Plant-derived angiogenin fusion protein’s cytoprotective effect on trabecular meshwork damage induced by Benzalkonium chloride in mice

Background Benzalkonium chloride (BAK), commonly used in glaucoma treatment, is an eye drop preservative with dose-dependent toxicity. Previous studies have observed the multi-functional benefits of angiogenin (ANG) against glaucoma. In our study, we evaluated ANG’s cytoprotective effect on the trabecular meshwork (TM) damage induced by BAK. Additionally, we developed a plant-derived ANG fusion protein and evaluated its effect on TM structure and function. Methods We synthesized plant-derived ANG (ANG-FcK) by fuzing immunoglobulin G’s Fc region and KDEL to conventional recombinant human ANG (Rh-ANG) purified from transgenic tobacco plants. We established a mouse model using BAK to look for degenerative changes in the TM, and to evaluate the protective effects of ANG-FcK and Rh-ANG. Intraocular pressure (IOP) was measured for 4 weeks and ultrastructural changes, deposition of fluorescent microbeads, type I and IV collagen, fibronectin, laminin and α-SMA expression were analyzed after the mice were euthanized. Results TM structural and functional degeneration were induced by 0.1% BAK instillation in mice. ANG co-treatment preserved TM outflow function, which we measured using IOP and a microbead tracer. ANG prevented phenotypic and ultrastructure changes, and that protective effect might be related to the anti-fibrosis mechanism. We observed a similar cytoprotective effect in the BAK-induced degenerative TM mouse model, suggesting that plant-derived ANG-FcK could be a promising glaucoma treatment.

158 using three different methods: three underwent ultrastructural analysis, three underwent 159 immunohistochemical analysis, and three underwent microbead injection to analyze the outflow 160 pathway. Mice were treated with ANG 3 days before BAK administration and the two 161 substances were administered at 10-minute intervals. 162 IOP was measured at 6 pm daily without sedation, and mice were euthanized 4 weeks after 163 BAK and/or ANG treatment. Their eyes were then prepared for electron microscopy or 172 Immunohistochemical and ultrastructural analyses 173 We embedded and froze 36 eyes in Optimal Cutting Temperature Compound (Tissue-Tek, Cat 174 #4583; Sakura Americas, Torrance, CA, USA). Sagittal cryosectioning was performed through 175 the entire anterior-posterior extension of the globe at a thickness of 10-μm. Sections were stored 176 at −80°C and dried for 10 minutes at room temperature. After we washed the sections three times 177 with PBS (Welgene, Gyeongsangbuk-do, Korea) for 10 minutes each, we drew circles along the 178 tissues using a PAP pen (Vector, Burlingame, CA, USA). The sections were fixed with 4% 213 Results 214 215 BAK effect on intraocular pressure 216 The mean IOP changes in response to various BAK concentrations are shown in Fig. 1A. 217 After 2 weeks of treatment twice a day, 0.1% and 0.2% BAK increased the mean IOP. The IOP 218 was higher in these groups than in the sham-operated control group over the 16-week period, 219 although treatment was performed for 4 weeks. At 4 weeks, 0.1% BAK treatment significantly 220 induced an increase in IOP by approximately 36% (17.3 ± 1.0 mmHg) compared to that of the 221 control group (12.7 ± 0.6 mmHg, P < 0.01). This group's IOP remained higher at 6 weeks (23.8 222 ± 1.2 mmHg) and 12 weeks (24.6 ± 2.3 mmHg) than the control group at the same points in time 223 (14.8 ± 3.0 mmHg and 16.1 ± 5.1 mmHg, P < 0.01).

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The mean IOP was significantly higher in the 0.2% BAK group than in the control group at 4 225 weeks (16.5 ± 2.4 mmHg, P = 0.02) and at 6 weeks (21.1 ± 1.3 mmHg, P < 0.01). The IOP under 226 0.01% and 0.02% BAK treatments and subconjunctival 0.1% BAK injection was not 227 significantly different from that of the control group, except for 0.02% BAK at 1 week (10.3 ± 228 1.3 mmHg vs. 14.9 ± 1.6 mmHg in the control group; P = 0.03). In the toxic BAK-induced TM 229 degeneration group, we administered a 0.1% BAK treatment twice a day for 4 weeks. The mean IOP after 0.1% BAK treatment continued to increase and was significantly higher 242 than that of the other groups at 3 weeks (15.2 ± 2.1 mmHg, P < 0.01) and 4 weeks (15.7 ± 1.7 243 mmHg, P < 0.01) ( Fig. 2C and 2D). There were few IOP differences between the single Rh-244 ANG or ANG-FcK treatments and the control group, and inter-and intra-group variability was 245 low (Fig. 2E). For treatments with Rh-ANG or ANG-FcK with BAK, the mean IOP was similar 246 to those of single Rh-ANG and ANG-FcK treatments and the control group at 3 weeks. However 247 at 4 weeks, Rh-ANG with BAK was elevated to 12.1 ± 1.8 mmHg (P < 0.01) and ANG-FcK 248 with BAK to 11.6 ± 0.4 mmHg (P < 0.05), although these values were lower for the single BAK 249 group (P < 0.01) (Fig. 2C, 2ED and 2E).
250 251 Immunohistochemical analysis of the effects of ANG on BAK response 252 We observed that type I collagen's Cy3 labeling in the outflow tissue along the iridocorneal 253 angle was more pronounced in BAK-treated eyes than in the single ANG and control groups 254 (Fig. 3A). The type Ⅰ collagen labeling in the TM region adjacent to the corneal endothelium In this study, we examined the toxicity of chronic BAK exposure on the TM and ANG's 296 defenses against changes in the trabecular outflow pathway. We induced the structural and 297 functional degeneration of the TM through BAK treatment in a mouse model. Co-treatment with 298 ANG successfully preserved the outflow function of the TM, suggesting that ANG prevents 299 fibrosis. Manuscript to be reviewed 317 Baudouin et al. (2012) injected 100 μL of BAK into the subconjunctival space of rats weighing 318 300 to 350 g. In our study, we performed a single injection of 10 μL into the subconjunctival 319 space of mice weighing 21 to 24 g. This might explain the lack of a significant change in IOP 320 despite a higher BAK concentration. In a mouse model, it is difficult to inject 10 μL into the 321 subconjunctival space without any losses. Moreover, the administration of topical drops is more 322 suitable for a chronic exposure model.

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The IOP of the combination of ANG and BAK was lower than that the IOP of BAK alone, but 324 was greater than that of eyes not exposed to BAK at 4 weeks. Our results are in agreement with 325 the findings of an earlier experimental study (Kim et al., 2016) where ANG lowered IOP in 326 both normal and elevated rat models using the vortex vein cauterization method. ANG also 327 conserved the conventional outflow of aqueous humor via the TM after BAK treatment.
328 Fluorescent beads were deposited along the outflow tract in the ANG and control groups, but 329 were sparse in the BAK-induced toxicity model. The cumulative distribution of microbeads was 330 sparse across the parallel sections of all three single BAK-treated mouse models (Fig. S2).     with Rh-ANG and BAK maintained the initial mean IOP over 3 weeks; however, IOP was elevated at 4 weeks, although it was lower than that for the single BAK treatment group. (D) The change in mean IOP for co-treatment with ANG-FcK and BAK was similar to that for Rh-ANG. IOP was higher than that in the control group, but lower than that in the single BAK treatment group at 4 weeks. (E) There was no significant difference in mean IOP between Rh-ANG and ANG-FcK for the combined use with BAK. Error bars represent standard errors of the mean. *P < 0.05 and **P < 0.01