Yang Kyung Cho

Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity

Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the corneas proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.

Comparison of Autologous Serum Eye Drops with Different Diluents

Purpose: To compare the effect of autologous serum eye drops with different diluents in patients with dry eyes and persistent epithelial defects. Methods: Patients of Sjögren’s syndrome (Group I), non-Sjögren’s syndrome (group II) with dry eye, and persistent epithelial defects (Group III) were included. The eyes of each group were randomly treated with one of the following autologous serum eye drops: 100% serum (AS100), 50% serum with normal saline (AS50NS); 50% serum with sodium hyaluronate (AS50HA); or 50% serum with ceftazidime (AS50CEF). The differences in dry eye symptoms, Schirmer test I, tear break-up time (TBUT), corneal staining, and speed in epithelial healing were studied. Results: In Group I, AS100 showed fewer symptoms than AS50NS, AS50HA and AS50CEF (all p < 0.01). AS100 showed significantly better effect than AS50NS, AS50HA and AS50CEF in decreasing corneal staining at the time point of 12-week post-treatment (p = 0.041, p < 0.001 and p < 0.001, respectively). In Group II, AS100 was more effective than AS50CEF in decreasing symptoms and decreasing corneal staining (all p < 0.05). There was no significant difference in symptom and corneal staining between AS100 and AS50NS. In Group III, AS100 was the most effective in achieving quick epithelial closure. Conclusion: In the eyes with Sjögren syndrome and persistent epithelial defects, AS100 was the most effective in decreasing symptoms, corneal epitheliopathy and promoting fast closure of wound. In the eyes with non-Sjögren syndrome, AS100 and AS50NS have similar effects in decreasing symptoms and corneal epitheliopathy.

Flt23k nanoparticles offer additive benefit in graft survival and anti-angiogenic effects when combined with triamcinolone.

PURPOSE To determine if nanoparticles delivering plasmids expressing Flt23k (an anti-VEGF intraceptor) can enhance murine cornea transplant survival and whether their effect is synergistic with steroid therapy. METHODS Biodegradable PLGA Flt23k loaded or blank nanoparticles were prepared using the emulsion solvent evaporation METHOD Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the Flt23k nanoparticles, blank nanoparticles, triamcinolone acetonide, and PBS groups following subconjunctival injection in mice that underwent penetrating keratoplasty. Graft survival, corneal neovascularization, and corneal lymphangiogenesis in a group treated with both nanoparticles and steroid therapy were also analyzed. RESULTS The Flt23k nanoparticle group showed less neovascularization, lymphangiogenesis, and graft failure compared with the PBS control group (P < 0.01). The 2-month graft survival rate was 20% in the Flt23k nanoparticle group with no grafts surviving in the PBS group. When the Flt23k nanoparticle was combined with steroid therapy, a significant increase in graft survival was seen compared with both steroid treatment alone (P < 0.05) and steroid combined with blank nanoparticle treatment (P < 0.05). The 2-month graft survival rate was 91.6% in the combination group compared with 47.6% in the triamcinolone-only group and 42.4% in the triamcinolone plus blank nanoparticle group. CONCLUSIONS Anti-VEGF nanoparticles (Flt23k) have a significant effect on decreasing neovascularization and lymphangiogenesis, resulting in increased graft survival in penetrating keratoplasty. This beneficial effect is synergistically enhanced with steroid treatment.

Vascular Endothelial Growth Factor Receptor 1 Morpholino Increases Graft Survival in a Murine Penetrating Keratoplasty Model

PURPOSE This study sought to determine whether a Vascular Endothelial Growth Factor Receptor 1 (VEGFR1)-specific morpholino (MO) could decrease neovascularization, thereby enhancing murine cornea transplant survival, and if this effect is synergistic with steroid therapy. METHODS Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the VEGFR1_MO, STD MO and PBS groups following subconjunctival injection in mice that underwent normal risk penetrating keratoplasty (NR PK) and high-risk penetrating keratoplasty (HR PK). Graft survival, corneal neovascularization, and corneal lymphangiogenesis in groups treated with both VEGFR1_MO and steroid therapy were also analyzed in HR PK. RESULTS In NR PK, the VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the PBS group (P = 0.055, P = 0.003, P = 0.043, respectively). In HR PK, VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the STD MO (P = 0.000, P = 0.000, P = 0.029, respectively) and PBS groups (P = 0.004, P = 0.002, P = 0.024). In HR PK, when the VEGFR1_MO was combined with steroid therapy, a significant increase in graft survival was seen compared with steroid treatment alone (P = 0.045). The 2-month graft survival rate for HR PK was 27% in the combination group compared with 0% in the triamcinolone only group. CONCLUSIONS VEGFR1_MO decreased angiogenesis and lymphangiogenesis, resulting in increased graft survival in both NR PK and HR PK. This beneficial effect is synergistically enhanced with steroid treatment in HR PK.

Vascular Endothelial Growth Factor Receptor 1 Morpholino Decreases Angiogenesis in a Murine Corneal Suture Model

PURPOSE This study sought to determine whether a vascular endothelial growth factor receptor 1 (VEGFR1)-specific morpholino could induce the alternative splicing of Flt-1 pre-mRNA to downregulate membrane-bound Flt-1 (mFlt-1) and increase the production of soluble Flt-1 (sFlt-1), thereby limiting angiogenesis and inflammation in a mouse corneal suture injury model. METHODS A murine corneal suture model was used to investigate the effects of a VEGFR1-specific morpholino in vivo. Western blot analysis and RT-PCR were used to compare the impact of the Flt morpholino on mFlt-1 and sFlt-1 levels. For vascular regression modeling, two corneal sutures were placed and injected with Flt morpholino, standard morpholino, and PBS on days 8 and 10. Corneas were harvested on day 14. The grade of neovascularization (graded 0-5; 0, no neovascularization; 5, thick tortuous new vessel growth over the suture and toward the center of the cornea) was compared on days 8, 10, and 14. Immunohistochemistry, fluorescent microscopy, and confocal microscopy were used to digitally quantify the area and volume of neovascularization and inflammatory infiltration. RESULTS Western blot analysis revealed that the Flt morpholino decreased mFlt-1 levels while increasing sFlt-1 levels. An increased sFlt-1/mFlt-1 ratio in the Flt morpholino group was seen with RT-PCR. Based on the neovascularization grading, there was a decrease in neovascularization area in the Flt morpholino group (3.29 ± 0.19 to 2.92 ± 0.13) from day 8 to 14 (P < 0.05) compared with that in both the standard morpholino (2.68 ± 0.19 to 3.14 ± 0.22) and in the PBS (2.96 ± 0.14 to 3.42 ± 0.19) groups, both of which showed an increase in neovascularization (P < 0.05). The Flt morpholino group also showed reduced neovascularization volume compared with that of the PBS (P = 0.001) and STD morpholino groups (P = 0.000). CONCLUSIONS Flt morpholinos decrease mFlt-1 and increase sFlt-1 levels, resulting in decreased neovascularization in a murine corneal suture model.

Vimentin knockdown decreases corneal opacity.

PURPOSE Wound induced corneal fibrosis can lead to permanent visual impairment. Keratocyte activation and differentiation play a key role in fibrosis, and vimentin, a major structural type III intermediate filament, is a required component of this process. The purpose of our study was to develop a nonviral therapeutic strategy for treating corneal fibrosis in which we targeted the knockdown of vimentin. METHODS To determine the duration of plasmid expression in corneal keratocytes, we injected a naked plasmid expressing green fluorescent protein (GFP; pCMV-GFP) into an unwounded mouse corneal stroma. We then injected pCMV-GFP or plasmids expressing small hairpin RNA in the corneal wound injury model (full-thickness corneal incision) to evaluate opacification. RESULTS GFP expression peaked between days 1 and 3 and had prominent expression for 15 days. In the corneal wound injury model, we found that the GFP-positive cells demonstrated extensive dendritic-like processes that extended to adjacent cells, whereas the vimentin knockdown model showed significantly reduced corneal opacity. CONCLUSIONS These findings suggest that a nonviral gene therapeutic approach has potential for treating corneal fibrosis and ultimately reducing scarring.

Presurgical Corticosteroid Treatment Improves Corneal Transplant Survival in Mice

Purpose: To examine the effects of presurgical corticosteroid treatment for normal-risk penetrating keratoplasty (NRPK), high-risk penetrating keratoplasty (HRPK), and high-risk penetrating keratoplasty plus lensectomy. Methods: We used 3 corneal transplantation models (NRPK, HRPK, and high-risk penetrating keratoplasty plus lensectomy). For each model, we tried to compare the effect of corticosteroid treatment according to different timetables as follows: The first trial began with a corticosteroid injection given 2 weeks before the PK and continued until 4 weeks after the PK (group 1). The second trial started with a corticosteroid injection given on the day of the PK and continued for 4 weeks after the PK (group 2). The third trial started with a corticosteroid injection administered on the day of the PK and continued for 8 weeks after the PK (group 3). After harvesting and immunostaining of corneas, graft survival, neovascularization (NV), and lymphangiogenesis (LY) were compared among the groups. A P value <0.05 was considered as being statistically significant. Results: With respect to graft survival, group 1 had improved graft survival compared with that of group 3 in the HRPK model (P = 0.025). In all the 3 PK models, groups 2 and 3 demonstrated a similar graft survival (P > 0.05). With respect to NV and LY, in NRPK, group 1 showed less NV than did group 2 (P < 0.001) and group 3 (P = 0.016). In HRPK, group 1 also demonstrated less NV and LY than did group 3 (P = 0.045 and 0.044, respectively). Conclusions: The initiation time point of the corticosteroid treatment is important for graft survival. Corticosteroid pretreatment is an effective means to increase graft survival for HRPK and to decrease NV and LY for both NRPK and HRPK.

Effect of glucocorticoid (triamcinolone acetonide) pretreatment in a murine penetrating keratoplasty and suture model.

Purpose: To evaluate the effect of glucocorticoid (triamcinolone acetonide injectable suspension) pretreatment on corneal neovascularization, lymphangiogenesis, and inflammation in a murine penetrating keratoplasty (PK) and corneal suture model. Methods: For the PK model, BALB/c mice were used as recipients and C57BL/6 mice were used as donors. A group pretreated with subconjunctival glucocorticoid and a combination of post-subconjunctival and topical glucocorticoids (group I) was compared with two groups that did not receive glucocorticoid pretreatment [one group received a combination of subconjunctival and topical glucocorticoids postoperatively (group II) and the other group received only topical glucocorticoid treatment postoperatively (group III)]. All groups were treated with subconjunctival glucocorticoid on the day of surgery. For the corneal suture model, BALB/c mice were used. A group receiving only pre-suture glucocorticoid treatment (group A) and a group receiving only post-suture glucocorticoid treatment (group C) were compared with a control group that did not receive glucocorticoid therapy (group B). The degree of neovascularization, lymphangiogenesis, and inflammatory infiltration was compared in each of these models. Results: In the PK model, the group receiving glucocorticoid pretreatment (group I) showed less neovascularization compared with the posttreatment-only groups (group II, P = 0.043; group III, P = 0.020) and less lymphangiogenesis compared with group III (P = 0.005). In the corneal suture model, the glucocorticoid pretreatment group showed a similar level of neovascularization, lymphangiogenesis, and inflammatory infiltration as the posttreatment-only groups (P > 0.05). Conclusions: Glucocorticoid pretreatment before PK decreases neovascularization and lymphangiogenesis compared with posttransplant glucocorticoid treatment alone.

Comparison of the Anti-angiogenic and Anti-inflammatory Effects of Two Antibiotics: Clarithromycin Versus Moxifloxacin

Abstract Purpose: Clarithromycin is a 14-membered ring macrolide antibiotic with anti-inflammatory as well as antibacterial activity, and has been used worldwide. Moxifloxacin is a leading fourth generation quinolone antibiotic that has been used worldwide perioperatively. We intended to evaluate whether clarithromycin can suppress angiogenesis and inflammation in the cornea, and to compare the anti-inflammatory and anti-angiogenic effects of the two antibiotics, clarithromycin and moxifloxacin. Methods: We made a murine corneal suture model and tested the anti-inflammatory and anti-angiogenic effects of clarithromycin (5 mg/ml) and moxifloxacin (5 mg/ml) in two randomly divided groups. Dexamethasone (5 mg/ml) was used as a positive control. After making two sutures on the cornea, we performed subconjunctival injections (10 μl) on each group on the day of suture, and every day thereafter until the 8th day post-suture. After harvesting corneas on the 8th post-suture day for immunohistochemical staining, we compared neovascularization (NV), lymphangiogenesis (LY) and inflammatory cell infiltration among the groups. Results: Clarithromycin suppressed NV, LY and inflammatory infiltration, compared with phosphate-buffered saline (PBS). However, moxifloxacin did not suppress NV, LY, or inflammatory infiltration, compared with PBS. Comparison between clarithromycin and moxifloxacin, clarithromycin showed a tendency of decreasing LY (p = 0.063) and had less inflammatory cell infiltration (p < 0.05) than did the moxifloxacin group. The anti-(lymph)angiogenic and anti-inflammatory effects of clarithromycin were as high as those of dexamethasone. Conclusion: Clarithromycin suppressed LY and inflammation in the cornea, and its anti-inflammatory effect was significantly superior to that of moxifloxacin.

Dry eye predisposes to corneal neovascularization and lymphangiogenesis after corneal injury in a murine model.

Purpose: Dry eye disease is becoming recognized as an immune-inflammation mediated disorder. Surgical insults such as corneal incision or suture can aggravate dry eye. We sought to determine whether underlying dry eye aggravates corneal inflammatory infiltration, hemangiogenesis, and lymphangiogenesis (LY) induced by surgical injury in a murine model. Methods: We used treatment arms; one, normal eye (non–dry eye) and the other, a scopolamine-induced dry eye model. We first compared the corneas of both groups on which no surgery was performed with confocal and fluorescent microscopy. In subgroups of each treatment arm, we made a corneal incision followed by 2 corneal sutures to approximate the wound. After harvesting the cornea on postoperative day 9 and immunohistochemical staining, we compared corneal neovascularization (NV), LY, and CD11b+ inflammatory cell infiltration between non–dry eye and dry eye groups. Results: In corneas in which no surgery was performed, the dry eye group showed more CD11b+ cell infiltration than did the non–dry eye group (P < 0.05). With respect to corneas after injury, there was significantly more hemangiogenesis, LY, and inflammatory infiltration in the dry eye group than in the non–dry eye group (all P < 0.05). Conclusions: The underlying status of the cornea, whether it is dry or not, plays a significant role in the development of NV, LY, and inflammation after corneal injury. Dry eye can aggravate post-injury NV, LY, and inflammation.