Endothelial Graft Research Articles

Anterior Segment Oct Evaluation of a Caucasian Population to Assess Posterior Corneal Arc Length as a Utility for Planning Endothelial Keratoplasty

Purpose: To provide the first data of posterior corneal arc length (PCAL) in a British Caucasian population. To assess the correlation of PCAL with white to white distance (WTW), anterior chamber (AC) diameter and depth. To establish whether PCAL measurements may be of use in determining graft diameter in corneal endothelial transplant surgery. Methods: Patients were recruited from a database of anterior segment optical coherence tomography images (Tomey Casia). Patients were included if their demographic information confirmed that they were White British. Patients were excluded if they had previously had corneal surgery. 192 eyes from 100 patients were used for the study. Together with Tomey, we developed image analysis software to measure PCAL. This was used to measure PCAL, WTW and AC depth and diameter. Statistical analyses were performed using SPSS 14.0. Results: 54 patients were female (54%) and 46 were male (46%). The average age of the patients was 62 (range 22-88). Mean PCAL was 13.38mm (95% CI 13.48 and 13.68) (SD 0.71mm). Mean WTW was 11.74mm (95% CI 11.67 11.82) (SD 0.51mm). PCAL correlated extremely well with WTW distance (Pearson’s r = 0.924, p<0.01). Mean WTW to PCAL ratio was 1.16 (1.08-1.21). PCAL also correlated well with ACD (Pearson’s r = 0.541, p<0.01) but the ratio of PCAL to ACD was much less consistent than that of PCAL to WTW. Conclusion: This is the first data documenting PCAL measurements in a White Caucasian population. PCAL correlates closely with WTW and may be extrapolated using a conversion factor of 1.16. PCAL may have a role in sizing of endothelial grafts but further analysis and larger patient numbers are required.

Anterior Chamber Stabilization in Combined Penetrating Keratoplasty With Scleral-Sutured Posterior Chamber Intraocular Lens Implantation.

To describe a novel technique for a stabilized penetrating keratoplasty procedure with implantation of a scleral-sutured posterior chamber intraocular lens in which the anterior chamber (AC) is maintained and the globe stabilized without using a Flieringa ring. In this procedure, a deep anterior corneal lamella separation was created. Four small incisions were made in each quadrant along the outer edge of the trephination groove of the residual stromal bed (RSB). The first incision at the 9-o'clock position was advanced with scissors in both clockwise and counterclockwise directions. This procedure was repeated in the remaining 3 quadrants, creating 4 small bridges between the RSB and corneal rim. A donor corneal button coated with a viscoelastic material was sutured at 3 incision sites leaving 1 quadrant sutureless to allow sufficient incision width for IOL insertion for scleral fixation. Scleral fixation of a 1-piece polymethyl methacrylate (PMMA) IOL was performed. After sequential cutting of the bridges, the RSB was removed from the AC. Ten eyes of 10 patients underwent this procedure without significant complications. There were no complications related to the open-sky procedure. Globe stabilization was achieved in all surgeries. Endothelial cell loss, visual acuities, and graft failure rates were comparable with the reports of AC-protecting penetrating keratoplasty surgeries. This technique provides a secured AC and stabilized globe without the use of a Flieringa ring and increases the surgical safety without a significant reduction of the graft endothelial cell density.

Eye Bank-Prepared Femtosecond Laser-Assisted Automated Descemet Membrane Endothelial Grafts.

The aim of this study was to investigate the use of a femtosecond laser (FL) in the eye bank preparation of corneas for Descemet membrane (DM) automated endothelial keratoplasty (fDMAEK) and to compare endothelial cell death in graft preparations between fDMAEK, Descemet stripping endothelial keratoplasty (DSEK), and DM endothelial keratoplasty (DMEK). Twenty cadaveric tissues were used to test the fDMAEK method. A 9.0-mm-diameter lamellar incision was made using the FL with a 6.0-mm perpendicular anterior ring cut that enabled a stromal rim by acting as a venting incision for bubble expansion. DM was pneumodissected off the central 6.0 mm of the tissue. The fDMAEK grafts were trephined and stained with a viability dye, calcein AM. The entire stained endothelial surface was digitally captured and the endothelial cell loss (ECL) was calculated using trainable segmentation software. For comparison, a series of 6 DSEK grafts and 8 DMEK grafts were created and analyzed. Six of 20 tissues (30%) were lost during fDMAEK preparation. In the 14 successful tissues, the average ECL was 30.4% [95% confidence interval (CI), 25.3-35.6] compared with 21.1% (95% CI, 13.2-28.9, P = 0.09) in the 6 DSEK grafts and 22.5% (95% CI, 18.0-27.0, P = 0.04) in the 8 DMEK grafts. FLs are useful in preparing DMAEK tissue at the eye bank and may promote predictable and precise big bubbles and stromal rims. The fDMAEK preparation success improved with experience and laser adjustments. In fDMAEK, the ECL is higher than was previously reported in DMEK and DSEK, likely due to greater tissue manipulation, although not significantly higher than DSEK controls.

Prevalence of guttae in the graft following corneal transplantation

AimTo evaluate the prevalence of guttae in donor grafts following corneal transplantation and to examine the possible effect of guttae on postoperative results.MethodsRetrospective cohort study. We reviewed the medical records...

Randomized, prospective, single-masked clinical trial of endothelial keratoplasty performance with 2 donor cornea 4°C storage solutions and associated chambers.

The aim of this study was to compare endothelial cell loss and graft success 6 months after endothelial keratoplasty (EK) with paired donor corneas stored in Optisol GS and Life4°C solutions and their associated storage chambers. Donor pairs were stored, one in Optisol GS and the other in Life4°C, and prepared for Descemet stripping automated EK or Descemet membrane EK. Matched pairs of recipients with Fuchs dystrophy were randomized to 1 member of each donor pair. Clarity of recipient stroma, intraocular pressure, and complications were followed for 6 months. Central endothelial images of the donor cornea at screening and 3 and 6 months after EK were analyzed by a masked central reading center. Dual grading of endothelial cell density was performed using the center method. Differences in endothelial cell density and cell loss were examined by paired analysis. Thirty-two pairs were enrolled, and 27 were analyzed (5 had improper matching or loss to follow-up). Donor age was 59 ± 14 years, median death-to-surgery time was 4 days (range, 2-9 days), 6 recipient pairs had Descemet membrane EK, and 21 had Descemet stripping automated EK. Recipient age was comparable in the Optisol GS and Life4°C groups (70 vs. 68 years, respectively, P = 0.46). Six-month central endothelial cell loss did not differ significantly between the Life4°C and Optisol GS groups (18 ± 18% vs. 20 ± 20%, respectively, P = 0.55). All recipient corneas were clear at 6 months in both groups. Endothelial cell loss and graft success were comparable at 6 months for paired donor corneas stored in Optisol GS and Life4°C.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01657500.

Femtosecond laser cutting of multiple thin corneal stromal lamellae for endothelial bioengineering.

To assess the feasibility of cutting multiple thin stromal lamellae in human donor corneas using a commercial femtosecond laser (FSL) to provide cell carriers for future endothelial graft bioengineering. Eight edematous organ-cultured corneas not suitable for grafting for endothelial reasons were mounted on a Ziemer anterior chamber and cut with a Z6 FSL with 6 successive parallel cuts, from depth to surface. Target thickness of each lamella ranged from 100 to 150 μm depending on initial corneal thickness. Thickness was measured using anterior segment optical coherence tomography before and after cutting on mounted corneas, and on each stromal lamella after detachment. Scanning electron microscopy observation was performed on 4 lamellae and histological cross sections on 1 cornea before detachment. A median of 5 (minimum 3, maximum 7) lamellae was obtained per cornea. All lamellae still attached were the most posterior ones, suggesting that FSL was less efficient because of light scattering by edematous stroma. Cut precision and postdetachment swelling were correlated with anterior-posterior position within the cornea. Median lamella thickness was 127 μm (56-222 μm) before detachment and 196 μm (80-304 μm) after detachment. Surface state was consistent with previously reported FSL lamellar cuts during Descemet stripping automated endothelial keratoplasty. Up to 7 thin lamellae can be cut in stored corneas with an FSL. This method, once optimized primarily by using deswelled, more transparent corneas, could prove effective for recycling unsuitable donor corneas in corneal bioengineering processes.

Influence of graft size on graft survival following Descemet stripping automated endothelial keratoplasty

PurposeTo evaluate graft size on outcome following Descemet stripping automated endothelial keratoplasty (DSAEK)MethodsConsecutive patients who had undergone a DSAEK for Fuchs endothelial dystrophy (FED) and pseudophakic bullous keratopathy (PBK) with...

Preconditioning by cilostazol protects against cold hepatic ischemia-reperfusion injury.

In liver transplantation, prolonged cold storage and post-transplant reperfusion are associated with endothelial inflammation, organ dysfunction, and graft failure. We herein studied whether cilostazol, a phosphodiesterase-3-inhibitor, attenuates post-ischemic liver injury after prolonged cold storage. Sprague-Dawley rats were assigned to 5 groups (n=6 each): sham animals (cold storage time (CST): 1 h), vehicle-treated (NaCl 0.9%) controls (CST: 24 h), and animals receiving 0.1, 1.0, or 10.0 mg/kg body weight (BW) cilostazol pre-treatment (CST: 24 h). After organ explantation, all livers were stored at 4°C in HTK solution, followed by 60 min of reperfusion with 37°C Krebs Henseleit buffer in a non-recirculating ex situ system. Bile flow was measured to evaluate liver function. To analyze inflammation and morphology, liver tissue samples were taken and histology, immunohistochemistry, and Western blotting were conducted. In vehicle-treated controls, prolonged cold storage and warm reperfusion induced inflammation, organ dysfunction, and cell death. This was indicated by an increase of hepatocellular vacuolization, endothelial ICAM-1 expression, and apoptotic cell death compared to sham animals. Cilostazol pre-treatment protected against cold hepatic ischemia-reperfusion injury by preventing hepatocellular disintegration, ICAM-1-associated endothelial inflammation, and apoptotic death. Inhibition of PDE3 reduces endothelial cell activation and hepatocellular injury in cold ischemia/reperfusion of the liver.

Endothelial approach ultrathin corneal grafts prepared by femtosecond laser for descemet stripping endothelial keratoplasty.

To investigate the quality of the ultrathin corneal grafts prepared by femtosecond laser from the endothelial side for Descemet stripping endothelial keratoplasty. Thirty human corneoscleral buttons were cut from the endothelial side by laser Doppler velocimetry (LDV) with or without viscoelastic materials coating. Two cutting depths were selected: 70 and 90 μm. The postcut endothelial count was determined by specular microscopy, and the graft thickness was evaluated by anterior segment optical coherence tomography. The endothelial viability was determined using Trypan blue/Alizarin red staining, calcein-AM/EthD-1 live/dead cell assay, and scanning electron microscope (SEM). The graft interface smoothness was evaluated by SEM. Another 18 corneoscleral buttons were used as controls for the comparisons. The overall targeted cutting depth and achieved cutting depth were significantly highly correlated (r = 0.84). The central to peripheral corneal thickness ratio was 0.976 and 0.998 for the 70- and 90-μm grafts. The percentage of the damaged endothelial cells assessed by vital staining and SEM showed the 70-μm grafts had noticeably more endothelial damage compared with the 90-μm grafts. But the damage was significantly reduced, to the control corneas level, after coating the endothelium with Viscoat. The 90-μm grafts had a slightly rougher graft interface than the 70-μm grafts, but all the grafts dissected by a Chansue dissector exhibited a generally smooth interface. The corneal endothelial grafts prepared by LDV femtosecond laser with endothelial approach produced consistently ultrathin grafts in uniform shape with high accuracy and good endothelial and stromal interface quality.

Intraocular lens opacification after descemet stripping automated endothelial keratoplasty.

Intraocular lens (IOL) opacification is a rare but serious complication that may necessitate its exchange. The use of intraocular gases is a known precipitant. Descemet stripping endothelial keratoplasty (DSAEK) involves injecting air into the anterior chamber. IOL opacification has been described after this procedure; however its incidence is currently unknown. A retrospective review of case notes from a single center of all patients undergoing DSAEK, who were either already pseudophakic or had simultaneous cataract surgery. Cases with IOL opacification were analyzed, and any risk factors were identified. One hundred sixty-eight DSAEK were performed on 154 eyes of 137 patients. Fifty-four cases had simultaneous cataract surgery with implantation of an IOL. Fifteen (9.7%) eyes developed IOL opacification. This had a distinctive pattern, being limited to the anterior lens surface, in the pupillary zone. Median time interval from keratoplasty to the first observation of IOL opacification was 17 months (range, 4-34 months). The only statistically significant risk factor was rebubbling of detached endothelial grafts. Rebubbling was performed in 62.5% (10/15) of cases with IOL opacification, compared with 23% (32/139) with no opacification (P = 0.0009). This is the first study to report the incidence of IOL opacification after undergoing DSAEK. Although the mechanism is unknown, multiple injections of air into the anterior chamber statistically increase the risk of IOL opacification.

New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops.

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.

Repeat Descemet Membrane Endothelial Keratoplasty after Complicated Primary Descemet Membrane Endothelial Keratoplasty

To describe the clinical outcome and complications of repeat Descemet membrane endothelial keratoplasty (re-DMEK). Retrospective case series study at a tertiary referral center. From a series of 550 consecutive DMEK surgeries with ≥ 6 months follow-up, 17 eyes underwent re-DMEK for graft detachment after initial DMEK (n = 14) and/or endothelial graft failure (n = 3). The outcomes were compared with an age-matched control group of uncomplicated primary DMEK surgeries. The re-DMEK eyes were evaluated for best-corrected visual acuity (BCVA), densitometry, endothelial cell density (ECD), pachymetry, and intraoperative and postoperative complications. Feasibility and clinical outcome of re-DMEK. In all eyes, re-DMEK was uneventful. At 12 months, 12 of 14 eyes (86%) achieved a BCVA of ≥ 20/40 (≥ 0.5); 8 of 14 eyes (57%) achieved ≥ 20/25 (≥ 0.8), 3 of 14 eyes (21%) achieved ≥ 20/20 (≥ 1.0), and 1 eye (7%) achieved 20/17 (1.2); 5 eyes were fitted with a contact lens. Average donor ECD decreased from 2580 ± 173 cells/mm(2) before to 1390 ± 466 cells/mm(2) at 6 months after surgery, and pachymetry from 703 ± 126 μm to 515 ± 39 μm, respectively. No difference in densitometry could be detected between re-DMEK and control eyes (P = 0.99). Complications after re-DMEK included primary graft failure (n = 1), secondary graft failure (n = 2), graft detachment requiring rebubbling (n = 1), secondary glaucoma (n = 2), cataract (n = 1), and corneal ulcer (n = 1). One eye received tertiary DMEK. In the management of persistent graft detachment and graft failure after primary DMEK, re-DMEK proved a feasible procedure. Acceptable BCVA may be achieved, albeit lower than after DMEK in virgin eyes, and some cases may benefit from contact lens fitting. Complications after re-DMEK may be better anticipated than after primary DMEK because graft detachment and graft failure tended to recur, suggesting that intrinsic properties of the host eye play a role in graft adherence and graft failure.

Comparison of ultrathin manual and automated stripping of the endothelium in Fuchs endothelial dystrophy

AbstractPurpose This study compares two techniques of preparation of the endothelial graft: the DSAEK (Descemet Stripping Automated Endothelial Keratoplasty) and an ultrathin endothelial graft manually prepared.Methods A randomised prospective study is being conducted at the Hospital of Rouen, France. Patients presenting a Fuchs dystrophy are included in two groups. Patients from Group A receive an ultrathin manually prepared endothelial graft and patients from Group B receive a DSAEK. In order to improve the power of our study, one patient from each group is grafted on the same day, with a corneal endothelium coming from the same donor. Improvement of visual acuity is the main outcome measure. Vitality of the graft, refractive modifications induced by surgery and complications are also studied.Results Currently, 22 patients with a follow up of 6 months have been included. At inclusion, patients from Group A and B were not statistically different. Six months after surgery, mean best corrected visual acuity has significantly improved in both groups but is statistically better in Group A (0,18 logMar in Group A versus 0,31 logMar in Group B (p&lt;0,05)). Mean endothelial cell density is 1686 cells in Group A statistically more than in Group B (1260 cells) p&lt;0,05. There were no refractive modifications in either group. There was no episode of graft rejection.Conclusion Ultrathin endothelial graft seems to give better results than DSAEK in Fuchs dystrophy in terms of visual acuity and endothelial cell density. It could be a good alternative to DMEK (Descemet membrane endothelial keratoplasty) because it is easier to prepare and inject than DMEK and should then be favoured when DMEK is not possible.

Validation of a novel device to easy the preparation of endothelial graft for DMEK

AbstractPurpose The DMEK is the only technique for endothelial graft without interface. It is however difficult to perform, especially the preparation of the graft. We present in this study a new simple device facilitating the preparation of grafts for DMEK.Methods The device, named “Muraine Punch” is a circular trephine with a blade which was open in two places and a guard to 300 micronmeters deep. The blade was pressed against the endothelial surface so as to sever Descemet's membrane and cut part of stroma. The preparation of descemetic graft was then continued on an artificial anterior chamber using a 27 gauge cannula , reversed upwardly corneal endothelium. Three methods of assessing injury of endothelial cells (ECs) were then performed: 1 / conventional measurement of the endothelial cell density (ECD). 2 / laboratory measurement of the viable ECD by image analysis after staining of ECs by alizarin red, followed an counterstaining using Dioc 6 (cytoplasm) and Hoechst (nucleus). 3/ post operatory ECD of 47 patients.Results Histological analysis confirmed this “Muraine technique” offered a pure Descemet membrane grafts for DMEK. The ECD analysis showed consistent with that of the grafts for transfixant keratoplasty.Conclusion The new technique with “Muraine Punch” facilitates the preparation of grafts for DMEK and causes only a minimal cell loss. Commercial interest

Bioengineering of endothelial grafts using femtosecond Laser cut corneal lamellae or collagen lenticules, endothelialized with immortalized or hIPSC‐derived endothelial cells

AbstractPurpose The bioengineering of endothelial grafts appears as a realistic solution to reduce the global scarcity for corneal donation worldwide. We focused our strategies of endothelial bioengineering on the endothelialization of human corneal stromal lamellae with hiPSC‐derived endothelial cells.Methods Human corneas unsuitable for graft but with a normal stroma, were cut into 4 to 7 lamellae of &lt;100 μm thickness with a femtosecond laser (FsL), decellularized from their keratocytes using an optimized protocol (Ethanol/DNAse/SDS) and stored at +4°C. They were compared with collagen I compressed thin discs (RAFT) already described as carrier. Endothelialization was performed with immortalized endothelial cells (ECs) (BAG12) and hIPSC‐derived ECs (also presented during this congress). Efficiency was assessed by determining endothelial cell density (ECD), morphology after alizarin red staining and SEM and immunolabeling for Na/K‐ATPase expression. Transparency was measured using our customized transparometerResults both carriers were evenly endothelialized by B4G12 with similar alizarin red staining and Na/K ATPase expression patterns but higher ECD for stromal lamellae. Transparency was better for stromal lamellae. hIPSC‐derived ECs gave similar characteristicsConclusion Decellularized Fsl cut stromal lamellae seem better than collagen I compressed lenticules for the endothelial bioengineering using hISPC‐derived ECs. Unsuitable human cornea could be therefore “recycled” to increase the pool of available grafts. Grants: ABM2013, AIRE2013

Development and evaluation of porcine atelocollagen vitrigel membrane with a spherical curve and transplantable artificial corneal endothelial grafts.

To develop a collagen vitrigel (CV) optimized as a corneal endothelial cell (CEC) carrier and create an artificial corneal endothelial graft. We first developed a flat-shaped collagen vitrigel for regenerative medicine (CV-RM) using porcine atelocollagen and ultraviolet (UV) irradiation. The optimal UV amount was determined by measuring the CV-RM transparency under various irradiating conditions. The collagen vitrigel for corneal endothelial regenerative treatment (CV-CERT), a transparent porcine atelocollagen with a curved shape, was made using spherically curved molds and UV irradiation. The membrane permeability of the CV-CERT was tested in vitro. The biocompatibility, transparency, and adhesiveness of the CV-CERT were evaluated in rabbit eyes. We also developed a culture technique for distributing human CECs on the curved CV-CERT. The optimal amount of UV irradiation for CV-RM transparency was 2400 mJ/cm(2). Membrane permeability of CV-CERT at day 5 was higher than that of commercially available CV (P = 0.032). The CV-CERT was transparent and biocompatible in rabbit corneas for up to 4 months. The CV-CERT remained attached to the rabbit corneal posterior surface, whereas the flat-shaped CV-RM, differing only in shape from the CV-CERT, dislocated soon after surgery. Human CECs seeded on the CV-CERT using our technique were evenly distributed with a single layer structure and a mean cell density of 2650 ± 100 cells/mm(2). We developed a transparent and biocompatible porcine-derived atelocollagen vitrigel membrane with a spherical curvature. A transplantable artificial endothelial graft was created by combining cultured human CECs and the CV-CERT.

Endothelial keratoplasty in children.

Descemet stripping endothelial keratoplasty has superseded penetrating keratoplasty (PKP) in adults as the procedure of choice for treating endothelial dysfunction, but endothelial keratoplasty despite the multitude of advantages has undergone a much slower adoption in pediatric patients. This review focuses on the recent advances made in the field of pediatric endothelial keratoplasty and the associated surgical challenges. Pediatric endothelial keratoplasty does not require as many sutures as PKP, and the wound size is smaller and more stable. Endothelial keratoplasty grafts clear faster, possibly allowing for a more effective management of amblyopia. Visual outcomes appear to be comparable with PKP but with a lower complication rate. As corneal surgeons grow more and more familiar with the nuances of pediatric endothelial keratoplasty, our understanding of this procedure will become more complete. The limited data available suggest that endothelial keratoplasty is a suitable surgical alternative to PKP in children.

Prone Positioning to Facilitate Graft Adherence in the Late Postoperative Period After Descemet Stripping Automated Endothelial Keratoplasty

The aim of this study was to report the use of prone positioning to facilitate graft adherence in the late postoperative period after Descemet stripping automated endothelial keratoplasty (DSAEK). A 75-year-old woman with Fuchs dystrophy had repeated endothelial graft detachments after simultaneous DSAEK and phacoemulsification cataract surgery. Multiple attempts in the operating room to reposition the tissue, including placement of air into the anterior chamber, postoperative face-up positioning, and the use of new donor tissue, were unsuccessful. In the late postoperative period, the patient was placed in a prone (face-down) position at home to promote graft adherence. After the maneuver, the endothelial graft was well attached, although it was slightly decentered. The remainder of the patient's postoperative course was routine, and her best-corrected visual acuity is 20/25 with a clear cornea 9 months later. Prone positioning can be used as a conservative maneuver to promote graft adherence after DSAEK when there is difficulty in achieving attachment postoperatively, especially when a return trip to the operating room may not be practical.

DMEK lenticule preparation from donor corneas using a novel ‘SubHyS’ technique followed by anterior corneal dissection

PurposeTo describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas.Methods30 human...

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.