Kimihiko Fujisawa

Possible benefits of triamcinolone-assisted pars plana vitrectomy for retinal diseases.

Purpose To study the advantages and complications of triamcinolone acetonide (TA)–assisted pars plana vitrectomy (PPV) for various retinal diseases. Methods This report is an interventional case series and nonrandomized study. One hundred seventy-seven eyes from 158 patients underwent PPV with or without TA. Group TA(+) consisted of 94 eyes and group TA(−) consisted of 83 eyes. The improvement in vision and postoperative complications were prospectively studied. Results Sixty-two percent of the eyes in group TA(+) and 49% of the eyes in group TA(−) had improved vision after surgery (P = 0.34). Twelve eyes in group TA(+) and 12 eyes in group TA(−) had an intraocular pressure higher than 21 mmHg after the operation, with no statistically significant difference (P = 0.63). Four eyes with proliferative diabetic retinopathy in group TA(+) and five eyes with proliferative diabetic retinopathy in group TA(−) needed an additional filtering surgery. Group TA(+) (five eyes) had a lower incidence (P = 0.041) of reoperation caused by preretinal fibrous membrane formation than group TA(−) (13 eyes). No apparent corneal disorder or infectious signs were found in any eyes. Conclusions Triamcinolone acetonide–assisted PPV appears to be potentially useful to reduce the incidence of reoperation owing to preretinal fibrosis with no serious complications.

Mechanism of intraocular pressure elevation during hemodialysis

PURPOSE To detect the mechanism of intraocular pressure elevation during hemodialysis. METHODS We measured intraocular pressure, as well as serum osmolality and plasma CO2 pressure, every 30 min during hemodialysis, in 5 eyes with severely compromised aqueous outflow facility (Group A) from 4 renal failure patients. The same measurements were repeated on the same eyes using intravenous hyperosmotic Glyceol to prevent a rapid change in serum osmolality. We also measured the same parameters on 8 eyes with normal aqueous outflow facility (Group B) from 5 patients. The mean +/- SE of percent changes in each parameter was used for the statistical analysis. RESULTS In Group A, the mean percent change of intraocular pressure increased significantly after 90 min, with the exception of the change at 180 min. The mean percent change of serum osmolality decreased significantly after starting dialysis. A negative correlation in the mean percent change of intraocular pressure with serum osmolality was detected (r = -0.759, r < 0.0001). The administration of intravenous hyperosmotic agent prevented significant changes in not only serum osmolality but also intraocular pressure. In Group B, the mean percent change in intraocular pressure showed no significant difference at any time, although the change in serum osmolality decreased significantly. CONCLUSION A remarkable rise in intraocular pressure occurs during hemodialysis in eyes with an impaired aqueous outflow, when serum osmolality decreases rapidly.

Capillary Blood Flow Velocity Measurements in Cystoid Macular Edema With the Scanning Laser Ophthalmoscope

Using a scanning laser ophthalmoscope, we calculated the velocity of retinal blood flow in a juxta-foveolar capillary during the course of cystoid macular edema after partial central retinal vein occlusion in a 53-year-old woman. The mean velocity of the fluorescent dots in the macular capillary of the right eye with cystoid macular edema was 1.59 +/- 0.08 mm/sec at the initial examination. Despite the systemic administration of indomethacin (75 mg/day for three weeks), best-corrected visual acuity decreased from 20/30 to 20/70, and the velocity became 0.82 +/- 0.13 mm/sec. Prednisolone (30 mg/day orally for one week) improved the cystoid macular edema, and the velocity was 0.96 +/- 0.06 mm/sec 12 days after initiation of the drug. The velocity gradually improved; one year later it was 1.65 +/- 0.17 mm/sec and visual acuity was 20/22. Velocity in the left eye, which did not have cystoid macular edema, was 2.16 +/- 0.16 mm/sec. Thus, scanning laser ophthalmoscopy proved useful for measuring the velocity of retinal blood flow.

TEM7 (PLXDC1) in neovascular endothelial cells of fibrovascular membranes from patients with proliferative diabetic retinopathy.

PURPOSE Proliferative diabetic retinopathy (PDR) results from the formation of fibrovascular membranes (FVMs) in the posterior fundus that can lead to a severe decrease of vision. Tumor endothelial marker 7 (TEM7) is a protein that is highly expressed in the endothelial cells of tumors, but whether it plays a role in FVMs is unknown. The purpose of this study was to determine whether TEM7 is associated with the formation of FVMs. METHODS FVMs were obtained during vitrectomy from patients with PDR. RT-PCR was performed to determine the level of expression of the mRNA of TEM7. The splice variants of TEM7 were identified by direct sequencing. Immunohistochemical analyses and in situ hybridization was performed to determine the sites of TEM7 in the FVMs. RESULTS The level of the mRNA of TEM7 was high in 10 of 10 FVMs but was barely detectable in the five idiopathic epiretinal membranes. Direct sequencing of subcloned TEM7 PCR products revealed several splice variants (intracellular, secreted, and membrane-bound forms of TEM7) in the FVMs. Immunohistochemical analysis showed a colocalization of TEM7 and CD34, an endothelial cell marker, in most of the neovascular endothelial cells in the FVMs. Immunoelectron microscopy revealed that membrane-bound TEM7 was expressed on the luminal surfaces of the vascular endothelial cells of FVMs. CONCLUSIONS This study indicates that TEM7 may play a significant role in the proliferation and maintenance of neovascular endothelial cells in the FVMs. If correct, TEM7 may be a molecular target for new diagnostic and therapeutic strategies for PDR.

Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic Macular Edema

This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry–based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor–induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.

Elevated adrenomedullin in the vitreous of patients with diabetic retinopathy.

Adrenomedullin (AM) is a multifunctional peptide with various physiological actions, including vasodilatation, a defense mechanism against microorganisms, the regulation of growth and the regulation of insulin and glucose. In this study, we measured the vitreous AM levels in patients with diabetes mellitus to determine its potential involvement in the pathogenesis of diabetic retinopathy (DR). We used an immunoradiometric assay to measure the vitreous AM concentrations in a total of 28 eyes: 13 with DR and 15 with macular holes (15 men and 13 women, 62.9 ± 10.4 years old). The AM levels in the vitreous fluid of patients with DR (22.9 ± 7.9 fmol/ml) were found to be significantly higher than the corresponding AM levels in patients with macular holes (4.7 ± 1.1 fmol/ml) (p < 0.05). These results indicate that the increase in the vitreous AM is related to DR.

Increased Expression of Angiotensin-converting Enzyme in Retinas of Diabetic Rats

PURPOSE The aim of this study was to examine the localization and the changes in the amount of angiotensin-converting enzyme (ACE) and the relationship between the renin-angiotensin (RA) system and vascular endothelial growth factor (VEGF)/VEGF-receptor system in the retinas of diabetic rats. METHODS Immunohistochemical localization of ACE, VEGF, and VEGF-receptor fetal liver kinase-1 (Flk-1) was examined in cryosections of the retinas of streptozotocin-injected diabetic rats. A semi-quantitative comparison of diabetic rats with age-matched controls was also performed by counting the ACE- or Flk-1-positive vessels per microscopic field. RESULTS ACE immunoreactivity was localized in the retinal vessel walls, and the percentages of ACE-positive vessels were significantly increased in the retinas of diabetic rats maintained 3 to 5 months. Both VEGF and Flk-1 signals increased simultaneously with the increment of ACE immunoreactivity. CONCLUSIONS ACE, expressed in the retinal vessel walls, increases simultaneously with the increment of both VEGF and Flk-1 in the retinas of diabetic rats, suggesting that upregulation of ACE might play some role in the progression of diabetic retinopathy through the VEGF/VEGF receptor system.

Distribution and characterization of proteoglycans associated with exfoliation material

PURPOSE To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials. METHODS The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy. RESULTS Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue. CONCLUSIONS Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.

Evaluation of cellular adhesions on silicone and poly(methyl methacrylate) intraocular lenses in monkey eyes: an electron microscopic study.

Purpose: To assess the biocompatibility of intraocular lens (IOL) material by studying the number of cells adhering to IOLs in monkey eyes. Setting: Department of Ophthalmology, Kyushu University, Fukuoka, Japan. Methods: Silicone or poly(methyl methacrylate) (PMMA) IOL implantation was performed in 21 monkeys. One eye of each animal had surgery. The IOL‐implanted eyes were enucleated 1, 2, 3, 5, and 7 days and 1, 3, 4, 5, and 9 months after the procedure. One eye was studied at each time. Cells on the anterior IOL surface were photographed using a scanning electron microscope, counted and assessed with NEC, Graphtec, and Nikon equipment, and then observed using a transmission electron microscope. Results: Leukocytes, macrophages, and giant cells were found on the anterior IOL surface. Numerous cells were observed on the PMMA IOLs in the early postoperative period; they gradually decreased. Few cells were seen on the silicone IOLs during the course of the study. The giant cells became larger during the postoperative period. Conclusion: The foreign‐body reaction to silicone IOLs in monkey eyes was less than that to PMMA IOLs.

Low-frequency coding variants in CETP and CFB are associated with susceptibility of exudative age-related macular degeneration in the Japanese population.

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. Previous sequencing studies of AMD susceptibility genes have revealed the association of rare coding variants in CFH, CFI, C3 and C9 in European population; however, the impact of rare or low-frequency coding variants on AMD susceptibility in other populations is largely unknown. To identify the role of low-frequency coding variants on exudative AMD susceptibility in a Japanese population, we analysed the association of coding variants of 34 AMD candidate genes in the two-stage design by a multiplex PCR-based target sequencing method. We used a total of 2,886 (1st: 827, 2nd: 2,059) exudative AMD cases including typical AMD, polypoidal choroidal vasculopathy, and retinal angiomatous proliferation and 9,337 (1st: 3,247 2nd: 6,090) controls. Gene-based analysis found a significant association of low-frequency variants (minor allele frequency (MAF) < 0.05) in CETP, C2 and CFB. The association of CETP remained after conditioned with all known genome-wide association study (GWAS) associated variants. In addition, when we included only disruptive variants, enrichment of rare variants (MAF < 0.01) was also observed after conditioned with all GWAS associated variants (P = 1.03 × 10−6, odds ratio (OR) = 2.48). Haplotype and conditional analysis of the C2-CFB-SKIV2L locus showed a low-frequency variant (R74H) in CFB would be individually associated with AMD susceptibility independent of the GWAS associated SNP. These findings highlight the importance of target sequencing to reveal the impact of rare or low-frequency coding variants on disease susceptibility in different ethnic populations.