Mouse Corneal Endothelial Cells Research Articles

Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.

Recent studies from Slc4a11 −/− mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)—Golgi, we asked whether ER function is affected by mitochondrial ROS in Slc4a11 KO corneal endothelial cells. In mouse Slc4a11 −/− corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP and GADD153, as well as reduced ER Ca2+ release as compared to WT. Induction of mitoROS by ETC inhibition also led to ER stress in WT cells. Treatment with the mitochondrial ROS quencher MitoQ, restored ER Ca2+ release and relieved ER stress markers in Slc4a11 KO cells in vitro. Systemic MitoQ also reduced BIP expression in Slc4a11 KO endothelium. We conclude that mitochondrial ROS can induce ER stress in corneal endothelial cells.

RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice

Slc4a11 KO mice show significant corneal edema, altered endothelial morphology, and mitochondrial ROS at an early age without a decrease in endothelial cell density. We examined the differential gene expression profile between wild type (WT) and KO with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function that can explain the corneal edema. Freshly dissected Corneal Endothelium-Descemet's Membrane (CEDM) and cultured Mouse Corneal Endothelial Cells (MCEC) were obtained from WT and Slc4a11 KO mice. RNA sequencing Ingenuity Pathway Analysis (IPA) predicted activation, inhibition or differential regulation of several pathways. QPCR and Western analysis validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. Functional testing revealed decreases in endothelial lactate production, Extracellular Acidification Rate (ECAR), glutaminolysis, and Oxygen Consumption Rate (OCR) of KO CEDM in the presence of Glutamine (Gln) that was not compensated by fatty acid oxidation. Stromal lactate was significantly elevated in KO along with decreased expression of MCT1 and MCT4 lactate transporters in endothelial cells. ATP levels were 2x higher in KO CEDM, concomitant with a 3-fold decrease in Na-K-ATPase activity and reduced basolateral membrane localization. Genes for cholesterol biosynthesis, glutathione metabolism and tight and adherens junctions were elevated. Alteration of tight junction structure and cortical cytoskeleton is evident in KO corneal endothelium with a significant increase in trans-endothelial fluorescein permeability. We conclude that Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, lactate transport and Na-K-ATPase activity. These changes together with an altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema.

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.

STAT3 signaling maintains homeostasis through a barrier function and cell survival in corneal endothelial cells

The cornea protects the eye from inflammation, which is one of the leading causes of blindness. Severe inflammation in the anterior chamber disrupts the barrier function of corneal endothelial cells (CECs) leading to severe visual loss. However, the mechanism by which such inflammation affects CEC function and survival is unknown. Activation of STAT3 signaling regulates various intracellular responses through inflammation and generally mediates expression of the barrier function marker zonula occludens-1 (ZO-1). In this study, we investigated the relationship between the corneal endothelial barrier function and activation of STAT3 signaling through a variety of cytokines in human CECs. Phosphorylated STAT3 (pSTAT3) was expressed in human and mouse CECs. Inhibition of pSTAT3 remarkably decreased the expression of the ZO-1 protein, reduced the trans-endothelial electric resistance, and induced cell apoptosis. The expression level of ZO-1 mRNA was correlated with that of STAT3 mRNA in the human corneal endothelium. pSTAT3 was increased with the addition of LIF, IL-6, and IFN-γ. LIF expressed in CECs suppressed pSTAT3 activation as observed experimentally using an anti-LIF antibody. Promoter regions of ZO-1 and SOCS3 were directly regulated by transcriptional activation of STAT3. These findings suggest that regulation of the STAT3 pathway is essential for corneal endothelial homeostasis via barrier function and may protect from various inflammatory factors.

173 - SLC4A1111 provides NH3 sensitive mitochondrial uncoupling and ROS prevention that facilitates glutamine catabolism

Mutations in SLC4A11 can cause CHED (Congenital Hereditary Endothelial Dystrophy) a disorder of the Corneal Endothelium that produces corneal edema and poor vision in early childhood. Slc4a11-/- (KO) mice recapitulate CHED showing corneal edema, evidence of oxidative stress, and eventual death of corneal endothelial cells. SLC4A11, which is highly expressed in the corneal endothelium, functions as an NH3-sensitive electrogenic H+ transporter. Loss of SLC4A11 depresses glutamine (Gln) catabolism, yielding lower [ATP] and NH3 production relative to wild-type. SLC4A11 has been localized to the basolateral membrane of polarized cells and undetermined cytoplasmic locations. In the current study, we found that SLC4A11 co-localizes to mitochondria, and is associated with inner mitochondrial membrane proteins. To test if SLC4A11 is acting as an NH3-activated mitochondrial uncoupler, we used Mouse Corneal Endothelial Cell (MCEC) lines derived from WT and Slc4a11 KO mice. We found that application of NH3 depolarized WT mitochondrial membrane potential (MMP), but not KO. Relative to KO, WT cells had a higher oxygen consumption rate (OCR), greater proton leak, greater NAD/NADH ratio, and higher [ATP] in the presence of 0.5 mM Gln. Moreover, mitochondrial ROS after 24 hours Gln-incubation was significantly higher in KO with concomitant MMP depolarization, low [ATP], and mPTP opening leading to increased apoptosis. Inhibiting Gln derived NH3 production by GLS1 inhibitors BPTES or CB839 or Dimethyl-α-Ketoglutarate ameliorated these effects. NH3 (25mM) alone increased ROS, decreased [ATP], and increased apoptosis in KO without affecting WT cells, indicating that KO cells are more sensitive to NH3. Slc4a11 KO cells were rescued from the effects of Gln or NH3 by MitoQ ROS scavenging or the mitochondrial uncoupler BAM15. In summary, these results indicate that mitochondrial Slc4a11 NH3 sensitive H+ influx is providing mitochondrial uncoupling that lowers Gln-dependent ROS production, facilitating Gln catabolism and protecting mitochondria.

Slc4a11 Facilitates Glutamine Metabolism, Mitochondrial Function and ROS Prevention

PurposeSLC4A11 codes for an electrogenic NH3‐sensitive H+ transporter. Slc4a11−/− mice show significant oxidative damage within corneal endothelial cells and an alteration of glutamine metabolism. Here, we asked if NH3 derived from glutamine (Gln) catabolism induces oxidative stress and whether Slc4a11 is protective.MethodsCorneal endothelial tissue from Slc4a11−/− mice was evaluated for oxidative stress (MitoSox, protein nitrosylation and OHdGuanine), mitochondrial morphology (MitoTracker Green) and autophagy (LC3B). Conditionally immortalized cell lines of Slc4a11−/− and WT mouse corneal endothelial cells (MCEC) expressing temperature sensitive Large T antigen were generated. In MCEC, cell death was quantified by AnV‐FITC + PI, mitochondrial anion superoxide (O2−) by MitoSox, mitochondrial membrane potential (MMP) measured by TMRE, and ATP was determined by luciferin‐luciferase assay. NH3 production was measured by colorimetric assay. Relative glucose and glutamine flux was quantified by GC‐MS.ResultsKO corneal endothelium from 12 and 40 week old mice show greater production of mitochondrial O2−, oxidized DNA and protein, induction of autophagy, and alteration of mitochondrial morphology compared to WT. In WT MCEC, Gln (0.5 mM) increases [ATP] concomitant with increased mitochondrial O2−, and increased apoptosis (compared to Gluc only). In contrast, in KO MCEC Gln decreases [ATP], with significantly greater mitochondrial O2− and apoptosis (compared to WT) that can be rescued by MitoQ. Application of NH3 alone increased mitochondrial O2−, depolarized MMP and increased apoptosis significantly more in KO. Gln derived NH3 toxicity can be partially reduced by GLS1 inhibitors BPTES or CB839 and totally inhibited by supplementation with Dimethyl‐α‐Ketoglutarate (α‐KG) or by Cyclosporin A (inhibitor of the opening of the mitochondrial transition pore). In MCEC stained with TMRE, 2 population of were observed: a population of smaller cells with depolarized MMP (TMRE− cells) and a population of normal size cells with hyperpolarized MMP (TMRE+ cells). Even though Gln increases the percentage of TMRE− cells the remainder TMRE+ cells are hyperpolarized as expected as Gln fuels the TCA cycle. This hyperpolarization is much higher in KO and suggest that it is the cause of O2− production followed by PTP opening and then secondary MMP depolarization. Gln induced hyperpolarization and NH3 derived Gln are the cause of high O2− production in KO cells.ConclusionWe conclude that Slc4a11 protects cells from Gln and NH3 induced toxicity.Support or Funding InformationNIH RO1 EY008834This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

PurposeTo establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11−/− mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.MethodsWe intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11−/− C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS).ResultsThe immortalized Slc4a11+/+ and Slc4a11−/− mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11−/− MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11−/− mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs.ConclusionsThis is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11−/− MCECs. Furthermore, Slc4a11−/− MCECs recapitulate the glutaminolysis defects observed in Slc4a11−/− mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.

Low temperature induces cryoinjury in mouse corneal endothelial cells by stimulating the Stk11-p53 signal pathway.

Cryoinjury, or injury caused by extremely low temperatures, can occur in corneal endothelial cells (CECs) and lead to visual impairment. However, the mechanism of cryoinjury in CECs is not clear. The Stk11-p53 signaling pathway regulates the proliferation and division of cells. Activity of the Stk11-p53 signaling pathway arrests the cell cycle at the G0/G1 phase and induces apoptosis. In this study, a mouse model of cryoinjury in CECs was used. Following injury, significant mouse CEC death and shedding were observed. In addition, the mRNA and protein levels of core factors from the Stk11-p53 signaling pathway (Stk11, p21 and p53) were elevated and Caspase-3 was activated following cryoinjury. In addition, chromatin immunoprecipitation revealed that Stk11 catalyzed p53 serine 15 phosphorylation, and the Stk11-p53 complex bound to the p21 promoter and stimulated gene transcription. Thus, the results of the present study suggest that cryoinjury leads to the damage and apoptosis of mouse CECs by activation of the Stk11-p53 signaling pathway, phosphorylation of p53 serine 15 and p21 gene transcription.

Mesothelial cells: a cellular surrogate for tissue engineering of corneal endothelium.

To evaluate whether mouse adipose tissue mesothelial cells (ATMCs) share morphologic and biochemical characteristics with mouse corneal endothelial cells (CECs) and to evaluate their capacity to adhere to the decellularized basal membrane of human anterior lens capsules (HALCs) as a potential tissue-engineered surrogate for corneal endothelium replacement. Adipose tissue mesothelial cells were isolated from the visceral adipose tissue of adult mice, and their expression of several corneal endothelium markers was determined with quantitative RT-PCR, immunofluorescence, and Western blotting. Adipose tissue mesothelial cells were cultured in a mesothelial retaining phenotype medium (MRPM) and further seeded and cultured on top of the decellularized basal membrane of HALCs. ATMC-HALC composites were evaluated by optical microscopy, immunofluorescence, and transmission electron microscopy. Mesothelial retaining phenotype medium-cultured ATMCs express the corneal endothelium markers COL4A2, COL8A2, SLC4A4, CAR2, sodium- and potassium-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase), β-catenin, zona occludens-1, and N-cadherin in a pattern similar to that in mouse CECs. Furthermore, ATMCs displayed strong adhesion capacity onto the basal membrane of HALCs and formed a confluent monolayer within 72 hours of culture in MRPM. Ultrastructural morphologic and marker characteristics displayed by ATMC monolayer on HALCs clearly indicated that ATMCs retained their original phenotype of squamous epithelial-like cells. Corneal endothelial cells and ATMCs share morphologic (structural) and marker (functional) similarities [corrected]. The ATMCs adhered and formed structures mimicking focal adhesion complexes with the HALC basal membrane. Monolayer structure and achieved density of ATMCs support the proposal to use adult human mesothelial cells (MCs) as a possible surrogate for damaged corneal endothelium.

L450W and Q455KCol8a2Knock-In Mouse Models of Fuchs Endothelial Corneal Dystrophy Show Distinct Phenotypes and Evidence for Altered Autophagy

We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues. In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using total RNA, and protein extracted from mouse CECs and human CECs obtained from FECD and autopsy patients. Both Col8a2 mouse models exhibited hallmarks of FECD; however, the Col8a2(L450W/L450W) mice exhibited a milder phenotype compared to the Col8a2(Q455K/Q455K) mice. Both models exhibited upregulation of the unfolded protein response (UPR) as evidenced by dilated rough endoplasmic reticulum (RER), and upregulation of UPR-associated genes and proteins. Real-time PCR of Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) CECs at 40 weeks revealed a 2.1-fold (P < 0.05) and a 5.2-fold (P < 0.01) upregulation of the autophagy marker Dram1, respectively. Real-time PCR of human FECD endothelium revealed a 10.4-fold upregulation of DRAM1 (P < 0.0001) compared to autopsy controls. The Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) mouse models of FECD showed distinct endothelial cell phenotypes. Dram1 was associated with activation of the UPR and increased autophagy. Overexpression of this gene in mouse and human FECD endothelial cells suggested a role for altered autophagy in this disease.

Functional Corneal Endothelium Derived from Corneal Stroma Stem Cells of Neural Crest Origin by Retinoic Acid and Wnt/β-Catenin Signaling

Corneal endothelial dysfunction remains a major indication for corneal transplantation. Both corneal endothelial cells and stromal cells originate from the neural crest, but have distinct phenotypes and function in the adult cornea. We previously reported that stem cells isolated from the adult corneal stroma [cornea-derived precursors (COPs)] show characteristics of multipotent neural crest-derived stem cells. In this study, we report the induction of functional tissue-engineered corneal endothelium (TECE) from mouse and human COPs. TECE was engineered from Wnt1-Cre/Floxed EGFP mouse COPs in a medium containing retinoic acid and glycogen synthase kinase (GSK) 3β inhibitor (activator of Wnt/β-catenin signaling). The expression levels of major markers characterizing corneal endothelial function (Atp1a1, Slc4a4, Car2, Col4a2, Col8a2, and Cdh2) were significantly upregulated. Both retinoic acid and GSK 3β inhibitor upregulated the expression of Pitx2, a homeobox gene involved in the development of the anterior segment of the eye. GSK 3β inhibitor increased Atp1a1 expression and Na,K-ATPase pump activity of TECE, which was significantly higher than COPs or control 3T3 cells, and 2.6-fold higher than cultured mouse corneal endothelial cells. Mouse TECE transplanted into rabbit corneas maintained transparency and corneal thickness, whereas control corneas without TECE showed marked edema and increased corneal thickness. Furthermore, we successfully induced TECE from human COPs, and human TECE transplanted into rabbit corneas also maintained corneal transparency and thickness. This protocol enables efficient production of corneal endothelium from corneal stromal stem cells by direct induction, which may lead to a novel stem cell therapy for corneal endothelial dysfunction.

Hormonal Regulation of Na+/K+-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

Role of Insulin in Regulation of Na+-/K+-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression, design optimization, uptake mechanism and nuclear localization

Gene transfer to the corneal endothelium has potential in preventing corneal transplant rejection. In this study, we transfected mouse corneal endothelial cells (MCEC) with a class of novel arginine-rich oligopeptides. The peptides featured a tri-block design and mediated reporter gene expression in MCEC more efficiently than the commercial polyethylenimine standard. The functionality of each block was demonstrated to critically influence the performance of the peptide. Results from confocal imaging and flow cytometry then showed that energy-dependent endocytosis was the dominant form of uptake and multiple pathways were involved. Additionally, uptake was strongly dependent on interactions with cell-surface heparan sulphate. Fluorescence resonance energy transfer studies revealed that the peptide/DNA entered cells as an associated complex and some will have dissociated by 8.5 h. Large-scale accumulation of uncondensed DNA within the nucleus can also be observed by 26 h. Finally, as a proof of biological relevance, we transfected MCEC with plasmids encoding for the functional indoleamine 2,3-dioxygenase (IDO) enzyme. We then demonstrated that the expressed IDO could catalyse the degradation of l-tryptophan, which in turn suppressed the growth of CD4+ T-cells in a proliferation assay.

Role of protein kinase C in regulation of Na+- and K+-dependent ATPase activity and pump function in corneal endothelial cells

Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

The Effects of Dexamethasone on the Na,K-ATPase Activity and Pump Function of Corneal Endothelial Cells

Purpose: The Na+- and K+-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Methods: Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase α1-subunit. Results: Dexamethasone (1 or 10 μ M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase α1-subunit. Conclusions: Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase α1-subunits.

Characteristics and Outcome of AIDS-Related Hodgkin Lymphoma Before and After the Introduction of Highly Active Antiretroviral Therapy

Corneal endothelial cell (CEC) transplantation should become clinically applicable in the near future. However, the immunologic changes after allo-CEC transplantation are poorly understood at present. We tried to establish a mouse model of allogeneic CEC transplantation for immunologic studies. Benzalkonium chloride was injected into the anterior chamber of the eyes of recipient BALB/c mice to create bullous keratopathy. Full-thickness corneal transplantation was performed by using 4 types of corneas: BALB/c corneas (isograft group), BALB/c corneas denuded of CEC (no endothelium group), C3H/He mouse corneas (allograft group), and corneas reconstituted by seeding immortalized C3H/He mouse CECs onto BALB/c corneas denuded of endothelium (CEC allograft group). Eyes were observed with an operating microscope for 4 weeks after transplantation and were subjected to histologic examination and fluorescein microscopy. All corneal grafts were transparent in the isograft group (n = 12), whereas none of the grafts were clear by 4 weeks after transplantation in the no endothelium group (n = 13). Corneal grafts were transparent at 4 weeks in 75% of the CEC allograft group (n = 12). The histologic rejection rate was 0% in the CEC allograft group, which was significantly lower than in the allograft group (67%; n = 18; P < 0.01). We established a mouse allo-CEC transplantation model by using cultured cells. This model should be useful for studying the immunologic processes after CEC transplantation.

P-selectin, an important mediator of angiogenesis in females: the role of estradiol

Angiogenesis, or new blood vessel formation, is key in vasculoproliferative disorders including rheumatoid arthritis. Here we report that the mediation of angiogenesis by P-selectin, an important member of the selectin adhesion molecule family, is unexpectedly sex dependent. In vivo angiogenesis measured in a Matrigel plug, in a sponge granuloma, and in a corneal micropocket was consistently impaired in P-selectin gene deficient mice compared with wild-type mice, but only if the P-selectin-deficient mice were female. Estrogen appeared to be one significant mediator of P-selectin sensitivity. In vitro the chemotactic activity of soluble P-selectin for human dermal microvascular endothelial cells was augmented by 17β-estradiol (E2). Conversely, an antibody blockade of P-selectin reduced E2-induced sprout formation in female mouse corneal endothelial cell morphogenesis assays, inhibited E2-induced endothelial cell signaling via the Src and mitogen-activated protein kinase pathways, and decreased E2-induced secretion of angiogenic basic fibroblast growth factor. In addition to its classical roles in leucocyte extravasation, P-selectin may also contribute to a variety of inflammatory diseases as a sex-restricted angiogenic intermediary.

Human Corneal Organotypic Cultures

Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.

Characterization of a Novel Cis-element That Regulates Fas Ligand Expression in Corneal Endothelial Cells

Constitutively expressed Fas ligand in the cornea, Sertoli cell of the testes, Paneth cell of the intestines, and Clara cell of the airway protect surrounding parenchymal tissue by inducing apoptosis of Fas(+) immune cells during inflammatory reactions. Indeed, the action of corneal Fas ligand has been suggested to facilitate successful allogeneic cornea transplantation. To study the transcriptional regulation of Fas ligand in the eye, we employed an immortalized mouse corneal endothelial cell line (C3H3) that constitutively expresses Fas ligand. By deletion analysis of the mouse Fas ligand promoter, gel mobility shift assays, and site-directed mutagenesis, we found that a TCCT motif located -299 base pairs upstream from the transcriptional start site served as a major positive regulatory cis-element in C3H3 cells. In contrast, this element was not required for Fas ligand transcriptional activity in Sertoli cells and airway epithelial cells. By UV cross-linking analysis, we found that an approximately 30-kDa corneal nuclear protein binds to the Fas ligand promoter TCCT box and, thus, likely plays an important role in Fas ligand expression in corneal endothelial cells.