Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells.

Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

ROCK inhibitor enhances adhesion and wound healing of human corneal endothelial cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Pan-corneal endothelial viability assessment: application to endothelial grafts predissected by eye banks.

PURPOSE: To present an experimental method for determining the viable cell pool of corneal endothelia and its application to assessing predissected endothelial grafts. METHODS: The endothelial cell density (ECD) of five pairs of human organ cultured corneas was determined using a standard counting method with a calibrated image analysis system. A thin posterior graft (30-50 µm) was manually predissected from a cornea chosen at random. Predissected and control corneas were shipped to the remote center, where standard ECD determination was repeated and was immediately followed by a triple Hoechst/ethidium/calcein labeling coupled with image analysis of the whole graft surface. Numeration of nuclei (H+), dead cells (E+), and total area covered by viable cells (C+) allowed the calculation of viable ECD corresponding to the cell density that the cornea may have after redistribution of viable cells over the whole Descemet surface. RESULTS: The median (range) viable ECD was lower than the standard ECD determined immediately earlier in predissected and control corneas: 1628 (1138-2379) and 2065 (1492-2876) cells/mm(2) (P = 0.043), corresponding to -20% (-1%-38%) and -12% (-3%-26%), respectively (P = 0.08). CONCLUSIONS: Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.

Ex vivo gene electrotransfer to the endothelium of organ cultured human corneas.

AIMS: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. METHODS: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (beta-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas beta-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions' integrity. RESULTS: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with beta-gal ranged from 0.1 to 54% of the transfected ECs (mean +/- SD: 7 +/- 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. CONCLUSIONS: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

Use of poloxamers for deswelling of organ-cultured corneas.

PURPOSE: Dextran T500, routinely used as a deswelling supplement in organ culture (OC), has been suspected of being toxic to corneal endothelial cells (ECs). This study was conducted to evaluate the innovative use of poloxamers compared with dextran for deswelling OC corneas. METHODS: Five poloxamers (P124, P188, P237, P338, and P407) were dissolved respectively in a standard OC medium to reach 350 mOsmol/kg. In vitro cytotoxicity of these media was tested by MTT assay on human corneal epithelial and endothelial cell lines and on primary human corneal fibroblasts. Paired human corneas stored in OC for at least 21 days were assigned for 48 hours to a poloxamer medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, visualization, mortality, stromal thickness, transparency, and folding were evaluated before and after deswelling. Corneas were finally cut into three parts for histologic and ultrastructural observation. RESULTS: Besides similar corneal transparency improvement and thickness deswelling, poloxamers (except P124) reduced EC loss and facilitated endothelial visualization, but improved stromal folding less than dextran. The similar ultrastructures observed in the two groups were epithelial shedding, normal collagen fiber diameter and organization, uptake of deswelling agents by ECs, vacuolization but normal organelles in ECs and keratocytes, and endothelial surface modifications. CONCLUSIONS: P188, P237, P338, and P407 performed similarly in preserving ECs, improving EC visualization, deswelling corneal stroma and inducing moderate injuries to corneal ultrastructure. They appear superior to dextran for corneal deswelling in OC.