Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation

PURPOSE: It would be advantageous if cultivated human corneal endothelial cells (cHCECs) could be transplanted for the treatment of diseases caused by corneal endothelial disorders. To achieve this, a matrix that can serve as a carrier for cHCECs is needed. The present study was conducted to examine the feasibility of using amniotic membrane (AM) as a carrier for this application. METHODS: HCECs obtained from peripheral corneal tissue were cultivated, passaged, and transplanted onto denuded AM. The cell density and morphology of the resultant cHCECs on AM were examined by light, scanning electron, and transmission electron microscopy. To determine whether these cHCEC sheets on AM carrier were functional in vivo, the cHCEC sheets on AM were transplanted onto rabbit corneas whose Descemet's membrane and endothelial cells had been completely removed. After transplantation, the corneal appearance was examined by slit lamp biomicroscopy, and corneal thickness was measured daily by pachymetry. At 7 days after surgery, the grafts were examined by light, scanning electron, and transmission electron microscopy. RESULTS: The density of the cHCECs on AM was greater than 3000 cells/mm(2). Morphologically, the cHCEC sheets consisted of a fairly continuous layer of flat squamous polygonal endothelial cells that appeared uniform in size with tightly opposed cell junctions in vitro and in vivo after transplantation. The corneas that received transplanted cHCEC sheets had little edema and retained their thinness and transparency. CONCLUSIONS: The cell density and morphology of cHCECs on AM were similar to those of normal corneas, and cHCECs on AM were functional in vivo. These results indicate that AM maintains HCEC morphology and function and could serve as a carrier for cHCEC transplantation.     

Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium

PurposeSLC4A11, an electrogenic H⁺ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown.MethodsThe presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11–transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking.ResultsSLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11–transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11–transfected fibroblasts.ConclusionsHSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.     

Exploring the Mechanism of the miRNA-145/Paxillin Axis in Cell Metabolism During VEGF-A–Induced Corneal Angiogenesis

PurposePaxillin (PXN) is a key component of focal adhesions and plays an important role in angiogenesis. The aim of the present study was to investigate the effect of PXN in vascular endothelial growth factor A (VEGF-A)–induced angiogenesis in human umbilical vein endothelial cells (HUVECs).MethodsHUVECs were transfected with PXN overexpression and PXN interference vectors. Biochemical detection was used to detect adenosine triphosphate and lactic acid production. The morphology of mitochondria was observed under an electron microscope, and flow cytometry was conducted to measure mitochondrial membrane potential. Transwell experiments were used to detect the migration and tube formation ability of each group of cells. The expression of hexokinase (HK)1, HK2, glucose transporter 1 (GLUT1), phosphorylated phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, and phosphorylated mechanistic target of rapamycin (mTOR) was evaluated by western blot.ResultsPXN silencing reduced the levels of lactic acid and adenosine triphosphate, downregulated HK1, HK2, and GLUT1, suppressed PI3K/AKT/mTOR signaling activation, and inhibited VEGF-A–induced mitochondria injury in VEGF-A–induced HUVECs. We also determined that miR-145-5p decreased the VEGF-A–induced expression of PXN and inhibited the invasion and angiogenesis of HUVECs. Also, miR-145-5p inhibition blocked the protective effect of PXN interference on VEGF-A–induced HUVEC injury. Furthermore, PXN interference significantly decreased lactic acid and adenosine triphosphate levels, inhibited PI3K/AKT/mTOR activation, and decreased the levels of HK1, HK2, and GLUT1 in VEGF-A-treated mouse corneal.ConclusionsThe results indicate that PXN silencing inhibited the VEGF-A–induced invasion and angiogenesis of HUVECs via regulation of cell metabolism and mitochondrial damage, suggesting that PXN may be a potential target for antiangiogenic therapies.     

Two-year clinical outcome after Descemet membrane endothelial keratoplasty using a standardized protocol

Purpose:The purpose of this study is to evaluate 2-year clinical outcome after Descemet membrane endothelial keratoplasty (DMEK) in a variety of endothelial dysfunctions using a standardized protocol.Methods:From a group of 230 eyes which underwent DMEK for Fuchs'' endothelial corneal dystrophy (FECD), aphakic and pseudophakic bullous keratopathy, failed full thickness corneal transplants, ICE syndrome, failed DSEK, and TASS the clinical outcomes [best spectacle-corrected visual acuity (BSCVA), central endothelial cell density (ECD)] were evaluated before, and at 6, 12, and 24 months and the success rate, failure rate and postoperative complications were also analyzed.Results:Out of 230 eyes, 144 eyes (70%) had BSCVA 6/9 or better 2 years postoperatively. Mean donor ECD was 2692.23 (range, 2300–3436) cells/mm² preoperatively, which was reduced to 1433.64 (range, 619.0–2272.0) cells/mm² 2 years after DMEK surgery, indicating a mean reduction of 1258 cells/mm² (46%) in ECD.Conclusion:DMEK is a highly successful surgical procedure when following a standard protocol for treating diseases of the corneal endothelium providing a near perfect anatomic restoration and a high degree of visual rehabilitation.     

Proliferative capacity of the corneal endothelium

Corneal endothelium is the single layer of cells forming a boundary between the corneal stroma and anterior chamber. The barrier and "pump" functions of the endothelium are responsible for maintaining corneal transparency by regulating stromal hydration. Morphological studies have demonstrated an age-related decrease in endothelial cell density and indicate that the endothelium in vivo either does not proliferate at all or proliferates at a rate that does not keep pace with the rate of cell loss. Lack of a robust proliferative response to cell loss makes the endothelium, at best, a fragile tissue. As a result of excessive cell loss due to accidental or surgical trauma, dystrophy, or disease, the endothelium may no longer effectively act as a barrier to fluid flow from the aqueous humor to the stroma. This loss of function can cause corneal edema, decreased corneal clarity, and loss of visual acuity, thus requiring corneal transplantation to restore normal vision. Studies from this and other laboratories indicate that corneal endothelium in vivo DOES possess proliferative capacity, but is arrested in G1-phase of the cell cycle. It appears that several intrinsic and extrinsic factors together contribute to maintain the endothelium in a non-replicative state. Ex vivo studies comparing cell cycle kinetics in wounded endothelium of young (< 30 years old) and older donors ( > 50 years old) provide evidence that cells from older donors can enter and complete the cell cycle; however, the length of G1-phase appears to be longer and the cells require stronger mitogenic stimulation than cells from younger donors. In vivo conditions per se also contribute to maintenance of a non-replicative monolayer. Endothelial cells are apparently unable to respond to autocrine or paracrine stimulation even though they express mRNA and protein for a number of growth factors and their receptors. Exogenous transforming growth factor-beta (TGF-beta) and TGF-beta in aqueous humor suppress S-phase entry in cultured endothelial cells, suggesting that this cytokine could inhibit proliferation in vivo. In addition, cell-cell contact appears to inhibit endothelial cell proliferation during corneal development and to help maintain the mature endothelial monolayer in a non-proliferative state, in part, via the activity of p27kip1, a known G1-phase inhibitor. The fact that human corneal endothelium retains proliferative capacity has led to recent efforts to induce division and increase the density of these important cells. For example, recent studies have demonstrated that adult human corneal endothelial cells can be induced to grow in culture and then transplanted to recipient corneas ex vivo. The laboratory work that has been conducted up to now opens an exciting new door to the future. The time is right to apply the knowledge that has been gained regarding corneal endothelial cell proliferative capacity and regulation of its cell cycle to develop new therapies to treat patients at risk for vision loss due to low endothelial cells counts.     

Evaluation of changes in corneal endothelium in chronic kidney disease

Purpose:Chronic kidney disease (CKD) is an emerging health problem worldwide. In CKD corneal endothelial changes also occur probably due to accumulation of inflammatory cytokines and increased multiple toxic products. The aim of this study was to analyze the effect of CKD on corneal endothelium and correlate the findings with severity of disease with help of noninvasive technique.Methods:The study comprised 75 eyes of 75 cases divided into three groups with group A comprising of CKD cases on dialysis, group B of nondialysis CKD cases, and group C of controls. Each group had 25 cases each of either sex and between 15–80 age groups. All patients were investigated for blood urea, serum creatinine, and blood sugar and underwent complete ophthalmic examination of both eyes along with wide-field specular microscopy examination.Results:The majority of patients (33.3%) belonged to age range of 61–70 years with male predominance and the most common cause of CKD was found to be diabetes with 17 (34%) cases. We found normal corneal endothelial cell density (ECD) with the mean ECD of 2364.52 ± 397.72 mm² in the dialysis group, 2467.8 ± 352.88 mm² in nondialysis group, and 2521.68 ± 250.26 mm² in the control group of patients. However, we found significant increase in coefficient of variation (CV) with 36 ± 5.8% in dialysis group, 37 ± 4.5% in nondialysis group and 32 ± 0.8% in controls (P = 0.001) and decreased hexagonality (Hx) with 47 ± 7.3% in dialysis group, 46 ± 4.7% in nondialysis group and 51 ± 6.7% in the controls (P = 0.031). This showed increased tendency of pleomorphism and polymegathism in corneal endothelial cells in CKD cases. No correlation was found between blood urea or serum creatinine levels with endothelial parameters in any group.Conclusion:CKD causes morphological changes like polymegathism and pleomorphism in corneal endothelium and hence these cases are more vulnerable and special care should be taken before any intraocular surgical procedure.     

Mushroom keratoplasty in pediatric patients

Objective

To report the outcome of mushroom keratoplasty for the treatment of full thickness corneal disease in pediatric patients with healthy endothelium.

Methods

A retrospective analysis of pediatric patients who underwent mushroom keratoplasty. The medical records of pediatric patients suffering from full thickness corneal stromal disease with normal endothelium who underwent mushroom keratoplasty at our Institution were included. A two-piece donor graft consisting of a large anterior stromal lamella (9.0 mm in diameter and ±250 μm in thickness) and a small posterior lamella (5–6.5 mm in diameter) including deep stroma and endothelium, prepared with the aid of a microkeratome had been transplanted in all cases. Ophthalmic examination including slit lamp examination, best corrected visual acuity, and corneal topography was performed preoperatively and at each postoperative visit on all patients. The endothelial cells were assessed by specular microscopy in these patients.

Results

Six eyes of six patients (five males and one female) were included. The mean age was 9.3 years (range 5–15 years). Average follow-up was 17.8 months (range 9–48 months).There were no early or late complications recorded. All corneas were clear at the last follow up visit.Preoperative best corrected visual acuity (BCVA) was worse than 20/70 in all six eyes. Postoperatively, four eyes achieved BCVA of 20/40 or better.Endothelial cell loss (n eyes = 3 averaged 24% (range 19–31%). The mean endothelial cell loss was 24% (range 19–31%) among these patients.

Conclusions

Microkeratome assisted mushroom keratoplasty is a viable surgical option for pediatric eyes with full thickness corneal stromal disease and healthy endothelium. Mushroom keratoplasty combines the refractive advantage of a large penetrating keratoplasty with the survival advantage of a small penetrating keratoplasty. Furthermore, mushroom keratoplasty exhibits the mechanical advantage of a shaped keratoplasty.     

Heterologous transplantation versus enhancement of human corneal endothelium

The ability to successfully transplant human corneal endothelium would offer a significant advance in the treatment of many corneal diseases. To investigate the feasibility of this, we established cultures of endothelial cells derived from neonatal human corneas. Eye bank donor corneas were either enhanced with a suspension of cultured endothelial cells or underwent endothelial cell removal and subsequent replacement with cultured endothelium. Following a 48-h incubation, the corneas were transplanted into the eyes of nonhuman primates. Over a 12-month period, 67% of the corneas with complete endothelial cell replacement thinned and remained clear, with a mean corneal thickness of 0.57 mm. Enhanced corneal buttons demonstrated a significantly lower success rate (35%), with opacified and thickened corneas. Control eyes in which the native endothelium was removed demonstrated advanced corneal edema and vascularization, with a mean corneal thickness in excess of 1 mm. By utilizing established tissue-culture techniques, we have demonstrated that human corneal endothelium, when cultured and subsequently transplanted, retains its in vivo pump function. Although further studies are warranted, these results indicate that transplanted human corneal endothelial cells can function normally and suggest the possibility of endothelial cell replacement for therapeutic purposes.     

Cytochrome oxidase activity of Fuchs' endothelial dystrophy

The normal human corneal endothelial monolayer maintains stromal water equilibrium and thus, transparency, by means of a pump-leak mechanism. Water leaks into the stroma through non-tight lateral cell junctional complexes and is drawn out by an energy dependent cell membrane ion pump. We investigated the histochemical localization of cytochrome oxidase activity (CO), an important energy-deriving mitochondrial enzyme in dysfunctional corneas with Fuchs' endothelial dystrophy (ED), which is a regionally distributed disease. Keratoconus corneas were used as controls for functional control endothelium. In the central area of the corneal button, decreased CO activity was demonstrated which correlated clinically with central corneal edema. This reflects decreased metabolic activity and/or decreased numbers of mitochondria in the attenuated dysfunctional cells. In the mid-periphery, CO activity was increased in the cellular rosettes surrounding guttata, which may be related to increased synthesis of abnormal Descemet's membrane and guttata. Peripherally, the large polygonal cells resembled functional endothelium in their morphology and CO activity. We have, therefore, demonstrated regional differences in energy metabolism in endothelium from Fuchs' ED patients which may be related to decreased numbers of mitochondria in the dysfunctional cells, and/or to synthesis of abnormal Descemet's membrane material.     

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.      

Donor organ cultured corneal tissue selection before penetrating keratoplasty

AIMS—Donor organ cultured corneal tissue selection before penetrating keratoplasty is carried out by taking into account different variables. The objective was to identify preoperative variables which are significantly and independently associated with transplant outcome and should effectively be taken into account before transplantation.
METHODS—231 consecutive penetrating keratoplasties were prospectively studied using organ cultured tissue. Morphometric analysis of the donor corneal endothelium was performed before transplantation. Graft survival and endothelial cell density, during the second year following transplantation, were studied both at a univariate and multivariate level.
RESULTS—Recipient age, recipient rejection status, and preoperative diagnosis significantly influenced graft survival. Graft survival was higher when using corneal tissue from donors older than 80 years. Postoperative endothelial density decreased with preservation time and coefficient of variation after preservation. It increased with endothelial cell density after preservation and deswelling time, and correlated with preoperative diagnosis.
CONCLUSION—Organ cultured corneas with endothelial cell density after preservation <2000 cells/mm², and high coefficient of variation, may be discarded before transplantation. Corneas should be preserved for less than 3 weeks, and allowed to deswell before transplantation for 2 or 3 days rather than 1 day.

Keywords: corneal transplantation; endothelium; graft survival; morphometry     

The Corneal Endothelium: Normal and Pathologic Structure and Function

A summary of normal and abnormal endothelial structure and function is presented. Endothelium originates from neural crest and it elaborates a banded basement membrane in utero. It is involved in mesenchymal dysgenesis of the anterior segment, like the central defect of Peters' anomaly. Cytoplasmic organelles include mitochondria that provide energy for the metabolic pump, rough endoplasmic reticulum that participate in secretion of extracellular matrix, and a terminal web that may participate in cell migration. The endothelium's main function is to control corneal hydration and nutrition with a leaky barrier formed by the apical gap and macula occludens junctions that keep some water out of the stroma but allow nutrients to pass, and with an ATPase-dependent metabolic pump that is located in the lateral plasma membranes. Endothelial wound healing involves flattening and enlargement of cells to maintain an intact monolayer as well as production of abnormal collagenous material posterior to Descemet's membrane. HLA antigens located in the plasma membrane may participate in corneal endothelial graft rejection. Clinical assessment of the endothelium involves three modalities: specular microscopy to study endothelial morphology, fluorophotometry to measure barrier function, and pachymetry to measure corneal thickness.     

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway

PurposePrevious work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H₂) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism.MethodsMNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H₂. The effect of H₂ was examined using morphological and functional assays.ResultsIt was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na⁺/K⁺-ATPase, whereas H₂ improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H₂ could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway.ConclusionsThis study suggests that topical application of H₂ could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.     

What does the future hold for the treatment of Fuchs endothelial dystrophy; will ‘keratoplasty' still be a valid procedure?

Fuchs endothelial corneal dystrophy (FECD) is a well recognized corneal disorder characterized by the presence of collagenous warts extending from Descemet membrane (guttae) and endothelial cellular dysfunction due to cell loss and/or degeneration. Because of the characteristic abnormal cell morphology as seen with specular microscopy as well as the limited regenerative capacity in vivo, the endothelial cells were considered to be ‘dystrophic''. Hence, FECD is commonly managed by replacement of the endothelium with donor tissue by means of a penetrating or endothelial keratoplasty. The latter procedure has now been refined to the isolated transplantation of a donor Descemet membrane and its endothelium, referred to as Descemet membrane endothelial keratoplasty (DMEK). Unexpectedly, clinical observation made after DMEK seemed to challenge the current concept of the state of the endothelium in FECD; we actually observed an important role for the ‘dystrophic'' host endothelium in re-endothelialization of the denuded DM, and subsequent corneal clearance. In addition, recent studies regarding the pathophysiology of FECD made us realize that the endothelial cells are not ‘dystrophic'' per se, but in the course of time may have acquired a dysfunction instead. This paper describes the rationale behind this new concept and based on this, discusses the possibilities for future, less invasive treatment modalities for FECD.     

Morphometric analysis of corneal endothelial giant cells in normal and traumatized corneas

Corneal endothelial cells from normal and traumatized human, primate, cat and rabbit eyes were studied by specular microscopy. Morphometric analysis was performed on micrographs of corneal endothelium using a semi-automated image analysis system. The results showed that under normal conditions the corneal endothelium of all four species exhibit major morphological similarities (mean cell areas: human 317 ± 32 μm², primate 246 ± 22 μm², cat 357 ± 25 μm², rabbit 308 ± 35 μm²). The normal corneal endothelium in man was found to be more polymegethous than that of the other species. Trauma to cat, primate and human corneas resulted in a long-term reduction in endothelial cell density and enhanced polymegethism. In contrast, the reparative response of the rabbit ensured the reformation of an essentially normal monolayer following injury. Endothelial giant cells were a normal inclusion in the rabbit corneal endothelium but were only significant in cat, primate and man following trauma. The presence of corneal endothelial giant cells in amitotic corneas may therefore represent a compensatory response in the absence of mitotic potential.     

Effects of posterior chamber lens implantation on the endothelium of transplanted corneas

AIMS—The morphological changes of the corneal endothelium after posterior chamber lens implantation in the transplanted corneas were investigated.
METHODS—36 patients underwent extracapsular cataract extraction with posterior chamber lens implantation. Among these, penetrating keratoplasty had been performed in 18 patients before cataract surgery. The indications for penetrating keratoplasty in these cases included keratoconus, herpetic keratitis, and macula cornea. 18 cataract patients with normal corneas were also studied as controls. The central corneal endothelium in each subject was examined with a wide field specular microscope at a few days before and 3 months after cataract surgery.
RESULTS—Although the transplanted corneas showed lower endothelial cell densities, marked polymegethism, and pleomorphism in the baseline variables, the endothelial morphological changes in the transplanted corneas after posterior chamber lens implantation were comparable with those in the normal corneas. Also, there was no clinical evidence, especially, of corneal epithelial and/or endothelial rejections and corneal decompensation in all corneas.
CONCLUSION—Even though the transplanted corneas have a lower endothelial cell density and marked polymegethism, it is believed that cataract surgery does not induce corneal decompensation in cases where the peripheral recipient endothelium can be considered to have normal morphology.

     

Corneal endothelium in a case of mitochondrial encephalomyopathy (Kearns-Sayre syndrome)

A case of mitochondrial encephalomyopathy (Kearns-Sayre syndrome) with corneal endothelial abnormality is reported. A 22-year-old woman had retinitis pigmentosa, external ophthalmoplegia, complete heart block, ataxia, muscle weakness, dementia, sensorineural hearing loss, and was of short stature. Renal dysfunction, diabetes mellitus, and amenorrhea were also observed. Biopsy revealed decreased cytochrome c oxidase (complex IV) activity in muscle mitochondria. The corneal endothelium examined by specular microscope showed decreased cell density, severe polymegathism, and pleomorphism in both eyes. To our knowledge, this is the first report concerning primary corneal endothelial abnormality in a case with mitochondrial encephalomyopathy. The corneal endothelium is one of the tissues that could be affected by the enzyme deficiency present in this disease.     

Contact lens-induced corneal endothelial polymegathism: functional significance and possible mechanisms

The corneal endothelium is principally responsible for maintenance of corneal deturgescence. Therefore, compromise of corneal endothelial functional integrity can result in corneal swelling and opacification. Contact lenses constitute a potential insult to the cornea because their wear reduces the oxygen available to that tissue. It has been reported that contact lens wear induces transient as well as permanent morphologic changes in the corneal endothelium. One of the permanent changes reported is referred to as polymegathism, which is a variation in cell size within the endothelial monolayer. Several investigators have suggested that polymegathism reflects a compromised endothelial functional status. Mechanisms proposed to explain contact lens-induced polymegathism include lactate accumulation, changes in pH, and elevation in CO2 content. We discuss these possibilities as well as speculate that these polymegathous shape changes may be a result of decreased endothelial ATP (adenosine triphosphate) levels and disturbed calcium homeostasis due to corneal endothelial hypoxia.     

Prospects for endothelial transplantation

BACKGROUND: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells (HCEC), transplantation of these cells may be an alternative therapeutic option. MATERIALS AND METHODS: In this review methods for the in vitro cultivation of HCEC and their transplantation onto the Descemet membrane of donor corneas are described. RESULTS: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors. Dependent on the culture conditions, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behaviour. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation onto donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation status of the cells in vitro. Highest cell numbers up to 3000 cells/mm2 were achieved using a SV40-transformed HCEC-cell line. Monolayer integrity could be demonstrated by positive staining for integrins and light junction proteins, and pump function of the newly established endothelium was proven by perfusion studies. CONCLUSIONS: Methods to transplant HCEC onto human denuded corneas have been successfully established to reconstruct human corneas. Recent developments in genetic manipulation of cells and tissue engineering will be of great help in constructing suitable corneas for keratoplasty. Thus corneal endothelial cell transplantation is one of the promising future possibilities to provide corneas of high quality for patients. Furthermore, improvement of the transplantation technique may lead to a method to directly manipulate the diseased endothelium of patients with corneal endothelial dystrophies.