Ultracytochemical characteristics of cultured goat brain microvascular endothelial cells [corrected]

This ultrastructural study was undertaken to determine the localization of cytochemically demonstrable blood-brain barrier (BBB)-associated enzymatic activities and of some nonenzymatic constituents in goat [corrected] brain microvascular endothelial cells (ECs) growing in vitro. Positive reactions for alkaline phosphatase (AP), 5'-nucleotidase (5'N), transport ATPase (Na+,K(+)-ATPase), and adenosine diphosphatase (ADPase) were present on both apical and basolateral plasma membranes (PMs) of the ECs. The reaction for calcium-dependent ATPase (Ca(2+)-ATPase) was less intense and was restricted to basolateral PM and associated plasmalemmal pits. These cells also revealed an abundance of anionic sites labeled with cationic colloidal gold (CCG) and Ricinus communis agglutinin 120 (RCA)-binding sites, specific for beta-D-galactosyl residues, on the apical PM. The labeling of the apical PM with Ulex europaeus agglutinin (UEA)-gold complex, specific for alpha-L-fucosyl residues, was negligible. When compared with results of cytochemical examination of the ECs of goat [corrected] brain capillary in vivo, these observations indicate that although cells cultivated in vitro retain at confluence the enzymatic activities typical for BBB-type ECS, they lose their characteristic (polar) localization. This loss is interpreted as a reflection of lost functional polarity of the microvascular endothelium in vitro resulting from deprivation of the normal influence of the components of brain parenchyma.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Direct regulation of Na(+)-dependent myo-inositol transport by sugars in retinal pigment epithelium: role of phorbol ester and staurosporin

An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, [MI]i, to external free MI concentration, [MI]i/[MI]o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media. The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [3H]phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 +/- 8% reduction in [3H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [3H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary cilia in vertebrate corneal endothelial cells

The presence of primary cilia in corneal endothelial cells of a range of species from six non-mammalian vertebrate classes (Agnatha, Elasmobranchii, Amphibia, Teleostei, Reptilia and Aves) is examined by scanning and transmission electron microscopy. Our aim is to assess whether these non-motile cilia protruding into the anterior chamber of the eye are a consistent phylogenetic feature of the corneal endothelium and if a quantitative comparison of their morphology is able to shed any new light on their function. The length (0.42-3.80 microm) and width (0.12-0.44 microm) of the primary cilia varied but were closely allied with previous studies in mammals. However, interspecific differences such as the presence of a terminal swelling in the Teleostei and Amphibia suggest there are functional differences. Approximately one-third of the endothelial cells possess cilia but the extent of protrusion above the cell surface varies greatly, supporting a dynamic process of retraction and elongation. The absence of primary cilia in primitive vertebrates (Agnatha and Elasmobranchii) that possess other mechanisms to control corneal hydration suggests an osmoregulatory and/or chemosensory function.     

Studies on avian erythrocyte metabolism. XVII. Kinetics and transport properties of myo-inositol in chicken reticulocytes

The uptake of myo-inositol was determined in a reticulocyte-enriched fraction prepared from chicken blood and compared with uptake in mature erythrocytes. While reticulocytes accumulated inositol at levels more than threefold that of the plasma concentration, erythrocyte levels were only slightly higher than that of the plasma concentration. The rate of uptake in reticulocytes was approximately 66 mumol/ml rbc/h compared to 5 mumol/ml rbc/h in mature erythrocytes when measured at an inositol medium concentration of 250 microM. The kinetic analysis of inositol influx by reticulocytes reveals a two component system: saturable and nonsaturable. The saturable component, which has a Km for inositol of approximately 222 microM, is Na-dependent. This Na-dependent saturable component, which presumably reflects active transport of inositol, accounts for 30-35% of the transport process. The saturable component is completely inhibited by amiloride but to a lesser extent by ouabain and bumetanide. Moreover, in the course of reticulocyte maturation, the saturable component is lost concomitantly with the completion of the synthesis of myo-inositol pentakisphosphate and the drastic decrease in the membrane permeability to inositol. In addition, phloretin and cytochalasin B, which bind to hexose carriers and inhibit hexose sugar transport, also inhibited inositol transport. The uptake of inositol was not affected by excesses of 3-O-methylglucose (100 mM) or by physiological concentrations of D-glucose. Thus, the transport mechanism of myo-inositol appears distinct from that of D-glucose.     

Kinetics of water transport in sickle cells

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.     

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4 °C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4–8 h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.     

Photosynthetic electron transport in guard cells of diverse species

Guard cells of plants representing 18 species were assayed qualitatively for potential to conduct photosynthetic linear electron transport. These plants included C₃ pteridophytes, C₃ and C₄ monocots, and C₃, C₄, and Crassulacean acid metabolism dicots. By use of a microfluorospectrophotometer, guard cell samples in epidermal peels were isolated optically. Chlorophyll fluorescence was monitored from the onset of excitation light. For guard cells of all these species, fluorescence intensity increased during illumination. When samples were preincubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, however, there was a more rapid increase in fluorescence. These results indicate that all tested guard cells conduct photosynthetic electron transport through the reaction center of photosystem II.     

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells

Previously we have shown that hyperosmolarity increasesNa⁺-myo-inositolcotransporter (SMIT) activity and mRNA levels in cultured endothelialcells. Because hyperosmolarity and cytokines, such as tumor necrosisfactor- (TNF-), activate similar signal transduction pathways, weexamined the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation. In contrastto the effect of hyperosmolarity, TNF- caused a time- andconcentration-dependent decrease in SMIT mRNA levels andmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was found in large-vessel endothelial cells (derived fromthe aorta and pulmonary artery) and cerebral microvessel endothelialcells. In bovine aorta and bovine pulmonary artery endothelial cells,TNF- activated nuclear factor (NF)-B. TNF- also increasedceramide levels, and C₂-ceramidemimicked the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation in bovineaorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibitNF-B activation, partially prevented the TNF--induced decrease inmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was also partially prevented by the protein kinase Cinhibitor calphostin C but not by staurosporine. These studiesdemonstrate that TNF- causes a decrease in SMIT mRNA levels andmyo-inositol accumulation in culturedendothelial cells, which may be related to the activation of NF-B.

     

角膜内皮细胞生理和再生特性研究进展

角膜内皮细胞膜上的Na -K -ATP酶、水通道蛋白-1和各类离子通道相辅相成,发挥离子和水液的跨膜转运功能,共同维持角膜的透明状态.影响角膜内皮细胞生理功能的因素,主要是通过影响Na -K -ATP酶、离子通道和水通道蛋白而发挥作用的.人类角膜内皮细胞再生能力具有年龄差异、区域差异、在体和离体差异,以及是否存在干细胞还存在争议等特点.成年人角膜内皮细胞失去有丝分裂能力,主要与细胞接触抑制、TGF-β2和p27kip1有密切关系.从分子水平和基因层面调控角膜内皮细胞的生理功能和再生能力,将为角膜病的防治提供新的思路.     

Cation transport in vascular endothelial cells and aging

Summary To understand the generation and maintenance of Na and K gradients in cultured vascular endothelial cells, net Na and K movements were studied. Ouabain-sensitive (OS) net Na gain and K loss were estimated as the difference between the cation content in the presence of ouabain and that in the control. Ouabain-and furosemide-resistant (OFR) fluxes were determined in the presence of the two inhibitors. When the normal medium bicarbonate and phosphate buffers were replaced by N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid both the OS ans OFR fluxes decreased more than 50%. Ouabain-sensitive and ouabain-and furosemide-resistant fluxes decreased with increasing cellular age (passage number) an effect not observed when the cation movements were studied in the absence of bicarbonate and phosphate. These results suggest that cultured vascular endothelial cells possess bicarbonate-and phosphate-dependent Na and K pathways which account for a significant portion of their passive movements. Furthermore, the behavior of cation permeabilities with passage number suggests that these modulations may be related to the cellular aging process.     

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.     

Gene transfer of cyto-protective molecules in corneal endothelial cells and cultured corneas: analysis of protective effects in vitro and in vivo

The loss of corneal endothelial cells plays a critical role in many corneal diseases and is a common phenomenon following cornea transplantation. In addition, the non-regenerative capacity of human corneal endothelial cells (HCEC) ultimately requires appropriate protection of corneal tissues during ex vivo storage to ensure vitality of the cells. However, only 70% of donor corneas can be used for grafting because of endothelial deficiencies. Corneal endothelial cell loss during storage is mainly induced by apoptotic cell death. This study was undertaken, for proof of principle, to investigate whether over-expression of cyto-protective molecules Bcl-x(L), Bag-1, and HO-1 prevents the loss of corneal endothelial cells both in vitro and in vivo. We demonstrate that gene transfer of both Bcl-x(L) and HO-1 has cyto-protective effects on HCEC in vitro. However, gene transfer of a single cyto-protective molecule does not prevent its rejection upon transplantation in a MHC class I/II disparate rat model.     

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Regulation and clinical implications of corneal epithelial stem cells

The corneal epithelium is known to have a rapid self-renewing capacity. The major advance in the field of cornead epithelial cell biology in the last decade is the establishment of the location of corneal epithelial stem cells at the limbus, i.e., the junctional zone between the cornea and the conjunctiva. This concept has helped explain several experimental and clinical paradoxes, produced a number of important clinical applications, and spawned many other research studies. This unique enrichment of epithelial stem cells at a site anatomically separated from their transient amplifying cells makes the ocular surface an ideal model to study the regulation of epithelial stem cells. The present review includes data from more recent studies and lays out other areas for future investigation, especially with respect to the role of apoptosis and cytokine dialogue between limbal epithelial stem cells and their stromal microenvironment.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - bFGF basic fibroblast growth factor - HGF hepatocyte growth factor - IGF-I insulin-like growth factor type I - IL-1 interleukin 1 - K3 or K12 keratin type 3 or 12 - KGF keratinocyte growth factor - LIF leukemia inhibitory factor - PDGF platelet-derived growth factor - PKC protein kinase C - TGF- transforming growth factor- - TGF- transforming growth factor- - TPA phorbol ester tumor promoting agents     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.