Human Non-pigmented Ciliary Epithelial Cells Research Articles

TRPP2 is located in the primary cilia of human non-pigmented ciliary epithelial cells

PurposeMechanosensitive channels (MSCs) and primary cilium possess a possible relevance for the sensation of intraocular pressure (IOP). However, there is only limited data on their expression and localization in the ciliary body epithelium (CBE). The purpose of this study was to characterize the expression and localization of TRPP2 in a human non-pigmented ciliary epithelial cell (HNPCE) line.MethodsThe expression of the TRPP2 was studied by quantitative (q)RT-PCR and in situ hybridization in rat and human tissue. Protein expression and distribution were studied by western blot analysis, immunohistochemistry, and immunoelectron microscopy. Cellular location of TRPP2 was determined in rat and human CBE by immunofluorescence and immunoblot analysis. Electron microscopy studies were conducted to evaluate where and with substructure TRPP2 is localized in the HNPCE cell line.ResultsThe expression of TRPP2 in rat and human non-pigmented ciliary epithelium was detected. TRPP2 was mainly located in nuclei, but also showed a punctate distribution pattern in the cytoplasm of HNPCE of the tissue and the cell line. In HNPCE cell culture, primary cilia did exhibit different length following serum starvation and hydrostatic pressure. TRPP2 was found to be colocalized with these cilia in HNPCE cells.ConclusionThe expression of TRPP2 and the primary cilium in the CB may indicate a possible role, such as the sensing of hydrostatic pressure, for the regulation of IOP. Functional studies via patch clamp or pharmacological intervention have yet to clarify the relevance for the physiological situation or aqueous humor regulation.

Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules In Vitro.

The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm2 significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.

A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells.

c-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein β (C/EBPβ) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPβ in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPβ as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPβ augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPβ was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPβ and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPβ appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.

A Rho-Associated Kinase Inhibitor Protects Permeability in a Cell Culture Model of Ocular Disease, and Reduces Aqueous Flare in Anterior Uveitis.

Recent clinical and experimental studies have reported favorable results when using Rho-associated kinase (ROCK) inhibitors for ocular disease, and in cell culture. Disruption of the human, nonpigmented ciliary epithelial cells (HNPCECs) that comprise the blood-aqueous barrier (BAB) induces anterior uveitis; these cells therefore provide a useful cell model of ocular disease. In this study, we examined the effects of ROCK inhibitors in anterior uveitis and in HNPCECs. Aqueous flare values and intraocular pressures (IOPs) were determined in patients with anterior uveitis, 2 weeks after administration of ripasudil hydrochloride hydrate, a commercial ROCK inhibitor used to treat glaucoma or ocular hypertension. We also investigated the effects of Y-27632, a second ROCK inhibitor, in HNPCECs following exposure to matrix metalloproteinases (MMPs) and human tumor necrosis factor-alpha (TNF-α). Patients with anterior uveitis, glaucoma, or ocular hypertension, referred to the Aichi Medical University from February to July 2015, were enrolled. Thirty eyes from 25 outpatients were studied. Aqueous flare values and IOPs were significantly decreased 2 weeks after ripasudil hydrochloride hydrate treatment, with no adverse events. In a cultured HNPCEC monolayer, permeability was markedly increased following exposure to MMPs-1, 3, 9, and TNF-α, with these effects attenuated by exposure to Y-27632. In cultured HNPCECs, Y-27632 provoked a marked alteration in cytoskeletal morphology without a significant change in expression levels of claudin-1 and occludin. ROCK inhibitors may confer favorable effects in anterior uveitis, possibly due to a reorganized BAB, although the relevant mechanisms remain unclear.

The Effect of Ultraviolet B on Fibrillin-1 and Fibrillin-2 in Human Non-pigmented Ciliary Epithelial Cells <i>In Vitro</i>

The ciliary zonules link the lens to the ciliary body in the eye, controlling the thickness of the lens for focusing through their characteristic elasticity. The ciliary zonules are composed of oxytalan fibers. Physiological or pathological damage to the ciliary zonules, including exposure to ultraviolet (UV)-A and UV-B components, can lead to lens dislocation. However, no studies have shown whether UV affects the ciliary zonule. Here, we assessed the effects of UV light on human nonpigmented ciliary epithelial cells (HNPCECs). HNPCECs were cultured for 4 weeks, and expression of fibrillin-1 and fibrillin-2 was confirmed. In control cultures (0 mJ/cm2), some fibrillin-1-positive fibers were merged with fibrillin-2. After UV-A irradiation, the appearance of both fibrillin-1- and fibrillin-2-positive fibers was unchanged. However, after UV-B irradiation, fibrillin-1-positive fibers became thin at an irradiation level of 100 mJ/cm2, and the fiber structure became amorphous at 150 mJ/cm2. Fibrillin-2-positive fibers lost their continuity and disappeared after being exposed to 150 mJ/cm2 UV-B. UV-B irradiation did not affect cell viability, possibly because of the sensitivity of fibrillin-1 and fibrillin-2 to UV-B. Thus, dislocation of the lens with age may be attributable to cumulative exposure to UV-B.

Bimatoprost, latanoprost, and tafluprost induce differential expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases.

BackgroundDifferences in the increase in matrix metalloproteinase (MMP) and decrease in tissue inhibitor of metalloproteinase (TIMP) activity may contribute to the different characteristics observed clinically on decreased intraocular pressure in patients with glaucoma or ocular hypertension. The purpose of this study was to investigate differences in the expression profiles of MMPs and TIMPs induced by the prostaglandin analogs bimatoprost, latanoprost, and tafluprost in human non-pigmented ciliary epithelial cells (HNPCECs).MethodsHNPCECs were cultured for 24 h with 0, 10, 100, or 1000 μM of the free acid forms of bimatoprost, latanoprost, and tafluprost. We measured the expression levels of MMPs and TIMPs using real-time polymerase chain reaction, and compared the results. Enzyme activities of MMP-2 and −9 in conditioned media were measured by gelatin zymography.ResultsAll prostaglandin analogs we examined dose-dependently increased expression levels of MMP-1, −2, −3, −9, and −17, whereas expression levels of TIMP-1 and −2 decreased with increasing concentrations of each analog. Each prostaglandin analog induced different levels of increases in MMPs and decreases in TIMPs.ConclusionsUnique expression profiles of MMPs and TIMPs induced by bimatoprost, latanoprost, and tafluprost, as shown in HNPCECs, may contribute to clinically different effects on intraocular pressure decreases in patients with glaucoma or ocular hypertension.

TRPV4 activation triggers the release of melatonin from human non-pigmented ciliary epithelial cells

Melatonin is a neurohormone mainly produced in the pineal gland; nevertheless, various ocular structures such as the ciliary body, lens and the retina produce it. One of the roles of melatonin in the eye is the modulation of intraocular pressure, although little is known about the mechanisms that causes its presence in the aqueous humour. TRPV4 is a membrane channel which is activated by both physical and chemical stimuli. Therefore, this channel is sensitive to osmotic and hydrostatic pressure. As a consequence, TRPV4 results as an interesting candidate to study the relation between the activation of the TRPV4 channel and the production of melatonin. In this sense we have studied the role of the TRPV4 agonist GSK1016790A to modulate the production of melatonin in a cell line derived from human non-pigmented ciliary epithelial cells. The stimulation of the TRPV4 produced an increase in the extracellular melatonin levels changing from 8.5 ± 0.6 nM/well/30 min (control) to 23.3 ± 2.1 nM/well/30 min after 10 nM GSK1016790A application, this action being blocked by the selective antagonist RN 1734. The activation of the TRPV4 by GSK1016790A permitted to observe a melatonin increase which was concentration-dependent, and provided a pD2 value of −8.5 ± 0.1 (EC50 of 3.0 nM). In conclusion, the activation of the TRPV4 present in human non-pigmented ciliary epithelial cells can modulate the presence of extracellular melatonin, this being of relevance since this substance controls the dynamics of the aqueous humour.

Microfibril-associated glycoprotein-1 controls human ciliary zonule development in vitro.

The ciliary zonule in the eye, also known as Zinn’s zonule, is composed of oxytalan fibers, which are bundles of microfibrils consisting mainly of fibrillin-1. However, it is still unclear which of the microfibril-associated molecules present in the ciliary zonule controls oxytalan fibers. Microfibril-associated glycoprotein-1 (MAGP-1) is the only microfibril-associated molecule identified in the human ciliary zonule. In the present study, we used siRNA against MAGP-1 in cultures of human non-pigmented ciliary epithelial cells to examine the extracellular deposition and appearance of fibrillin-1 employing Western blotting and immunofluorescence. MAGP-1 suppression led to a reduction of fibrillin-1 deposition. Immunofluorescence also confirmed that RNAi-mediated down-regulation of MAGP-1 led to suppression of fiber development. These results suggest that MAGP-1 plays a crucial role in the extracellular deposition of fibrillin-1 during formation of the human ciliary zonule.

Involvement of AP-1 and C/EBPβ in Upregulation of Endothelin B (ETB) Receptor Expression in a Rodent Model of Glaucoma

Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The −300 to −1 bp and −1258 to −600 bp upstream promoter regions of the ETB receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at −615 to −624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPβ enhanced the ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ETB receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPβ in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ETA and ETB receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ETB receptor in a rodent model of ocular hypertension.

Piceatannol suppresses endotoxin-induced ocular inflammation in rats.

Anti-inflammatory effect of piceatannol, a naturally occurring polyphenol and a potent free radical scavenger, on ocular inflammation is not known. We examined the anti-inflammatory role of piceatannol in ocular inflammatory response due to endotoxin-induced uveitis (EIU) in rats. EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS; 150 ug/rat). Piceatannol (30mg/kg body wt, i.p) was injected either 2h prior to or 1h post LPS induction. A significant increase in the number of infiltrating cells, total protein, and various cytokines and chemokines in AqH were observed in the EIU rat eyes as compared to control groups. However, pre- or post-treatment of piceatannol significantly blocked the LPS-induced changes. Further, piceatannol also suppressed the expression of cyclooxygenase-2 (Cox-2), inducible nitric oxide synthase (iNOS) and activation of NF-κB in the ciliary bodies as well as retina. Further, piceatannol also inhibited the expression of Cox-2, iNOS, and phosphorylation of NF-κB in primary human non-pigmented ciliary epithelial cells (HNPECs) treated with LPS. Similarly, piceatannol also diminished LPS-induced level of NO and prostaglandin E2 in HNPECs. Thus our results demonstrate an anti-inflammatory role of piceatannol in suppressing ocular inflammation induced by endotoxin in rats.

Infliximab counteracts tumor necrosis factor-α-enhanced induction of matrix metalloproteinases that degrade claudin and occludin in non-pigmented ciliary epithelium

Infliximab, a monoclonal antibody directed against human tumor necrosis factor-alpha (TNF-α), effectively treats anterior uveitis, which can accompany Behçet's disease. Here, we investigated the underlying mechanism of this action. We examined human, non-pigmented ciliary epithelial cells (HNPCECs), which make up the blood–aqueous barrier (BAB) in the uvea. We measured the expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the presence or absence of TNF-α using quantitative, real-time polymerase chain reaction and enzyme-linked immunosorbent assays. The expression of MMP-1, MMP-3, and MMP-9 increased in the presence of TNF-α, and the addition of infliximab reversed the increase. The TNF-α effects were more attenuated when infliximab was added before than when it was added after TNF-α exposure. Gelatin zymography demonstrated that the protease activity of these MMPs was also increased in the presence of TNF-α and attenuated with infliximab. Immunostaining showed that MMP-1, MMP-3, and MMP-9 degraded claudin-1 and occludin in HNPCECs and in non-pigmented ciliary epithelial cells of the swine ciliary body. In a monolayer of HNPCECs, we found that permeability was significantly increased with MMP treatment. Thus, TNF-α increased levels of MMPs in cells that form the BAB, and MMPs degraded components of the tight junctions in the BAB, which increased permeability through the cellular barrier. Furthermore, infliximab effectively attenuated the TNF-α-induced increases in MMP expression in cells that make up the BAB. These findings might suggest a basis for the clinical prevention of anterior uveitis.

Matrix Metalloproteinase-2 Degrades Fibrillin-1 and Fibrillin-2 of Oxytalan Fibers in the Human Eye and Periodontal Ligaments <i>In Vitro</i>

Oxytalan fibers are distributed in the eye and periodontal ligaments (PDL). The ciliary zonule, known as Zinn’s zonule, in the eye is composed of oxytalan fibers, which are bundles of microfibrils consisting mainly of fibrillin-1 and fibrillin-2. As turnover of oxytalan fibers is slow during life, their degradation mechanism remains unclarified. This study was performed to examine degradation pattern of fibrillin-1 and fibrillin-2 by experimental MMP activation. We cultured human non-pigmented ciliary epithelial cells (HNPCEC) and PDL fibroblasts for 7 days, then treated them with concanavalin A to activate matrix metalloproteinase (MMP)-2, and examined the degradation of fibrillin-1 and fibrillin-2 for 72 hr using immunofluorescence. At 7 days of HNPCEC culture, fibrillin-1-positive fibers were observed, some of which merged with fibrillin-2. After MMP-2 activation, fibrillin-1-positive fibers became thin and disappeared by 72 hr, while fibrillin-2-positive fibers disappeared almost completely within 24 hr. At 7 days of PDL fibroblast culture, fibrillin-1-positive fibers were mostly merged with fibrillin-2. After MMP-2 activation, fibrillin-1-positive fibers became thin by 24 hr and had almost disappeared by 48 hr, while fibrillin-2-positive fibers decreased constantly after 24 hr. A MMP-2 inhibitor completely suppressed these degradations. These results suggest that the patterns of fibrillin-1 and fibrillin-2 degradation differ between the eye and the PDL, possibly reflecting the sensitivity of fibrillin-1 and fibrillin-2 of each type of oxytalan fiber against MMP-2.

Melatonin receptor agonist-induced reduction of SNP-released nitric oxide and cGMP production in isolated human non-pigmented ciliary epithelial cells

The present study was designed to determine the effects of melatonin and its receptor agonists on SNP-released nitric oxide (NO) and cGMP production in aqueous humor producing cells of the ciliary body because these effects may play a role in melatonin receptor-mediated regulation of intraocular pressure (IOP). NO release protocols were carried out using human non-pigmented ciliary epithelial (hNPCE) cells treated in dye free DMEM containing l-arginine (10−3 M). The cGMP experimental protocols were performed using dye free DMEM containing 3-isobutyl-1-methylxanthine (IBMX, 10−4 M). The effects of varying concentrations (10−13, 10−11, 10−9, 10−7, and 10−5 M) of melatonin, 5-MCA-NAT (putative MT3 agonist), N-butanoyl-2-(2-methoxy-6H-isoindolo[2, 1-a]indol-11-yl)ethanamine (IIK7; selective MT2 agonist) or S-27633-1 (selective MT1 agonist) on sodium nitroprusside (SNP)-released NO or cGMP production were determined in separate experiments. NO and cGMP levels were measured using a colorimetric assay or enzyme immunoassay (EIA), respectively. Melatonin receptor selectivity was evaluated using luzindole (LUZ; nonselective MT1/MT2 antagonist) or 4-phenyl-2-propionamidotetralin (4P-PDOT; selective MT2 antagonist). Melatonin, 5-MCA-NAT, and IIK7 all caused concentration-dependent reduction of SNP-released NO and cGMP production. The inhibitory actions of melatonin, 5-MCA-NAT and IIK7 were either completely blocked at 10−13, 10−11, and 10−9 M concentrations of the agonists or partially at 10−7 and 10−5 M in the presence of luzindole or 4P-PDOT. Results from this study suggest that melatonin and its analogs, 5-MCA-NAT and IIK7 inhibit SNP-released NO and cGMP production via activation of MT2 receptors in human NPCE cells. These actions may play a role in melatonin agonist-induced regulation of aqueous humor secretion and IOP.

Antidiabetic Drug Metformin Suppresses Endotoxin-Induced Uveitis in Rats

To investigate the therapeutic effects of metformin, a commonly used antidiabetic drug, in preventing endotoxin-induced uveitis (EIU) in rats. EIU in Lewis rats was developed by subcutaneous injection of lipopolysaccharide (LPS; 150 μg). Metformin (300 mg/kg body weight, intraperitoneally) or its carrier was injected either 12 hours before or 2 hours after LPS induction. Three and 24 hours after EIU, eyes were enucleated and aqueous humor (AqH) was collected. The MILLIPLEX-MAG Rat cytokine-chemokine magnetic bead array was used to determine inflammatory cytokines. The expression of Cox-2, phosphorylation of AMPK, and NF-κB (p65) were determined immunohistochemically. Primary human nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of metformin. Compared with controls, the EIU rat AqH had significantly increased number of infiltrating cells and increased levels of various cytokines and chemokines (TNF-α, MCP-1, IL-1β, MIP-1α, IL-6, Leptin, and IL-18) and metformin significantly prevented the increase. Metformin also prevented the expression of Cox-2 and phosphorylation of p65, and increased the activation of AMPK in the ciliary bodies and retinal tissues. Moreover, metformin prevented the expression of Cox-2, iNOS, and activation of NF-kB in the HNPECs and decreased the levels of NO and PGE2 in cell culture media. Our results for the first time demonstrate a novel role of the antidiabetic drug, metformin, in suppressing uveitis in rats and suggest that this drug could be developed to prevent uveitis complications.

Fibrillin-1 and Fibrillin-2 Are Essential for Formation of Thick Oxytalan Fibers in Human Nonpigmented Ciliary Epithelial Cells In Vitro

The ciliary zonule, also known as Zinn’s zonule, is composed of oxytalan fibers. However, the mechanism by which epithelial cells in the ciliary body form these fibers in not fully understood. We examined human nonpigmented ciliary epithelial cells to determine the appearance and amount of oxytalan fibers in terms of positivity for their major components, fibrillin-1 and fibrillin-2. Examination of fibrillin-1 and fibrillin-2 expression by immunofluorescence revealed that thin fibers positive for fibrillin-1 on Day 2 changed to thick fibers by Day 8. The fibers positive for fibrillin-2 appeared on the thick fibrillin-1-positive fibers after Day 4. Northern blot analysis revealed that the level of fibrillin-1 did not change markedly, while induction of fibrillin-2 gene was evident on Day 5. Western blot analysis showed that fibrillin-1 deposition increased gradually, while that of fibrillin-2 increased markedly from Day 5 to Day 8. Fibrillin-1 suppression did not lead to the formation of fibrillin-2-positive thick fibers, whereas fibrillin-2 suppression led to the formation of fibrillin-1-positive thin fibers, but not thick fibers. These results suggest that both fibrillin-1 and fibrillin-2 are essential for the formation of thick oxytalan fibers in the ciliary zonule and are informative for clarifying the mechanism of homeostasis of the ocular matrix.

TGF-β signaling is required for maintenance of retinal ganglion cell differentiation and survival

To determine the role of TGF-β1 in the maintenance of retinal ganglion cell line (RGC-5) differentiation and integrity. RGC-5 cells were differentiated in media conditioned by human non-pigmented ciliary epithelial cells (HNPE) for 4 days before treatment with TGF-β1 for 24 h. Cells were examined for morphological changes and harvested for western blot and real-time PCR analysis. For study of apoptosis, differentiated RGC-5 cells were grown in serum-free medium for 24 h in the presence or absence of TGF-β1 and collected for Annexin V/Propidium iodide FACs analysis. The role of MAPK pathways in TGF-β1-dependent signaling was determined by treatment with specific inhibitors of ERK, JNK and p38. Differentiation of RGC-5 cells in HNPE-conditioned media (CM) increased the neural cell markers, Brn-3c, NF-160, Thy1.2, Tau and PGP9.5. Treatment with TGF-β1 significantly increased the length of neurites extended by differentiated RGC-5s, concomitant with increased expression of NF-160 and PGP9.5, but not Brn-3c, Thy1.2 or Tau. TGF-β1 also decreased RGC-5 cell apoptosis in serum-free medium. p38 phosphorylation, but not smad2/3, JNK or ERK phosphorylation, was increased in TGF-β1 treated cells. Specific inhibition of p38 signaling reversed TGF-β1 induced neurite growth. These findings demonstrate the induction of RGC-5 cell differentiation by HNPE-derived CM and illustrate a role for TGF-β1 in maintaining RGC-5 cell survival and promoting neurite outgrowth through p38 MAPK.

Protein Profiling of Human Nonpigmented Ciliary Epithelium Cell Secretome: The Differentiation Factors Characterization for Retinal Ganglion Cell line

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

Constitutive secretion of chemokines by cultured human trabecular meshwork cells

Trabecular meshwork endothelial (TME) cells secrete a number of factors, such as enzymes and cytokines, which modulate the functions of the cells and the extracellular matrix of the conventional aqueous outflow pathway. TME cells usually secrete these factors in response to stimuli such as mechanical stretching, laser irradiation and pro-inflammatory cytokines. Here, we report that cultured human TME cells isolated from two non-glaucomatous individuals secrete significant quantities of the chemotactic cytokines IL8, CXCL6 and MCP1 in the absence of any stimulation. The secretion of these chemokines was augmented by treatment with the pro-inflammatory cytokines TNFα and IL1β. By way of comparison, there was little or very low production of the three chemokines by human non-pigmented ciliary epithelial cells in the absence of stimulation. Our findings provide support to our recent observations that monocytes, presumably under the influence of chemotactic signals, circulate through the trabecular meshwork in the normal state and also that cytokines regulate the permeability of Schlemm’s canal endothelial cells. In addition, the fact that normal TME cells constitutively secrete chemotactic cytokines strengthens the notion that cytokines play a key role in the homeostasis of the outflow of the aqueous humor and, possibly, in the pathogenesis of glaucoma.

Inhibition of aldose reductase attenuates endotoxin signals in human non-pigmented ciliary epithelial cells

Chronic inflammatory diseases such as autoimmune and bacterial infections are associated with an elevated risk of ocular inflammation. Ciliary epithelial cells that play an important role in maintaining aqueous humor dynamics and homeostasis of anterior segment of eye are continuously exposed to inflammatory markers during infections and injury. Lipopolysachharide (LPS), a Gram-negative bacterial endotoxin, dysregulates aqueous humor (AqH) homeostasis by inducing inflammatory changes. We have investigated how inhibition of a polyol pathway enzyme, aldose reductase (AR), alters LPS-induced inflammatory changes in human non-pigmented ciliary epithelial cells (hNPECs). The stimulation of hNPECs with LPS (1 μg/ml) caused increased secretion of inflammatory markers such as PGE 2 and NO in the culture medium as well as increased expression of COX-2 and iNOS proteins in cell extracts. LPS also increased phosphorylation of MAPKs (ERK1/2) and SAPK/JNK and activation of redox-sensitive transcription factors NF-κB and AP-1 in hNPECs and inhibition of AR by zopolrestat and sorbinil ameliorated these changes. Further, LPS-induced decrease in the expression of Na/K-ATPase in hNPECs was restored by AR inhibitors. Similar results were observed in ciliary bodies of LPS-injected rats. Taken together, our results suggest that AR plays an important role in the LPS-induced inflammatory changes in hNPECs and that inhibition of AR could be a novel therapeutic approach for ocular inflammation.

Expression of Cytochrome P450 (CYP) Enzymes in Human Nonpigmented Ciliary Epithelial Cells: Induction of CYP1B1 Expression by TCDD

Cytochrome P450 (CYP) enzymes metabolize endogenous compounds such as steroid hormones, fatty acids, and xenobiotics, including drugs and carcinogens. Expression of CYP enzymes in ocular tissues is poorly known. However, mutations in the CYP1B1 gene have been linked to congenital glaucoma. The aim of the present study was to investigate the expression and regulation of cytochrome P450 enzymes in a human nonpigmented ciliary epithelial cell line. Expression of mRNAs for major xenobiotic metabolizing CYPs in families 1-3 and regulatory factors involved in the induction of CYPs was studied using reverse transcriptase-polymerase chain reaction. For induction studies, the cells were treated with dexamethasone or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 hours. RNA and immunoblotting analysis were used to study CYP induction. Transcriptional regulation of CYP1B1 gene was studied by transient transfection of reporter gene constructs. mRNAs of CYP1A1, CYP1B1, and CYP2D6 and of the regulatory factors aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator, and glucocorticoid receptor were expressed in the human nonpigmented ciliary epithelial cell line. CYP1B1 mRNA was strongly and dose dependently induced by TCDD. CYP1B1 protein was detected only after TCDD treatment of the human nonpigmented ciliary epithelial cells. CYP1B1 promoter was activated by TCDD. The major drug-metabolizing enzymes CYP1A2, CYP2Cs, and CYP3As were not detected in these cells, and dexamethasone treatment had no effect on CYP expression. TCDD potently induces CYP1B1 mRNA in human nonpigmented ciliary epithelial cells, suggesting the involvement of an AHR-mediated pathway in the regulation of ciliary CYP1B1 expression.