Tetsuya Toyono

Angiopoietin-like protein 2 is a potent hemangiogenic and lymphangiogenic factor in corneal inflammation

PURPOSE We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo. METHODS Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1β (IL-1β) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed. RESULTS Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1β were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas. CONCLUSIONS Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1β expression. Angptl2 may be a novel therapeutic target for preventing blindness.

Topical Use of Angiopoietin-like Protein 2 RNAi-loaded Lipid Nanoparticles Suppresses Corneal Neovascularization

Corneal neovascularization (CNV) is a sight-threatening condition that is encountered in various inflammatory settings including chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2) is a potent angiogenic and proinflammatory factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA) for topical application. In this study, we have further examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2 Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibited in both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2 Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased in corneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified RNA interference agent is clinically viable in a topical formulation for use against CNV.

Angiopoietin-Like 7 Is an Anti-Angiogenic Protein Required to Prevent Vascularization of the Cornea

Purpose We sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea. Methods Human umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and in vivo in mice. Results Areas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo, intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels. Conclusions ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.

Accelerated transepithelial corneal cross-linking for progressive keratoconus: a prospective study of 12 months

Background/aims To evaluate the clinical results of accelerated transepithelial corneal cross-linking (CXL) in Japanese patients with progressive keratoconus (KCN). Methods Thirty eyes of 19 patients (16 male, 3 female patients) with progressive KCN were included. The mean age was 24.9±7.0 (range 16–38) years. All patients received ultraviolet A radiation for 3 min at an irradiance of 30 mW/cm2. Patients were followed up on the first day, at 1 week and 2 weeks, and at 1 month, 3 months, 6 months and 12 months postoperatively. Clinical examinations included measures of uncorrected visual acuity, best corrected visual acuity (BCVA), average keratometry (AveK), maximum keratometry (Kmax), central corneal thickness, thinnest corneal thickness (TCT), endothelial cell density, intraocular pressure and non-mydriatic indirect fundus examination. Patients were asked to report any pain or discomfort at each visit. Results There were no intraoperative or postoperative complications. All 30 eyes finished the follow-up. After 12 months, there was a significant decrease in Kmax (p<0.0001), AveK (p=0.003) and TCT (p=0.002), and a significant improvement in BCVA (p=0.001). There were no other significant changes. Pain or foreign-body sensation following CXL appeared in the first 2 days, but lasted no more than 1 week in all cases. Conclusions There were no complications associated with accelerated transepithelial corneal CXL, and the clinical outcomes were appraisable in a 12-month follow-up. Trial registration number UMIN000009372.

Microrna-29b Overexpression Decreases Extracellular Matrix mrna and Protein Production in Human Corneal Endothelial Cells

Purpose: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. Methods: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b−transfected donor corneal endothelium was also evaluated by Western blot. Results: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b−transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%). Conclusions: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.

Stromal bed quality and endothelial damage after femtosecond laser cuts into the deep corneal stroma

Aim To evaluate the stromal bed quality and endothelial damage after femtosecond laser (FSL) cuts into the deep corneal stroma. Methods Using a 150-kHz FSL, a lamellar cut was aimed at a depth of 100, 300, or 500 μm in porcine corneas. Stromal bed smoothness was graded from light microscopy and scanning electron microscopy images. Rabbit corneas were cut at remaining thicknesses of 70, 100 and 150 μm using the FSL. The effects of peeling off the corneal flap and the distance between laser spots (2 or 4 μm) were examined. Results The ratio of damaged cells in the group with a remaining depth of 70 μm was significantly larger than that in the groups with a remaining depth of 150 μm. The ratio of damaged cells in the group with a 4-μm spot separation and the flap peeled off was significantly larger than that in the group with a 4-μm spot separation and the flap not peeled off. Conclusions Corneal endothelial damage is likely to increase when the remaining depth is less than 70 μm, and peeling off the flap damages corneal endothelial cells when the remaining depth is less than 100 μm.

Relationship between conjunctivochalasis and refractive error

Objectives: To assess the relation between the prevalence and grade of conjunctivochalasis and refractive error and to compare the grade of conjunctivochalasis between myopic and hyperopic patients. Methods: Consecutive patients aged from 3 to 94 years were chosen for this study. Exclusion criteria included a history of using contact lenses, ocular surgeries, infectious conjunctivitis, or corneal diseases. The age, gender, medical history, ocular history, the grade and other parameters of inferior conjunctivochalasis classified into three locations (nasal, middle, and temporal), and refractive error were determined in all subjects. Patients were divided into three groups as follows: a hyperopic group (≥0.0 D), an emmetropic group (<0.0 and ≥−2.0 D), and a myopic group (<−2.0 D). They were also divided into 10 groups according to age. One-way analysis of variance and the Scheffe multiple comparison test were used to compare the mean values among three groups. Relations among the variables were investigated by calculating Pearson correlation coefficients and partial correlation coefficients. Results: A total of 1,110 patients were included in the study. In each age group, the mean grade of conjunctivochalasis was higher in hyperopic patients than in myopic patients. There were no significant differences in both the downward gaze- and digital pressure-dependent changes of conjunctivochalasis between the myopic and hyperopic groups. The severity of conjunctivochalasis affecting the nasal and temporal bulbar conjunctiva, and parameters such as the changes of conjunctivochalasis caused by downward gaze or digital pressure, were correlated with the refractive error, especially in patients over 40 years old (P<0.05). Conclusions: This was the first assessment of the relationship between refractive error and the grade of conjunctivochalasis in a large consecutive series of patients. Our results suggest that the prevalence and grade of conjunctivochalasis are dependent on refractive error, with hyperopia being an important risk factor for conjunctivochalasis.

Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair

Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.