Murine Corneal Endothelial Cells Research Articles

Energy Shortage in Human and Mouse Models of SLC4A11-Associated Corneal Endothelial Dystrophies.

PurposeTo elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4.MethodsComparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11−/− MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations.ResultsCTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK–p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3− transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11−/− MCEnC.ConclusionsReduced steady-state ATP levels and subsequent activation of the AMPK–p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.

A new tool for the transfection of corneal endothelial cells: Calcium phosphate nanoparticles

Calcium phosphate nanoparticles (CaP-NP) are ideal tools for transfection due to their high biocompatibility and easy biodegradability. After transfection these particles dissociate into calcium and phosphate ions, i.e. physiological components found in every cell, and it has been shown that the small increase in intracellular calcium level does not affect cell viability. CaP-NP functionalized with pcDNA3-EGFP (CaP/DNA/CaP/DNA) and stabilized using different amounts of poly(ethylenimine) (PEI) were prepared. Polyfect®-pcDNA3-EGFP polyplexes served as a positive control. The transfection of human and murine corneal endothelial cells (suspensions and donor tissue) was optimized by varying the concentration of CaP-NP and the duration of transfection. The transfection efficiency was determined as EGFP expression detected by flow cytometry and fluorescence microscopy. To evaluate the toxicity of the system the cell viability was detected by TUNEL staining. Coating with PEI significantly increased the transfection efficiency of CaP-NP but decreased cell viability, due to the cytotoxic nature of PEI. The aim of this study was to develop CaP-NP with the highest possible transfection efficiency accompanied by the least apoptosis in corneal endothelial cells. EGFP expression in the tissues remained stable as corneal endothelial cells exhibit minimal proliferative capacity and very low apoptosis after transfection with CaP-NP. In summary, CaP-NP are suitable tools for the transfection of corneal endothelial cells. As CaP-NP induce little apoptosis these nanoparticles offer a safe alternative to viral transfection agents.

Cryopreservation and long-term culture of transformed murine corneal endothelial cells

To characterize the morphology and gene expression of transformed murine corneal endothelial cells. Primary immortomouse corneal endothelial cells were continuously cultured before and after cryopreservation. Morphologic assessment, real time-reverse transcriptase polymerase chain reaction ((RT)-PCR) and immunofluorescence studies were performed using newly cultured cells, cells that had been continuously in culture for 1 year, and cryopreserved cells, to assess for structural and functional integrity. The expression of corneal endothelial markers zonula occludens-1 (ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and myofibroblast markers Desmin, alpha smooth muscle actin (αSMA), and Vimentin was assessed and compared by both RT-PCR and immunofluorescence. Cells in culture formed a monolayer, and exhibited a polygonal shape after reaching confluence. Cells retained this morphology during the full observation time of 12 months and when reused after cryopreservation. Immunofluorescence experiments exhibited positive staining for NaK-ATPase and COL8A2 with low variability between the three groups. In RT-PCR experiments, ZO1, COL8A2 and Desmin were increased in fresh and thawed cells, αSMA was decreased, and NaK-ATPase and Vimentin remained unchanged, compared to 12-month-old cells. Comparing fresh and thawed cells, COL8A2 was increased in thawed cells, while Desmin was increased in fresh cells. Using the immortomouse strain, murine corneal endothelial cells can be propagated over a long time period and be used after cryopreservation. Cells retain the expression of NaK-ATPase, but show some decline in ZO1 and COL8A2 over time and after cryopreservation. The expression of myofibroblast markers suggests an endothelial-to-mesenchymal transformation process in culture.

Function of indoleamine 2,3‐dioxygenase in corneal allograft rejection and prolongation of allograft survival by over‐expression

Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-gamma and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.

Vergleich verschiedener viraler Vektoren zur Gentherapie von Hornhautendothelzellen

In this paper we compare the transduction efficiency, toxicity, and safety of retroviral vectors [equine infectious anemia virus (EIAV), human immunodeficiency virus-1 (HIV-1), human foamy virus (PFV] and adenovirus (Ad) for potential use in gene therapy of corneal endothelial cells. Murine corneal endothelial cells were transduced with EIAV, HIV-1, PFV, and Ad, resulting in the overexpression of a green fluorescent protein (eGFP) transgene marker. The transduction efficiency was assessed by flow cytometry, while cytotoxicity and apoptosis rate were detected by annexin V/propidium iodide (PI) stain. Ad had the highest transduction efficiency with 99% of the cells expressing the transgene, followed by EIAV (95%), HIV-1 (75%), and PFV (43%). However, the high transduction efficiency of Ad also resulted in the highest apoptosis rate (25%) in the corneal endothelial cells. There was no detectable difference in the toxicity between PFV and HIV-1 (10%). EIAV transduction had the lowest cytotoxicity, with only 3% of the cells being annexin V/PI positive. Compared to other vectors EIAV exhibited high transduction efficiency combined with low toxicity to corneal endothelial cells. Therefore, it is a powerful tool for gene therapy applications in selected corneal endothelial diseases.

Indoleamine 2,3-dioxygenase protects corneal endothelial cells from UV mediated damage

Indoleamine-2,3-dioxygenase (IDO) is an intracellular enzyme present in dendritic cells and macrophages. It is a known modulator of T-cell response and contributes to the UV protection of the lens. There yet is no information on IDO activity in the corneal endothelium, protecting the endothelial cells from light mediated damage. We exposed murine corneal endothelial cells (MCEC) with different doses of UV-B light 280–320 nm, probed for IDO mRNA (real-time PCR) and assessed apoptosis rate (flow cytometry) and caspase-3-activity in the cells. The metabolites of the IDO catalysed reaction, l-kynurenine, was also measured. Malondialdehyde was detected for quantification of UV-B-induced oxidative stress. To investigate specificity, IDO effects were blocked by 1-methyl-tryptophan. The effects of IDO overexpression in the MCEC were assessed by transfection of an expression vector. MCEC consistently express IDO at low levels. Exposure to UV-B light led to a dose-responding upregulation of IDO; IDO was found competent converting l-tryptophan into l-kynurenine. Irradiation led to increased apoptosis and caspase-3-activity of MCEC. Supplementation of l-kynurenine or overexpression of IDO in the MCEC could reduce apoptosis significantly following UV-B irradiation. Inhibition of IDO by 1-MT was potent to reverse this effect. IDO and its metabolite l-kynurenine can protect corneal endothelial cells from UV-B-induced oxidative stress and apoptosis. It may be an active protection mechanism against corneal endothelial damage.

Vitamins Inhibit Oxidant-Induced Apoptosis of Corneal Endothelial Cells

To determine the effects of vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells. Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. The concentration of antioxidative vitamins (ascorbic acid, tocopherol, and retinoic acid) in the cells and supernatant was determined using reversed-phase high-performance liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity, using annexin-V/propidium iodide stains for flow cytometry. Lipid peroxidation was assessed using the malondialdehyde method. Supplementation of antioxidative vitamins was tested in the setting of apoptosis. Increasing levels of free iron led to a rapid loss of antioxidative vitamins in the supernatant and corneal endothelial cells. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or alpha-tocopherol alone was not sufficient to prevent lipid peroxidation in the cells, whereas a combination of vitamins C and E was able to do so. In contrast, supplementation with vitamin A alone significantly reduced oxidative stress and apoptosis. We present an in vitro model to test the direct influence of vitamin supplementation on corneal endothelial cells with regard to lipid peroxidation and apoptosis. We show that supplementation with antioxidative vitamins of corneal endothelial cells significantly prevents the generation of free-radical injury, lipid peroxidation, and consequent apoptosis.

Verminderung der Lipidperoxidation und der Apoptoserate in kornealen Endothelzellen durch Vitamin A

The goal of this study was to determine the effects of lipid peroxidation-mediated toxicity of iron ions on corneal endothelial cells leading to apoptosis. Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. Retinoic acid in the cell supernatant and cytoplasm of these cells was determined using HPLC. The rate of apoptosis was assessed by quantification of caspase-3-like activity. The lipid peroxidation was measured using the malondialdehyde method. Supplementation of retinoic acid was tested in the setting of apoptosis. Free iron ions led to a rapid loss of retinoic acid in the supernatant and the corneal endothelial cells. This was correlated with rising levels of malondialdehyde following oxidative stress and increased apoptosis. Supplementation of retinoic acid alone significantly reduced oxidative stress and apoptosis in the respective cells. In this study the authors present a novel in vitro model to test the direct influence of pro-oxidative species on corneal endothelial cells. The authors also prove that supplementing corneal endothelial cells with retinoic acid sufficiently prevents free radical injury and apoptosis.

Anti-oxidative vitamins prevent lipid-peroxidation and apoptosis in corneal endothelial cells

To determine the effects of vitamin supplementation on the lipid-peroxidation-mediated toxicity of iron-ions on corneal endothelial cells (CECs) leading to apoptosis, murine CECs were maintained in tissue culture medium supplemented with increasing concentrations of free iron-ions, a treatment known to lead to increased lipid-peroxidation. The concentration of anti-oxidative vitamins (ascorbic acid, tocopherol and retinoic acid) in the cell supernatant and in the cells was determined by high-pressure liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity and by using annexin-V/propidium iodide stains for flow cytometry. Lipid-peroxidation was measured by the malondialdehyde method. Supplementation with anti-oxidative vitamins was tested for the ability to counteract the induction of apoptosis. The production of nitric oxide was assessed spectrophotometrically and the expression levels of inducible and endothelial nitric oxide synthase were determined by Western blot. Increasing levels of free iron led to a rapid loss of anti-oxidative vitamins in the supernatant and in the CECs. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or alpha-tocopherol alone did not prevent lipid-peroxidation in the cells. A combination of vitamins C and E (ascorbic acid, tocopherol) or solitary supplementation with vitamin A (retinoic acid) prevented lipid-peroxidation. We thus present a novel in vitro model for testing the direct influence of pro-oxidative species on CECs. We also show that supplementation with anti-oxidative vitamins to CECs significantly prevents the generation of free-radical-induced oxidative injury and apoptosis. These findings may have important implications for the storage of human corneae prior to transplantation and for the prolongation of corneal graft survival.