Mitochondrial Antioxidant SkQ1 Improves Hypothermic Preservation of the Cornea.

Diseases of the cornea are a frequent cause of blindness worldwide. Keratoplasty is an efficient method for treating severely damaged cornea. The functional competence of corneal endothelial cells is crucial for successful grafting, which requires improving the media for the hypothermic cornea preservation, as well as developing the methods for the evaluation of the corneal functional properties. The transport of water and ions by the corneal endothelium is important for the viability and optic properties of the cornea. We studied the impact of SkQ1 on the equilibrium sodium concentration in the endothelial cells after hypothermic preservation of pig cornea at 4°C for 1, 5, and 10 days in standard Eusol-C solution. The intracellular sodium concentration in the endothelial cells was assayed using the fluorescent dye Sodium Green; the images were analyzed with the custom-designed CytoDynamics computer program. The concentrations of sodium in the pig corneal endothelium significantly increased after 10 days of hypothermic preservation, while addition of 1.0 nM SkQ1 to the preservation medium decreased the equilibrium concentration of intracellular sodium (at 37°C). After 10 days of hypothermic preservation, the permeability of the plasma membrane for sodium decreased in the control cells, but not in the cells preserved in the presence of 1 nM SkQ1. Therefore, SkQ1 increased the ability of endothelial cells to restore the intracellular sodium concentration, which makes SkQ1 a promising agent for facilitating retention of the functional competence of endothelial cells during cold preservation.

3D map of the human corneal endothelial cell.

Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 µm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca(2+)-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Progenitors for the corneal endothelium and trabecular meshwork: a potential source for personalized stem cell therapy in corneal endothelial diseases and glaucoma.

Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE) and the anterior nonfiltering portion of the trabecular meshwork (TM), which is known as the Schwalbe's Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

Identification of alpha-Gal and non-Gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry.

PURPOSE: To investigate the expression of alpha-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry. METHODS: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immunochemical staining was added to confirm the non-Gal antigen expression in pig corneal cells. RESULTS: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of alpha-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed. CONCLUSIONS: As well as alpha-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the alpha-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.

Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas.

PURPOSE: The corneal endothelium is a monolayer of cells in the posterior cornea that is responsible for maintaining a clear cornea. Corneal endothelial cells may be induced to divide, but it has been held that they do not divide in the normal cornea of an adult human. Some studies have suggested that a stem cell population for the corneal endothelium exists. This population could give rise to mature corneal endothelial cells and may reside either in the peripheral corneal endothelium or in the adjacent posterior limbus. This study was initiated to demonstrate the presence of such stem cells in the region of the posterior limbus and to show the response of these cells to corneal wounding. METHODS: Unwounded and wounded corneas with their attached limbal sections were analyzed by immunofluorescence for the presence of nestin, telomerase, Oct-3/4, Pax-6, Wnt-1, and Sox-2. Alkaline phosphatase activity was observed with an enzyme-based reaction that produced a fluorescent product. RESULTS: In the unwounded cornea, stem cell markers nestin, alkaline phosphatase, and telomerase were found in the trabecular meshwork (TM) and in the transition zone between the TM and the corneal endothelial periphery (including Schwalbe's line). Telomerase was also present in the peripheral corneal endothelium. When wounded corneas and their attached limbii were tested, the same markers were found. However, after wounding, additional stem cell markers, Oct-3/4 (in the TM) and Wnt-1 (in both the TM and the transition zone), appeared. Moreover, the differentiation markers Pax-6 and Sox-2 were seen. Pax-6 and Sox-2 were also manifest in the peripheral endothelium post-wounding. CONCLUSIONS: Well documented specific stem cell markers were found in the TM and the transition zone of the human posterior limbus. Wounding of the corneas activated the production of two additional stem cell markers (Oct-3/4, Wnt-1) as well as two differentiation markers (Pax-6, Sox-2), the latter of which also appeared in the corneal endothelial periphery. It is suggested that stem cells reside in the posterior limbus and respond to corneal wounding to initiate an endothelial repair process. The stem cells may also contribute to a normal, slow replacement of corneal endothelial cells.

Caveolin-1 is up-regulated in transdifferentiated lens epithelial cells but minimal in normal human and murine lenses.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types. Caveolae play a role in lipid transport, endocytosis, signal transduction, and cell transformation. Expression of caveolin-1, the principal component of caveolae and a regulator of caveolae-dependent signaling and endocytosis, was investigated in lens epithelial cells and lens fiber cells in wild-type (wt) and SPARC-null mice and normal human donors in vivo and in vitro. RT-PCR, immunofluorescence and immunoblot analyses of human and murine ocular tissues revealed that caveolin-1 was expressed in the corneal epithelium, corneal endothelial cells, and blood vessels of iris, ciliary body and retina, but minimal in the normal lens epithelia or fiber cells of murine and human lens. In contrast, caveolin-1 was significantly up-regulated in mesenchymal-transdifferentiated lens epithelia in SPARC-null cataract lenses. In addition, lens epithelial cells from primary culture or from cultures of immortalized lens epithelial cell lines expressed significant amounts of caveolin-1. The lens epithelial cells expressed epidermal growth factor (EGF) receptor and were responsive to EGF-mediated cell proliferation, but they did not show EGF-dependent caveolin-1 tyrosine phosphorylation. Caveolin-1 might have a role in the process of epithelial-mesenchymal transdifferentiation (EMT) in the lens, the most common cause of vision loss in human secondary cataracts.

[Protective effect on the corneal endothelium and remaining effect at the anterior chamber for three different kinds of viscoelastic devices].

PURPOSE: The present study was performed to evaluate the corneal endothelium protection and anterior chamber stagnation abilities of three different types of viscoelastic substances (Healon, Viscoat, HealonV). METHODS: Viscoelastic substances were selected at random for 120 eyes with cataracts, and the postoperative reduction rates of the corneal endothelium cells were compared. The residual viscoelastic substances after filling of the anterior chamber of pig eyes and aspiration with a handpiece were measured by an anterior eye segment image analysis system. The same procedures were performed in rabbit eyes and the residual levels of viscoelastic substances on the corneal endothelium were photographed histologically. RESULTS: The reduction rate of endothelium corneal cells tended to decrease with Viscoat three months after surgery. The results obtained with the anterior eye segment image analysis system showed that the residual level in the anterior chamber was higher with Healon. Histological analyses demonstrated residual Viscoat at the center of the corneal endothelium after perfusion. CONCLUSION: HealonV was superior in terms of spatial retention and Viscoat had corneal endothelium protection potential.

A novel strategy for corneal endothelial reconstruction with a bioengineered cell sheet.

Cellular organization of foreign grafts constructed from cultivated cells is critical to successful graft-host integration and tissue repair. This study described a novel human corneal endothelial cell (HCEC) therapeutic method, where cultivated adult HCEC sheet with uniform orientation was prepared and transplanted to a rabbit cornea. Having a correct morphology and intact barriers, the HCEC sheet was made by the temperature-modulated detachment of monolayered HCECs from thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces and was delivered with proper polarity to the corneal posterior surface by a bioadhesive gelatin disc. Results of the in vivo studies, including the follow-up clinical observations and histological examinations, showed the laminated HCEC sheet was successfully integrated into rabbit cornea denuded with endothelial layer after the biodegradation of gelatin carrier. These data indicate the feasibility of the proposed procedure in cell therapy for corneal endothelial cell loss.

Transgenic studies on the role of optineurin in the mouse eye.

Mutations in the OPTN gene encoding for optineurin have been associated with primary open-angle glaucoma. The functional role(s) of optineurin in the normal and glaucomatous eye are unclear. As optineurin interferes with TNF-alpha mediated cell death in vitro, an involvement of optineurin in the regulatory pathways leading to apoptosis of retinal ganglion cells has been suggested. The goal of the present study was to study the molecular properties of optineurin and its capabilities to prevent apoptosis in vivo in the eyes of transgenic mice. The chicken betaB1-crystallin promoter was used to overexpress ectopic optineurin in the lenses of transgenic mice. The expression of transgenic mRNA was monitored by northern blot analysis. The localization of transgenic optineurin was investigated by one- and two-dimensional western blot analysis and by immunohistochemistry, and compared with that of endogenous optineurin. To assess effects of transgenic optineurin on apoptosis, betaB1-crystallin-OPTN mice were crossbred with betaB1-crystallin-TGFbeta1 mice that undergo substantial TGF-beta1-induced apoptotic cell death in the lens. Two independent betaB1-crystallin-OPTN transgenic lines were established, in which transgenic optineurin was expressed strictly lens-specific as assessed by Northern and Western blotting, and by immunohistochemistry. In contrast, endogenous optineurin was preferentially expressed in the retina, where retinal ganglion cells showed strong labeling. Immunostaining for endogenous optineurin in the anterior eye was considerably weaker than in the posterior eye and was seen in iris, ciliary epithelium, cells of corneal stroma and endothelium, and in the trabecular meshwork. Neither transgenic nor endogenous optineurin was found in the aqueous humor. Transgenic overexpression of optineurin did not have measurable effects on TGFbeta1-induced apoptosis in mixed betaB1-crystallin-OPTN/betaB1-crystallin-TGFbeta1 transgenic mice. Our results show that optineurin is a cytoplasmatic rather than a secretory protein that is preferentially expressed in retinal ganglion cells, and argue against a major role of optineurin for the modulation of apoptosis in vivo.

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.

Characterization of adenosine receptors in bovine corneal endothelium.

Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).

[The relationship between the postmortem interval and the changes of DNA content in rabbit's cornea epithelial and endothelium by the computerized image analysis].

OBJECTIVE: To study the relationship between the postmortem interval (PMI) and the changes of DNA content in rabbit's cornea epithelial and endothelium. METHODS: Using the computerized image analysis to measure the DNA content in the cornea epithelial and endothelium of 105 rabbit's at every six hours during 72 hours after death. RESULTS: The degradation rate of DNA in nuclear has an apparent relationship in 72 hours after death of the rabbits. CONCLUSION: The determination of the quantity of DNA in cornea nuclear is a new method to estimate the PMI accurately.

Localization of ocular P2Y2 receptor gene expression by in situ hybridization.

The objective of this study was to determine the cellular localization of P2Y(2) receptor gene expression in rabbit and primate ocular tissues using the technique of non-isotopic in situ hybridization. Fresh frozen whole eye from a New Zealand White rabbit and whole eye and eyelid from a rhesus macaque were cut into 5 microm thick sections and mounted onto glass slides. In situ hybridization was performed on ocular cryosections using digoxigenin-labeled P2Y(2) receptor riboprobes. Alkaline phosphatase-conjugated anti-digoxigenin antibody was used to localize riboprobe hybridization, which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate. Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y(2) receptor mRNA was observed in the palpebral and bulbar conjunctival epithelium, including goblet cells, the corneal epithelium, and in meibomian gland sebaceous and ductal cells. Staining was also observed in both layers of the ciliary body epithelium, subcapsular epithelium of the lens, and corneal endothelium. In the posterior eye, staining was observed in various layers of the retina, including ganglion cell, inner nuclear, inner segment and retinal pigment epithelium layers, in the optic nerve head, and in a variety of structures within the choroid. No specific staining of sense riboprobe was seen on any of the ocular structures. These results demonstrate that the P2Y(2) receptor gene is expressed in a variety of ocular cells types and suggest that P2Y(2) receptors are associated with diverse physiological functions throughout the eye.

Expression and distribution of the serum and glucocorticoid regulated kinase and the epithelial sodium channel subunits in the human cornea.

The sodium transporting capacity of the corneal endothelium is vital for preserving corneal transparency, and has traditionally been attributed to the endothelial pump transporting sodium and bicarbonate across the corneal endothelium, maintaining the cornea in a dehydrated state. Recent studies have shown that the enzyme, serum and glucocorticoid regulated kinase isoform 1 (SGK1), plays a pivotal role in the corticosteroid induction of epithelial sodium transport in tissues such as the distal nephron, through activation of the epithelial sodium channels (ENaC). This study was designed to identify whether these elements were present within the human cornea. In situ hybridisation studies were conducted on paraffin embedded sections from six human eyes, using in-house generated cRNA antisense probes for human SGK1 and ENaC subunits (alpha, beta, gamma), and confirmed expression of SGK1 and all ENaC subunits in the corneal endothelial cytoplasm. Although ENaC subunits were not demonstrated in the corneal epithelium, SGK1 mRNA was identified in the nuclear region of central basal cells of the corneal epithelium, and limbal epithelial cells. Minimal chromagen precipitation was seen in the Bowman's membrane, corneal stroma, or Descemet's membrane. Control experiments consisted of no antisense probe, competition of the labelled antisense cRNA probe by a 60-fold excess unlabelled antisense cRNA, and use of labelled sense cRNA probes, revealing minimal or no hybridisation signal throughout the corneal layers. These data define components of the mineralocorticoid regulatory pathways of sodium transport in human corneal endothelium, and provide evidence for an additional mechanism contributing to corneal transparency and the 'metabolic' sodium pump.

Gene expression in donor corneal endothelium.

OBJECTIVE: To report gene expression profiles of normal human corneal endothelium with microarray analysis and serial analysis of gene expression (SAGE). METHODS: Corneal endothelium was removed from normal human corneas obtained from eye banks. Total RNA was isolated and SAGE analysis was performed. The same RNA source was used to construct a complementary DNA library that was hybridized to microarrays containing 12 558 transcripts. RESULTS: A total of 9530 SAGE tags were sequenced, representing 4724 unique tags. Microarray analysis identified 542 distinct transcripts. A database of human corneal endothelial gene expression was compiled. Of the SAGE tags, 1720 matched known genes, 478 corresponded to expressed sequence tags, and 2526 had no known match to public databases. The 5 most abundantly expressed SAGE tags were cytochrome c oxidase subunit II, adenosine triphosphate synthase F(0) subunit 6, carbonic anhydrase XII, 12S ribosomal RNA, and ferritin, heavy polypeptide 1. Thirty-four percent of the transcripts (n = 1616) were specific to the corneal endothelium, when compared with other publicly available SAGE libraries. The 5 most abundant unique tags were keratin 12, angiopoietinlike factor, annexin A8, and 2 tags with no match to the database. Many endothelial pump function enzymes were confirmed, including several plasma membrane Na( +)/K(+) adenosine triphosphatases and a recently reported bicarbonate transporter. CONCLUSIONS: Corneal endothelial gene expression profiles by the current analysis provide an understanding of endothelial metabolism, structure, and function; enable comparisons to diseased endothelium; and provide baseline data that may lead to the discovery of novel endothelial genes.

Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells.

HCO-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed a single band at approximately 170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during Cl(-) influx in the absence and presence of forskolin (FSK). Apical and basolateral Cl(-) permeability increased 10- and 3-fold, respectively, in the presence of 50 microM FSK. FSK-activated apical chloride permeability was unaffected by H(2)DIDs (250 microM); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 microM) and glibenclamide (100 microM ) inhibited activated Cl(-) fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl(-) permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by H(2)DIDS. HCO permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HCO]. FSK (50 microM) increased apical HCO permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HCO permeability was unaffected by FSK. Genistein (50 microM) significantly increased apical HCO and Cl(minus sign) permeability by 1.8- and 16-fold, respectively. When 50 microM genistein was combined with 50 microM FSK, there was no further increase in Cl(-) permeability; however, HCO permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl(-) and HCO transport. Furthermore, there is a cAMP-activated Cl(-) pathway on the basolateral membrane that is not CFTR.

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells.

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.

Localization of thrombomodulin in the anterior segment of the human eye.

PURPOSE: To localize thrombomodulin (TM) in the anterior segment of the human eye. TM is a vascular endothelial cell surface glycoprotein that acts as a cofactor for the thrombin-catalyzed activation of the anticoagulant protease zymogen, protein C. METHODS: Immunohistochemical methods were used to detect TM expression in corneal epithelial cells, the lens epithelial cells, and other cells in the anterior segment of the eye. The expression of TM was also examined in cultured human corneal epithelial cells. RESULTS: TM was expressed in corneal epithelial cells, corneal endothelial cells, and nonpigmented ciliary epithelial cells, which are in direct contact with the aqueous humor. TM was also expressed in cultured corneal epithelial cells and showed cofactor activity. The amount of the antigen in the cultured corneal cells was approximately one tenth of that in human umbilical vein endothelial cells, but its specific cofactor activity (75%) was comparable to that of TM in human umbilical vein endothelial cells. The trabecular meshwork and endothelial cells lining Schlemm's canal also showed positive staining for TM. CONCLUSIONS: The TM in the cells that are in contact with the aqueous humor appears to be involved in maintaining the fluidity of the aqueous humor. In contrast, TM in cells that are not in contact with the aqueous humor may function in regulating cell proliferation and/or differentiation.

The similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma.

The purpose of the present investigation was to compare protein expression in various ocular cells and tissues including the human trabecular meshwork (TM) and the lamina cribrosa (LC). To conduct the comparisons, we primarily utilized autofluorography of one-dimensional (1D) and high resolution, two-dimensional (2D) polyacrylamide gels of proteins from radiolabelled tissues and cultured cells. Results from the investigations indicated that patterns of protein expression from TM and LC were the most similar among the ocular cells and tissues compared.Specifically, these autofluorographic ' fingerprints' indicated that proteins in TM and LC cultured cells and tissue were exceptionally similar (a) in band position and intensity (1D gels) and (b) in spot congruence (2D gels) as compared to other ocular cells and tissues. We conclude that the TM and the LC, two ocular tissues intimately linked to the pathogenesis of primary open-angle glaucoma, display remarkable similarity in protein expression. This finding may have implications for the molecular etiology of glaucoma.