHEK.

<p>DDB2 and XPC protein half-lives in NHEK and HCEC were assessed by cycloheximide chasse assay. (A) Representative image of Western immunoblots of DDB2 and XPC analysed from chromatin bound (CB) and nuclear soluble (NS) fractions of NHEK and HCEC cultures without (-) and with added cycloheximide to the medium for different times to inhibit <i>de novo</i> protein synthesis. (B), (C), (D), and (E) Quantitative analysis of Western blots shows a decrease of CB and NS fractions of DDB2 and XPC over time post cycloheximide exposure in both NHEK and HCEC. However, the decrease is faster in NHEK for both CB and NS fractions when compared to HCEC, indicating a greater half-lives of DDB2 and XPC proteins in HCEC. Percentages of protein remaining are calculated by relating the intensity of signals from cells exposed to cycloheximide to the signals from their unexposed counterparts. Data information: Data presented as means ± SEM. *<b><i>P</i></b> < 0.05, **<b><i>P</i></b> < 0.01, ***<b><i>P</i></b> <0.001; Student’s <b><i>t</i></b>-test. Experiment was repeated on 4 NHEK (N = 4) and 4 HCEC (N = 4) in duplicate (n = 2).</p

More CPD are found in corneal explants than in skin explants following a same dose of UV-irradiation but similar levels of CPD are detected in cultured NHEK and HCEC.

<p>UVB-induced CPD formation in skin and cornea was assessed in human skin biopsies, eyes and cultured NHEK and HCEC irradiated with 2,000 J/m² of UVB. CPD were detected with an anti-CPD antibody (green) and DNA was counterstained with DAPI (red). (A) Immunohistofluorescence of UVB-induced CPD in skin and cornea shows an important induction of CPD in both epidermis and corneal epithelium. (B) Quantitative analysis of immunohistofluorescence. Relative CPD/DNA ratio from the epidermis and the corneal epithelium indicates that 3.4 times more CPD are found in the corneal epithelium from explants than in the epidermis from skin explants. (C) Immunocytofluorescence of UVB-induced CPD in cultured HCEC and NHEK. (D) Quantitative analysis of immunocytofluorescence. Relative CPD/DNA ratio from HCEC and NHEK show there is not difference in CPD induction between both cell types. Data information: (A, C) Scale bar, 50μm. (B, D) Data presented as means ± SEM. ***<b><i>P</i></b> < 0.001; Student’s <b><i>t</i></b>-test. (A, B) Experiment was repeated on 5 skins (N = 5) and 4 corneas (N = 4) in duplicate (n = 2). (C, D) Experiment was repeated on 3 strains of NHEK and HCEC (N = 3).</p

NHEK are more sensitive to UV-induced cell death when compared to HCEC.

<p>Apoptosis and necrosis is assessed by Annexin V/FITC and PI assay in UVB-irradiated NHEK and HCEC with doses ranging from 1,000 J/m² to 5,000 J/m² of UVB at 16h post-irradiation. Dotted, dashed and plain bars represent necrosis, apoptosis and live cells, respectively. Data information: Data presented as means ± SEM. Percentage of necrotic, apoptotic and live cells are significantly different (p<0.05) between NHEK and HCEC irradiated at 5,000 J/m² UVB, according to the Student’s <b><i>t</i></b>-test. Experiment was repeated on 3 NHEK cultures (N = 3) and 3 HCEC cultures (N = 3) in triplicate (n = 3).</p

DDB2, XPC and p53 protein levels are higher in HCEC than NHEK.

<p>Representative Western immunoblots for DDB2 (A), XPC (B) and p53 (C) analysed in the chromatin bound (CB) and nuclear soluble (NS) protein extracts from NHEK and HCEC. Coomassie blue staining was used as a loading control. (A) The quantitative analysis of Western blots shows DDB2 protein levels higher in HCEC when compared to NHEK in both CB and NS fractions. (B) As for XPC, protein level is significantly higher in HCEC than NHEK only in the NS faction. (C) p53 level is significantly higher in NS fraction of HCEC when compared to NHEK. Relative HCEC protein levels are related to their NHEK protein levels counterparts. Data information: Data presented as means ± SEM. *<b><i>P</i></b> < 0.05, *<b><i>P</i></b> < 0.01; Student’s <b><i>t</i></b>-test. (A to F) Experiment was repeated on 5 NHEK (N = 5) and 9 HCEC (N = 9) in triplicate (n = 3).</p