Ji-Ae Ko

Neuropeptides released from trigeminal neurons promote the stratification of human corneal epithelial cells.

PURPOSE To examine the effects of neural cells on the stratification of and junctional protein expression by corneal epithelial cells with a coculture system. METHODS PC12 cells induced to undergo neuronal differentiation or rat trigeminal nerve cells were cultured together with simian virus 40-transformed human corneal epithelial (HCE) cells on opposite sides of a collagen vitrigel membrane. Stratification of HCE cells was examined by immunofluorescence analysis with antibodies to zonula occludens-1. Expression of junctional proteins in HCE cells was assessed by RT-PCR and immunoblot analyses. RESULTS The presence of neural cells (PC12 cells or trigeminal neurons) markedly promoted the stratification of HCE cells as well as increased the amounts of N-cadherin mRNA and protein in these cells. These effects of the neural cells were mimicked by conditioned medium prepared from differentiating PC12 cells or by the neuropeptides substance P and calcitonin gene-related peptide (CGRP). Furthermore, the stimulatory effects of trigeminal neurons on the stratification of and N-cadherin expression by HCE cells were inhibited by antagonists of substance P or of CGRP. CONCLUSIONS These results suggest that trigeminal neurons play an important role in the differentiation of corneal epithelial cells. Neuropeptides released from these neurons may thus regulate adhesion between corneal epithelial cells and thereby contribute to the establishment and maintenance of corneal structure and function.

Matrix metalloproteinase and cytokine expression in Tenon fibroblasts during scar formation after glaucoma filtration or implant surgery in rats

Failure of surgery for glaucoma is usually due to post‐surgical scarring (fibrosis), a process in which fibroblasts play a prominent role. We investigated the molecular mechanisms of such scarring by examining the expression of matrix metalloproteinases and cytokines in Tenon fibroblasts isolated from rats after glaucoma surgery. Filtration surgery was performed in one eye and implant surgery in the other; and Tenon fibroblasts were isolated from the tissue surrounding the bleb after surgery. The cells were cultured and examined for the expression of matrix metalloproteinases (MMPs) by reverse transcription‐polymerase chain reaction, immunoblot and gelatin zymographic analyses. Culture supernatants were also assayed for cytokines with a multiplex array. The amounts of MMP‐1 and MMP‐3 mRNAs and proteins were greater in cells isolated after implant surgery than in those isolated after filtration surgery, with the progression of scar formation being more complete after the former surgery. The secretion of interleukin‐6 (IL‐6) by cells isolated after filtration surgery was greater than that for cells isolated after implant surgery. Depletion of IL‐6 by RNA interference in cells isolated after filtration surgery increased the expression of MMP‐1 and MMP‐3 in these cells. These results thus suggest that the expression of MMP‐1 and MMP‐3 in Tenon fibroblasts is regulated by IL‐6 during, and may play an important role in, scar formation after glaucoma surgery. Copyright

Up-regulation of matrix metalloproteinase-1 and interleukin-6 expression in cocultures of corneal fibroblasts and neural cells

The cornea is the most sensitive tissue in the human body, with the dense nerve endings of the cornea being derived from the first division of the ophthalmic nerve. The existence of such organized nerve fibers reflects the role of neural regulation in corneal homeostasis, with the proper distribution and function of these nerve fibers thus being required for maintenance of a healthy cornea. We recently established an in vitro model, based on the coculture of human corneal epithelial cells and fibroblasts on opposite sides of a collagen vitrigel membrane. We have now examined the role of neural cells in corneal homeostasis with the use of a similar coculture system. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that the presence of neural cells (differentiated PC12 cells) increased the expression of matrix metalloproteinase-1 (MMP-1) in human corneal fibroblasts at both the mRNA and protein levels. The expression of MMP-2 and MMP-9 in corneal fibroblasts was not affected by PC12 cells. Furthermore, a multiplex assay showed that, among various cytokines assayed, only the release of interleukin-6 in cocultures of the two cell types was markedly greater than that in cultures of corneal fibroblasts alone. These results thus suggest that factors released from neural cells may play an important role in regulation of the function of corneal fibroblasts and thereby contribute to the maintenance of corneal structure and function.

Up-regulation of semaphorin 4A expression in human retinal pigment epithelial cells by PACAP released from cocultured neural cells.

Development and homeostasis of multicellular organisms require interactions between neighbouring cells. We recently established an in vitro model of cell–cell interaction based on a collagen vitrigel membrane. We have now examined the role of neural cells in retinal homeostasis by coculture of human retinal pigment epithelial (RPE) cells and neural cells on opposite sides of such a membrane. The neural cells (differentiated PC12 cells) induced up‐regulation of semaphorin 4A (Sema4A), a member of the semaphorin family of neural guidance proteins, in RPE (ARPE19) cells. This effect of the neural cells was mimicked by the neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) and was abolished by the PACAP antagonist PACAP(6–38). Coculture with neural cells or stimulation with PACAP also induced the phosphorylation of extracellular‐signal‐regulated kinase in ARPE19 cells, and this effect of the neural cells was inhibited by PACAP(6–38). Finally, among various cytokines examined, only the amount of interleukin‐6 released by cocultures of ARPE19 and neural cells differed from that released by ARPE19 cells cultured alone. Interleukin‐6 was not detected in culture supernatants of neural cells, and the reduction in the amount of interleukin‐6 released by the cocultures compared with that released by ARPE19 cells alone was prevented by PACAP(6–38). Our findings suggest that PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function.

Differential expression of semaphorin 3A and its receptors during mouse retinal development

Semaphorins not only function in axon guidance during development but also contribute to various other biological processes. We have now examined the expression of semaphorin 3A (Sema3A) and its receptor components neuropilin 1 (Npn1) and plexin A (PlxA) during development of the mouse retina. Immunohistofluorescence analysis revealed that the expression patterns of Sema3A and Npn1 were similar during embryonic and postnatal development. The expression pattern of PlxA was also similar to those of Sema3A and Npn1 during embryonic and early postnatal (before eye opening) developments. However, the pattern of PlxA expression changed markedly after eye opening, with the expression disappearing from the optic nerve and increasing in intensity in the retinal pigment epithelium. Immunoprecipitation analysis showed that Sema3A interacted with PlxA in the retinal pigment epithelial cell line ARPE19 but not in the retinal ganglion cell line RGC5, whereas the opposite pattern of association was apparent for Sema3A and Npn1. Given that atmospheric oxygen is thought to play a role in the differentiation and maintenance of various ocular cell types, our results suggest that Sema3A–PlxA signalling activated by an effect of ambient oxygen on PlxA expression may contribute to differentiation of the retinal pigment epithelium. Copyright

Role of macrophage migration inhibitory factor (MIF) in the effects of oxidative stress on human retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial‐mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H2O2 induced the contraction of RPE cells in a three‐dimensional collagen gel. Analysis of a cytokine array revealed that H2O2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription–polymerase chain reaction and immunoblot analyses showed that H2O2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H2O2 upregulated the expression of α–SMA and vimentin and downregulated that of ZO‐1 and N‐cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H2O2. The effects of oxidative stress on EMT‐related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen‐activated protein kinase signalling revealed that H2O2 specifically induced the phosphorylation of p38 mitogen‐activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line ARPE19. Our results thus implicate MIF in induction of the EMT and related processes by oxidative stress in RPE cells and the regulated expression of EMT markers. They further suggest that MIF may play an important role in the pathogenesis of PVR.

Down-regulation of semaphorin 3F in rat retinal ganglion cells in response to optic nerve crush.

Glaucoma is characterized by degeneration of optic nerve axons and death of retinal ganglion cells (RGCs). Nerve crush and axotomy of the optic nerve are studied as models of RGC death in glaucoma and of axon regeneration. The mechanisms underlying the response of RGCs to axonal injury remain unclear, however. We have now examined the effects of optic nerve crush on the expression of members of the semaphorin family of neuronal guidance proteins in the rat retina. The expression of semaphorin 3F (Sema3F) in the retina was down‐regulated at both the mRNA and protein levels at 7 days after optic nerve injury, whereas that of Sema3A, Sema3B or Sema3C remained unaffected. Immunohistofluorescence analysis and laser capture microdissection followed by reverse transcription–polymerase chain reaction analysis revealed that this loss of Sema3F expression occurred in the RGC layer of the retina. Furthermore, antibody‐mediated neutralization of secreted Sema3F in retinal organ culture resulted in down‐regulation of neuron‐specific βIII‐tubulin (Tuj‐1 antigen), a marker of RGCs. Our results suggest that Sema3F may contribute to the regulation of RGC function or survival and therefore warrants further investigation as a potential mediator of neuroprotection. Copyright

Oxidative stress regulates expression of claudin-1 in human RPE cells

Age-related macular degeneration (AMD) is a neurodegenerative disease associated with irreversible loss of central vision in the elderly. Disruption of the homeostatic function of the retinal pigment epithelium (RPE) is thought to be fundamental to AMD pathogenesis, and oxidative stress is implicated in the associated RPE damage. We examined the effects of oxidative stress on the expression of junctional proteins in cultured human retinal pigment epithelial (ARPE-19) cells. Reverse transcription-PCR and immunoblot analyses revealed that expression of the tight-junction protein claudin-1 was increased at both the mRNA and protein levels 8 to 12 h after exposure of ARPE-19 cells to H2O2, whereas that of the tight-junction protein ZO-1 or the adherens-junction protein N-cadherin was unaffected. Expression of both claudin-1 and N-cadherin was down-regulated by exposure of the cells to H2O2 for longer periods (24 to 48 h). Oxidative stress also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) with a time course similar to that apparent for the up-regulation of claudin-1 expression. Furthermore, the increase in the abundance of claudin-1 induced by H2O2 was blocked by the p38 inhibitor SB203580. Phosphorylation of the MAPKs ERK and JNK was not affected by H2O2. Our results suggest that modulation of claudin-1 expression in the RPE by oxidative stress may contribute to the pathogenesis of AMD.