Megumi Honjo

Retinal Thickness and the Structure/Function Relationship in the Eyes of Older Adults with Glaucoma.

Glaucoma is common and shows high prevalence in older adults. However, there are few studies on the structure/function relationship in older adults with glaucoma. This prospective, cross-sectional study (conducted between February and August 2014), enrolled 102 eyes of 102 subjects aged over 75 years, including 57 eyes with primary open angle glaucoma (POAG), 15 eyes with pseudoexfoliation glaucoma (PXG), and 30 healthy eyes. Multiple regression analysis was used to determine the correlation of circumpapillary retinal nerve fiber layer thickness (cpRNFLT) and macular parameters to mean deviation (MD) to and standard automated perimetry (SAP)-measured sensitivity, assessed with the 30–2 and 10–2 programs. In each 10–2 SAP test point, Spearman’s rank correlation coefficient was used to compare macular retinal nerve fiber layer thickness (mRNFLT), macular ganglion cell-inner plexiform layer thickness (GCIPLT), and mRNFL+GCIPL thickness (GCCT) with sensitivity after adjusting for retinal ganglion cell (RGC) displacement. In eyes with POAG and PXG, cpRNFLT was significantly correlated with 30–2 MD and 30–2 sensitivity. Multiple regression analysis revealed that the POAG had significantly lower cpRNFLT, mRNFLT, GCIPLT, and GCCT according to the severity of disease than control eyes after adjusting for sensitivity, age, sex, and axial length. The PXG eyes had significantly lower cpRNFLT, mRNFLT, and GCCT when compared with the early to moderate POAG eyes. GCCT was significantly correlated with 10–2 sensitivity, except in one juxtafoveal point, (r = 0.338–0.778) in the POAG eyes. The periphery of the central 10° area showed a good correlation between sensitivity and mRNFLT, while the central 5.8° showed a good correlation between sensitivity and GCIPLT. The correlation between structure and function was significant, and objective and quantitative method with OCT assessing glaucoma that does not require patient ability could be a possible parameter to assess diagnosis and progression in older patients with glaucoma.

Autotaxin–Lysophosphatidic Acid Pathway in Intraocular Pressure Regulation and Glaucoma Subtypes

Purpose To compare the levels of autotaxin (ATX), lysophosphatidic acid (LPA), and lysophosphatidylcholine (LPC) in the aqueous humor (AH) of healthy control subjects with those of patients with different subtypes of glaucoma, and also to investigate the relationship of the ATX-LPA pathway with IOP and subtype of glaucoma. Methods This study included 164 eyes of 164 consecutive cases of cataract and glaucoma surgery (37 healthy, 31 normal tension glaucoma, 49 primary open angle glaucoma, 28 secondary open angle glaucoma, and 19 exfoliation glaucoma). Aqueous levels of LPA, LPC, and ATX were quantified using liquid chromatography-tandem mass spectrometry and a two-site immunoenzymetric assay. The association between aqueous levels of ATX/LPA/LPC and IOP elevation in different glaucoma subtypes was investigated. The diagnostic values of indices of the ATX-LPA pathway were compared using receiver operating characteristic curve analysis. Results Notable increases in ATX/LPA/LPC levels in glaucoma patients were observed. The ATX-LPA pathway was significantly related to IOP elevation and the subtype of glaucoma, especially in SOAG and XFG patients, and the area under the curve was significant for discriminating glaucoma eyes from healthy eyes. Conclusions Bioactive ATX/LPA/LPC concentrations were present in aqueous humor, and higher ATX and LPA concentrations were significantly correlated with IOP in all study subjects. Furthermore, the ATX-LPA pathway was significantly related to glaucoma subtype. These results reveal the potentially important role of the ATX-LPA pathway for IOP regulation in healthy subjects and glaucoma patients.

Role of the Autotaxin-LPA Pathway in Dexamethasone-Induced Fibrotic Responses and Extracellular Matrix Production in Human Trabecular Meshwork Cells

Purpose Dexamethasone (Dex) regulates aqueous humor outflow by inducing reorganization of the cytoskeleton and extracellular matrix (ECM) production. Rho kinase (ROCK) has an important role in this process, but the upstream pathway leading to its activation remains elusive. The purpose of the study was to determine the role of autotaxin (ATX), an enzyme involved in the generation of lysophosphatidic acid (LPA), in the Dex-induced fibrotic response and ECM production in human trabecular meshwork (HTM) cells. Methods The expression of ATX in specimens from glaucoma patients was investigated by immunohistochemistry. Regulation of ATX expression and the changes in actin cytoskeleton, ECM production, myosin light chain (MLC) and cofilin phosphorylation, ATX secretion, and lysophospholipase D (lysoPLD) activity induced by Dex treatment in HTM cells were determined by immunofluorescence, real-time quantitative PCR, immunoblot, and the two-site immunoenzymetric and lysoPLD assays. Results Significant ATX expression was found in conventional outflow pathway specimens from glaucoma patients. Dex treatment induced increases in ATX mRNA levels, protein expression, and secretion in HTM cells in association with reorganization of cytoskeleton and ECM accumulation. Significant suppression of these aforementioned changes was observed after ATX/LPA-receptor/ROCK inhibition as well as suppression of fibrotic changes and MLC and cofilin phosphorylation in HTM cells. Conclusions The results of this study, including the robust induction of ATX by Dex treatment, in association with fibrotic changes and ECM production in HTM cells, collectively suggest a potential role for ATX-LPA pathway in the regulation of aqueous humor outflow and IOP in glaucomatous eyes.

The Anti-Inflammatory Effect of Ripasudil (K-115), a Rho Kinase (ROCK) Inhibitor, on Endotoxin-Induced Uveitis in Rats

Purpose To investigate the anti-inflammatory properties of ripasudil, a Rho kinase (ROCK) inhibitor, using endotoxin-induced uveitis (EIU) in rats. Methods Endotoxin-induced uveitis was induced by footpad injection of lipopolysaccharide (LPS). Ripasudil was administered intraperitoneally 1 hour before and after LPS injection. The aqueous humor was collected 24 hours after injection, and the infiltrating cells, protein concentration, and levels of monocyte chemotactic protein-1 (MCP-1) were determined. Infiltrating cells in the iris ciliary body (ICB) and adherent leukocytes in retinal vessels were evaluated. The mRNA levels of IL-1β, IL-6, TNF-α, and MCP-1 in the retina and ICB were determined. A mouse macrophage cell line, RAW264.7, was stimulated with LPS in the presence or absence of ripasudil, and the expression of MCP-1 and nuclear translocation of nuclear factor (NF)-κB was analyzed. Results Ripasudil significantly reduced infiltrating cells and protein exudation in the aqueous humor, as well as the number of infiltrating cells in the ICB and adherent leukocytes in retinal vessels in EIU. Additionally, the protein level of MCP-1 in the aqueous humor and mRNA levels of IL-1β, IL-6, TNF-α, MCP-1, and intercellular adhesion molecule-1 in the ICB and retina were suppressed by ripasudil. The production of MCP-1 and nuclear translocation of NF-κB in RAW264.7 cells were also suppressed by ripasudil. Conclusions The Rho/ROCK pathway plays a role in adhesion molecule expression and inflammatory cell infiltration in EIU, and ripasudil is a potent anti-inflammatory agent against ocular inflammatory diseases, including acute uveitis and possibly uveitic glaucoma.

Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma

Purpose To investigate levels of sphingosine-1-phosphate (S1P) in aqueous fluid samples taken before and after filtration surgery and S1P-induced human conjunctival fibroblast (HCF) responses. Methods Levels of S1P and its related sphingophospholipids in aqueous fluid obtained immediately before and after filtration surgery were determined by liquid chromatography-tandem mass spectrometry. HCFs were used for all in vitro experiments. The expression of five S1P receptor subtypes in HCFs was examined by quantitative real-time PCR. The effect of S1P and receptor-specific antagonists on HCF viability and cell migration was assessed by WST-1 assay and scratch migration assay, respectively. Differentiation to myofibroblasts and extracellular matrix production was evaluated by examining changes in F-actin, α-smooth muscle actin (αSMA), and collagen expression with immunocytochemistry, Western blotting, and collagen accumulation assay, respectively. Results No significant S1P levels in the aqueous fluid samples were detectable immediately before surgery, but postoperative levels of several lysophospholipids, including S1P, dehydro-S1P, and sphingosine, were significantly increased to bioactive concentrations in aqueous fluid in the blebs (P < 0.0001). mRNA expression of the three main S1P receptor subtypes was detected in HCFs. Although S1P levels did not influence HCF proliferation, S1P enhanced cell migration, which could be inhibited by the S1P2 antagonist JTE 013. F-actin, αSMA, and collagen expression was significantly increased by S1P stimulation and was reduced by JTE 013. Conclusions Bioactive S1P concentrations were present in the aqueous fluid at the end of filtration surgery. S1P activated HCFs via S1P2 receptors. These results revealed the potential of S1P2 antagonists in preventing scarring after glaucoma filtration surgery.

Apolipoprotein M Inhibits Angiogenic and Inflammatory Response by Sphingosine 1-Phosphate on Retinal Pigment Epithelium Cells

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammatory responses and proangiogenic factors. It has been suggested that S1P upregulates choroidal neovascularization (CNV) and may be deeply involved in the pathogenesis of exudative age-related macular degeneration (AMD). Recent studies have suggested that apolipoprotein M (ApoM), a carrier protein for S1P, modulates the biological properties of S1P in the pathogenesis of atherosclerosis. However, the role of ApoM/S1P in AMD has not been explored. We investigated the effect of S1P on proangiogenic factors in human retinal pigment epithelium (RPE) cell lines in vitro. S1P promoted the expression of vascular endothelial growth factor in RPE cells. Hypoxia inducible factor-1α expression was also upregulated. These S1P-induced enhancements in growth factors and chemotactic cytokines in RPE cells were significantly inhibited by ApoM treatment. Additionally, in vivo experiments using a laser-induced CNV murine model demonstrated that intravitreal ApoM injection significantly reduced the progression of CNV formation. Although the detailed mechanisms remain to be elucidated, the present results provide a novel potential therapeutic target for AMD, and demonstrate a suppressive role for ApoM and S1P in the pathology of CNV progression.

Increased aqueous autotaxin and lysophosphatidic acid levels are potential prognostic factors after trabeculectomy in different types of glaucoma

We explored the potential relevance of aqueous lysophosphatidic acid (LPA) and autotaxin (ATX) levels on postoperative outcomes of trabeculectomy, and the effects of ATX on fibrotic response in cultured human conjunctiva fibroblast (HCF) cells. We enrolled 70 glaucomatous eyes which underwent trabeculectomy, and quantified aqueous LPA and ATX. Those eyes were followed up for 12 months, and postoperative filtering blebs were evaluated using anterior segment optical coherence tomography. Also, the ATX-induced fibrotic changes in HCFs and the effects of an ATX inhibitor were assessed. Measured aqueous ATX and LPA levels were significantly different between glaucoma subtypes. In multivariate analyses, aqueous ATX levels were significantly correlated with the presence of needlings at 1, 3, 6 and 12 months after surgery. Exfoliative glaucoma, whose ATX level was significantly high, showed significantly increased numbers of needlings and a lower cumulative success rate without needlings. An in vitro study showed that fibrotic changes were upregulated by ATX treatment in HCFs, which was significantly suppressed by an ATX inhibitor. We presently demonstrate that aqueous ATX may be a prognostic factor affecting the fibrotic response in HCFs and bleb formation, and inhibition of ATX could be a therapeutic target after trabeculectomy.

Contribution of prostanoid FP receptor and prostaglandins in transient inflammatory ocular hypertension

We explored the involvement of FP receptor and endogenous prostaglandins (PGs) in transient ocular hypertension (OH) induced by PGE2 or PGF2α in mouse eyes. PGE2 and PGF2α were topically applied to induce transient OH in Wild-type (WT) and FP-, EP1-, EP2-, and EP3-deficient (knockout [KO]) mice. To suppress endogenous PG production, the non-steroidal anti-inflammatory drug nepafenac was applied topically before treatment. PGE2 and PGF2α induced significant OH in the WT, FPKO, and EP1–3KO mice compared to the control 30 min after instillation, and the increase in IOP at 30 or 60 min after instillation in FPKO mice was significantly higher than that in the WT mice. The effects of PGF2α on the increase in IOP were significantly weaker than those of PGE2, especially in EP1KO and EP3KO mice. Transient OH induced by PGE2 and PGF2α was significantly attenuated by nepafenac treatment in FPKO mice. Transient OH was induced by PGE2 and PGF2α in WT, FPKO, and EP1–3KO mice, which was enhanced in FPKO mice. This OH was significantly diminished by nepafenac treatment in FPKO mice, suggesting that FP receptor may have an important naïve physiological role in the eye, and could regulate IOP elevation during PG-associated ocular inflammation.

Impact of the clinical use of ROCK inhibitor on the pathogenesis and treatment of glaucoma

Rho-associated protein kinase (ROCK), a ubiquitously expressed signaling messenger and downstream effector of Rho, is activated by several bioactive factors in the aqueous humor (AH). Rho-ROCK signaling regulates a wide spectrum of fundamental cellular events, including cell adhesion, motility, proliferation, differentiation, and apoptosis. Previous studies, including our own, found that ROCK inhibitor lowers intraocular pressure (IOP) via a direct effect on the conventional AH outflow pathway, by regulation of contractile properties, fibrotic activity, and permeability of the trabecular meshwork (TM) and Schlemm’s canal (SC) tissues, influencing extracellular matrix (ECM) production. Recently, a novel ROCK inhibitor, ripasudil, has been introduced in Japan. Other ROCK inhibitors are now in clinical trials as new IOP-lowering drugs for glaucoma patients. To date, ripasudil, administered together with other glaucoma medications, has proved safe and efficient in lowering IOP as well as additional effects such as prostaglandin analogs, beta-blockers, and carbonic anhydrase inhibitors, all of which help lower IOP by different mechanisms. In addition, we found that long-term treatment with ripasudil exerted an additional IOP-lowering effect, especially in eyes with high IOP, suggesting that late-onset remodeling of the ECM in glaucomatous eyes may elicit mild and delayed changes in IOP levels. ROCK inhibitors have also shown several additional effects, including increased retinal blood flow, direct protection of neurons against various types of stress, and regulation of wound healing; these benefits may potentially be useful in glaucoma treatment.

Interaction Between Pilocarpine and Ripasudil on Intraocular Pressure, Pupil Diameter, and the Aqueous-Outflow Pathway

Purpose To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on IOP and pupil diameter in human eyes, and morphological and functional changes in outflow tissues in vitro. Methods IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which were topically applied in healthy subjects. Human trabecular meshwork (HTM) cells were used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemms canal endothelial (SCE) cells was evaluated by measuring transendothelial electrical resistance and fluorescein permeability. Results Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Ripasudil significantly inhibited gel contraction, TGFβ2-induced stress fiber formation, α-smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFβ2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced miosis. Conclusions Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow.