HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Effects of Ripasudil, a Rho-Kinase Inhibitor, on Scar Formation in a Mouse Model of Filtration Surgery.

PURPOSE: Glaucoma is a leading cause of blindness worldwide. Characteristic changes occur in the optic nerve and visual field of patients with glaucoma; optic nerve damage can be mitigated by lowering intraocular pressure. Treatment modalities include drugs and lasers; filtration surgery is necessary for patients with insufficient intraocular pressure reduction. Scar formation often contributes to glaucoma filtration surgery failure by increasing fibroblast proliferation and activation. Here, we examined the effects of ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, on postoperative scar formation in human Tenon's fibroblasts. METHODS: Collagen gel contraction assays were used to compare contractility activity among ripasudil and other anti-glaucoma drugs. The effect of Ripasudil in combination with other anti-glaucoma drugs and transforming growth factor-ß (TGF-ß), latanoprost and timolol-induce contractions were also tested in this study. Immunofluorescence and Western blotting were used to study the expression of factors relating scarring formation. RESULTS: Ripasudil inhibited contraction in collagen gel assay and reduced α-smooth muscle actin (SMA) and vimentin (scar formation-related factors) expression, which was inversely promoted by latanoprost, timolol or TGF-ß. Ripasudil also inhibited contraction on TGF-ß, latanoprost and timolol-induced contraction. Furthermore, we investigated the effects of ripasudil on postoperative scarring in a mouse model; ripasudil suppressed postoperative scar formation by altering the expression of α-SMA and vimentin. CONCLUSIONS: These results suggest that ripasudil, ROCK inhibitor may inhibit excessive fibrosis after glaucoma filtering surgery vis inhibition the transdifferentiation of tenon fibroblast into myofibroblast and may have a potential effect as anti-scarring for glaucoma filtration surgery.

Synergistic effect of TONS504-mediated photodynamic antimicrobial chemotherapy and additives widely contained in ophthalmic solutions: benzalkonium chloride and ethylenediaminetetraacetic acid.

TONS504 (C51H58N8O5I2), a chlorine derivative, effectively generates singlet oxygen by light activation and exhibits photodynamic antimicrobial effects (PAEs) on various pathogens. However, this photosensitizer has some limitations: a high tendency to self-aggregate and a relatively weak PAE for Gram-negative bacteria compared with Gram-positive bacteria. To overcome these limitations, the present study investigated the synergistic effects of the PAE of TONS504 and two additives commonly contained in ophthalmic solutions: benzalkonium chloride (BAC) or ethylenediaminetetraacetic acid (EDTA). Staphylococcus aureus and Pseudomonas aeruginosa were exposed to TONS504 and/or each additive. Photodynamic antimicrobial chemotherapy was performed with light irradiation centered at a wavelength of 665 nm with a total light energy of 30 J/cm2. Following incubation, the number of colonies formed was counted. Additionally, we examined the inhibitory effects of the additives on TONS504 self-aggregation by observing its absorption spectrum. Consequently, the PAEs of TONS504 on S. aureus were enhanced by both additives, and BAC displayed stronger synergistic effects on the bacteria than EDTA. By contrast, only EDTA increased the PAE on P. aeruginosa. The peak of the TONS504 absorption spectrum shifted to a longer wave length and the absorbance increased in the presence of BAC, suggesting that BAC inhibited the self-aggregation of the photosensitizer. In conclusion, the combination of BAC or EDTA and TONS504-mediated photodynamic antimicrobial chemotherapy exhibits a synergistic antimicrobial effect on S. aureus and P. aeruginosa. The optimal additive to enhance the PAE may differ between bacterial strains.

Molecular characteristics of the photosensitizer TONS504: Comparison of its singlet oxygen quantum yields and photodynamic antimicrobial effect with those of methylene blue.

TONS504 (C51H58O5I2) is a chlorin derivative that exhibits a photodynamic antimicrobial effect (PAE) on various infectious keratitis pathogens. However, the molecular characteristics of TONS504 are not well understood. This study aimed to investigate the molecular characteristics of TONS504 by comparing its singlet oxygen (1O2) quantum yields and PAE with those of methylene blue (MB). To measure the 1O2 quantum yields, TONS504 and MB were dissolved in phosphate-buffered saline and phosphate-buffered saline containing 1% Triton X-100. The solutions were then activated by a Nd:YAG laser with an average output power of 8 mW. Near-infrared 1O2 luminescence was detected as an indicator of the 1O2 quantum yields. To evaluate the PAE, TONS504 and MB were activated by a light-emitting diode with a total light energy of 30 J/cm2. We compared the minimum molar concentration of each photosensitizer to show apparent PAEs on Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. TONS504 exhibited higher 1O2 quantum yields than MB in PBS/Triton X-100 but not in PBS. S. aureus and C. albicans were reduced by TONS504 at lower concentrations than by MB, but this was not the case for P. aeruginosa. Our results provide insight on the molecular characteristics of TONS504 and suggest that TONS504 has excellent 1O2 quantum yields and PAE. Compared with MB, TONS504 in PBS has stronger efficacy toward some infectious keratitis pathogens but not others.

Efficacy of Photodynamic Anti-Microbial Chemotherapy for Acanthamoeba Keratitis In Vivo.

BACKGROUND AND OBJECTIVES: Acanthamoeba keratitis is a sight-threatening infectious disease that is difficult to treat. The aim of this study was to evaluate TONS504 (cationic chlorin derivative photosensitizer)-mediated photodynamic antimicrobial chemotherapy (PACT) in vivo as a potential treatment for Acanthamoeba keratitis. STUDY DESIGN/MATERIALS AND METHODS: Acanthamoeba keratitis was induced by soft contact lenses incubated with 1 × 105 /ml Acanthamoeba castellanii, which were placed over debrided corneas with temporary tarsorrhaphy. Thirty-eight male Japanese white rabbits were randomly divided into three groups (normal eye, no treatment, and treatment groups). TONS504 was administered as eye drops at 1 mg/ml, followed by light-emitting diode irradiation after the establishment of keratitis at 7 days after infectious contact lens exposure. All animals were evaluated under a slit-lamp microscope every 3 days for 6 days after the treatment. Clinical scores based on corneal epithelial defects detected by fluorescein staining, stromal opacity edema, and vascular infiltration into the cornea were determined. After 6 days, the eyes were enucleated for histopathological analysis. RESULTS: Clinical signs of infection in the treatment group were markedly reduced for up to 6 days after treatment. Histopathology showed a regular arrangement of stromal fibers and a small number of inflammatory cells in 58% of the corneas. However, 42% of corneas in the treatment group showed infiltrating neutrophils and irregular alignment of stromal collagen fibers. CONCLUSIONS: Our TONS504-PACT achieved complete recovery from keratitis in 58% of the rabbit models. Further studies are required to determine the conditions for the maximal effectiveness of our TONS504-PACT for Acanthamoeba keratitis. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Functional analysis of mesencephalic astrocyte-derived neurotrophic factor in retinal ganglion cells under oxidative stress.

Glaucoma is optic neuropathy that is characterized by progressive neurodegeneration of the retinal ganglion cells (RGCs) and axons. This condition will lead to visual impairment and bring glaucoma to become the second cause of blindness globally. Neuroprotection in glaucoma is needed to prevent the progression of optic neuropathy. In this study, we examined the effects of the superior colliculus (SC), and mesencephalic astrocyte-derived neurotrophic factor (MANF) secreted from the SC, on RGC survival after oxidative stress. SC slices and RGCs from rats (3-day old) were co-cultured using a 3D-transwell system. In addition, primary RGCs from 4 to 5-day-old rats were cultured and treated with 100 µM hydrogen peroxide (H2 O2 ), together with stimulation by MANF. Immunoblot and immunofluorescence analyses indicated down-regulated expression levels of several survival markers of RGCs. Extension of neurites was decreased in RGCs treated with 100 µM H2 O2 . Following co-culture with SC slices, or the addition of MANF, we found that both the down-regulated expression of neural markers and extension of neurites caused by oxidative stress in RGCs were blocked. Furthermore, we found a decrease in the expression of neural markers and extension of neurites after co-culture with MANF siRNA-treated SC slices compared with slices treated with mock siRNA, but, RGCs co-cultured with SC slices treated with MANF siRNA displayed no-changed about to apoptosis. These results suggest that MANF secreted from the SC may play an important role in maintenance of function and survival of RGCs. It is also possible that MANF is an important factor in neuroprotection of RGCs. SIGNIFICANCE OF THE STUDY: Glaucoma is a progressive neurodegenerative disease of the retinal ganglion cell (RGC) and their axons. Neuroprotection is aimed at protecting those neurons that are damaged glaucomatous optic neuropathy. We have now examined the effects of superior colliculus, or msencephalic astrocyte-derived neutrophic factor (MANF), secreted from superior colliculus, on RGC survival using co-culture system. Our results suggested that MANF may important key factor in neuroprotection of RGC.

Antimicrobial Photodynamic Therapy with the photosensitizer TONS504 eradicates Acanthamoeba.

BACKGROUND: Microbial keratitis is a potential cause of corneal blindness. We investigated the amoebicidal efficacy of photodynamic antimicrobial therapy with a light-emitting diode as the light source and the cationic chlorin derivative TONS504 as the photosensitizer for the elimination of Acanthamoeba, a causative organism of corneal infection and blindness. Acanthamoeba keratitis remains a challenge to treat because of limited available treatments. METHODS: Acanthamoeba castellani 50370 was exposed to TONS504 at various concentrations (0, 1, or 10 mg/L for trophozoites; 0, 1, 10, or 20 mg/L for cysts), irradiated at various light energies (0, 10, or 30 J/cm2 for trophozoites; 0, 30, or 60 J/cm2 for cysts), and incubated at 26 °C for 3 h. Assessment of cell viability by trypan blue staining revealed that photodynamic antimicrobial therapy attenuated the survival of trophozoites and cysts dependent on TONS504 concentration and light energy. RESULTS: Photodynamic antimicrobial therapy with 10 mg/L TONS504 and 30 J/cm2 light energy suppressed trophozoite viability by 77%, and 20 mg/L TONS504 and 60 J/cm2 light energy attenuated cyst survival by 42%. Staining with fluorescein isothiocyanate-conjugated annexin V and ethidium homodimer III revealed photodynamic antimicrobial therapy induced apoptosis and necrosis in trophozoites dependent upon the intensity of treatment, whereas apoptosis was the predominant form of cell death in cysts. CONCLUSIONS: Photodynamic antimicrobial therapy with TONS504 warrants further investigation as a potential treatment modality for Acanthamoeba keratitis.

An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y-27632, a Rho kinase inhibitor.

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y-27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation-related factors. We found that Y-27632 inhibited collagen gel contraction, as well as α-smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)-ß. To investigate the effect of Y-27632 in postsurgical scarring, we mimicked TGF-ß secretion by stimulating HTFs with TGF-ß prior to Y-27632 treatment. HTFs cultured in the presence of TGF-ß significantly increased gel contraction. In contrast, when HTFs were treated with 10µM Y-27632, contraction was significantly inhibited. Furthermore, Y-27632 reduced TGF-ß-induced phosphorylation of mitogen-activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF-ß signalling after surgery through mitogen-activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis. SIGNIFICANCE OF THE STUDY: Scar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y-27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y-27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y-27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF-ß and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis.

Antifungal efficacy of photodynamic therapy with TONS 504 for pathogenic filamentous fungi.

The pathogenic filamentous fungi Fusarium solani (F. solani) and Aspergillus fumigatus (A. fumigatus) are common causes of fungal keratitis. We have here evaluated the antifungal efficacy of photodynamic antimicrobial chemotherapy (PACT) with the novel chlorin derivative TONS 504 and a light-emitting diode (LED) with a wavelength of 660 nm for these fungal species. Isolated fungal spores were irradiated at LED energies of 10, 20, or 30 J/cm2 in the presence of TONS 504 at concentrations of 1 or 10 mg/L. As a control, spores were exposed to TONS 504 or LED radiation alone. The treated spores were then cultured on potato dextrose agar plates at 25 °C for 3 to 4 days before determination of colony formation as a measure of viability. Fungal growth was inhibited in a manner dependent on both LED energy and TONS 504 concentration. The inhibitory effect on F. solani was complete with TONS 504 at a concentration of 1 mg/L and LED irradiation at 30 J/cm2 as well as at a TONS 504 concentration of 10 mg/L and LED irradiation at 10, 20, or 30 J/cm2. In contrast, that on A. fumigatus was only partial at a TONS 504 concentration of 10 mg/L and LED irradiation at 20 or 30 J/cm2. The antifungal effect of PACT on A. fumigatus was thus inferior to that on F. solani. PACT with TONS 504 and an LED thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious fungal keratitis.

Time-dependent antimicrobial effect of photodynamic therapy with TONS 504 on Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major cause of infectious keratitis, which itself is a major cause of blindness worldwide. We have now evaluated the time-dependent effectiveness of photodynamic antimicrobial chemotherapy (PACT) with the chlorin derivative TONS 504 and a light-emitting diode (LED) on P. aeruginosa in vitro. PACT with TONS 504 (10 mg/L) and irradiation (30 J/m2) by an LED device that delivers light centered on a wavelength of 660 nm was applied to 1 × 106 colony-forming units of P. aeruginosa in liquid medium. The bacteria were then cultured at 37 °C for various times before assay of viability by determination of colony formation on agar plates. The effect of a second irradiation at 3 h after the initial LED exposure was also examined. Bacterial growth was markedly inhibited between 3 and 9 h after PACT with TONS 504, with the maximal effect being apparent at 3 h. Furthermore, a second exposure to LED irradiation at 3 h after the first treatment enhanced the inhibitory effect on bacterial growth. PACT with TONS 504 thus inhibited the growth of P. aeruginosa in a time-dependent manner, and an additional irradiation exposure applied 3 h after the first LED treatment greatly increased the effectiveness of PACT. This antibacterial system thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious keratitis.

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 H2 O2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H2 O2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H2 O2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H2 O2 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 H2 O2 . 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 H2 O2 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 ßIII-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 © 2016 John Wiley & Sons, Ltd.

Effects of topical adrenergic agents on prostaglandin E2-induced aqueous flare and intraocular pressure elevation in pigmented rabbits.

PURPOSE: To evaluate the effects of signals through adrenergic receptors on the changes in the aqueous flare and intraocular pressure (IOP) induced by topical prostaglandin E2 (PGE2) in pigmented rabbits. METHODS: Adrenergic agents were applied topically to pigmented Dutch rabbits, and PGE2 was then applied to induce an increase in the aqueous flare and IOP. The degree of aqueous flare was measured with a laser flare meter, and the IOP was measured with a rebound tonometer. Measurements were made every 30 min after the PGE2 had been applied for 2 h and at 4.0 and 4.5 h. Repeated measure analysis of variance and Dunnett's post hoc tests were used for the statistical analyses. RESULTS: The topical application of PGE-2 increased the aqueous flare for more than 4.5 h. The topical instillation of 1.0 % apraclonidine significantly inhibited the increase in the PGE2-induced aqueous flare by 75.1 %, of 0.1 % brimonidine by 57.2 %, of 0.04 % dipivefrin by 57.4 %, and a combination of 0.1 % brimonidine and 5 % phenylephrine by 78.9 %. Topical 5.0 % phenylephrine and 0.05 % isoproterenol had little effect on the aqueous flare elevation induced by PGE2. The IOP increased 0.5 h after the topical application of PGE-2. Topical 1.0 % apraclonidine, 0.1 % brimonidine, 0.1 % dipivefrin, and the combination of 0.1 % brimonidine and 5.0 % phenylephrine significantly inhibited the PGE2-induced IOP elevation. However, topical 5.0 % phenylephrine and 0.05 % isoproterenol did not significantly inhibit the IOP elevation caused by PGE2. CONCLUSIONS: Signaling by the α2 receptor inhibits both the PGE2-induced flare and IOP elevation caused by topical PGE2 application.

Inactivation of acyclovir-sensitive and -resistant strains of herpes simplex virus type 1 in vitro by photodynamic antimicrobial chemotherapy.

PURPOSE: To evaluate the efficacy of photodynamic antimicrobial chemotherapy (PACT) with the new porphyrin derivative TONS 504 and a light-emitting diode (LED) against acyclovir (ACV)-sensitive and -resistant herpes simplex virus type 1 (HSV-1). METHODS: Human FL cells infected with the viral strains were subjected to PACT with TONS 504 at various concentrations (0.01 to 10 mg/l) and irradiation at various light energies (10 to 30 J/cm(2)) and were then incubated for 24 h before analysis. RESULTS: Immunocytofluorescence analysis with antibodies to HSV-1 revealed that PACT eliminated HSV-1 and ACV-resistant HSV-1 in a manner dependent on the TONS 504 concentration and light energy. Complete eradication of both viruses was apparent at a TONS 504 concentration of 10 mg/l and light energy of 10 to 30 J/cm(2) as well as at a TONS 504 concentration of 1 mg/l and light energy of 20 or 30 J/cm(2). No antiviral effect was apparent with TONS 504 in the absence of irradiation or with irradiation in the absence of TONS 504. Staining of cell nuclei with 4', 6-diamidino-2-phenylindole revealed no apparent cytotoxicity of the PACT system, a finding that was confirmed by the system's failure to induce the release of lactate dehydrogenase from the host cells. CONCLUSIONS: We conclude that our PACT system based on TONS 504 and an LED is effective for eliminating HSV-1 and ACV-resistant HSV-1 without a harmful effect on host cells.

Antimicrobial action from a novel porphyrin derivative in photodynamic antimicrobial chemotherapy in vitro.

Efforts to identify improved treatments for corneal infection include the development of photodynamic antimicrobial chemotherapy (PACT). We evaluated the antimicrobial effect of PACT with a novel porphyrin derivative, TONS 504, and a novel light system on methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Bacteria were irradiated with a light-emitting diode (LED) at energies of 10, 20, or 30 J/cm(2) in the presence of various concentrations of TONS 504. Bacterial viability was assessed at 30 min and 24 h after irradiation by determination of colony formation on agar plates. PACT inhibited the growth of both MSSA and MRSA as early as 30 min after light exposure. Complete inhibition of bacterial growth was apparent at 24 h after irradiation at a TONS 504 concentration of 1 mg/L and LED energies of ≥10 J/cm(2) or a TONS 504 concentration of 0.5 mg/L and LED energies of ≥20 J/cm(2) for MSSA, and at a TONS 504 concentration of 10 mg/L and LED energies of ≥10 J/cm(2) or of a TONS 504 concentration of 1 mg/L and LED energies of ≥20 J/cm(2) for MRSA. Bacterial growth was unaffected by TONS 504 in the absence of irradiation or by irradiation in the absence of TONS 504. Our results thus demonstrate the antimicrobial efficacy of PACT with TONS 504 and a LED against both MSSA and MRSA in vitro, and they therefore provide a basis for further investigation of this system as a potential treatment for corneal infection.

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. Development and homeostasis of multicellular organisms require interactions between neighbouring cells. With the use of a coculture system based on a collagen vitrigel membrane, we have now shown that neural cells induce up-regulation of the neural guidance protein Sema4A in RPE cells. This effect of neural cells appears to be mediated by the neuropeptide PACAP. PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may thus regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function.

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.

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.

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.