| Author: | Xiao, Hu |
| Title: | The effects of apolipoprotein A-1 expression on eye growth of chicks |
| Advisors: | To, Chi Ho (SO) |
| Degree: | Ph.D. |
| Year: | 2020 |
| Subject: | Myopia Apolipoproteins Eye -- Growth Hong Kong Polytechnic University -- Dissertations |
| Department: | School of Optometry |
| Pages: | xvii, 302 pages : color illustrations |
| Language: | English |
| Abstract: | Aims: Myopia has become not only a public health issue, but also a socioeconomic problem affecting many youngsters worldwide (Zheng et al., 2013). Therefore, it is important to control the myopia epidemic so as to save people from sight-threatening diseases associated with myopia. Previous studies have identified a number of key signaling molecules in myopia development, such as dopamine (Zhang and Wildsoet, 2015, Mao et al., 2010b), apolipoprotein A-1 (apoA1) (Bertrand et al., 2006), retinoic acid (McFadden et al., 2004a, Summers et al., 2016), early growth response 1 (Fischer et al., 1999, Ashby et al., 2007a, Mathis and Schaeffel, 2007) and TGF-β (Rohrer and Stell, 1994). However, a full picture of how these signaling molecules may work together in biochemical cascades that eventually modulate eye growth is still unclear (Bertrand et al., 2006). Unravelling the biology of myopia could help reveal key molecular targets and develop novel therapeutics for myopia control in the future. The present study profiled the retinal differential protein expression in lens induced myopia (LIM) and hyperopia (LIH) in chicks. ApoA1 was thought to be one of key stop signals in eye development. We attempted to examine if altering apoA1 may be effective in controlling myopia development in chicks, and the mechanisms behind its action. Methods: White leghorn chicks (Gallus gallus) were used in our studies. There were four main experiments in this thesis. Experiment 1 was to explore the retinal apoA1 protein expression in LIH, LIM and control chick eyes. Experiment 2 studied the apoA1 mRNA differential expression in LIH, LIM and control chick eyes. Experiment 3 examined the direct effect of apoA1 on myopic eye growth when apoA1 protein is intravitreally injected. Experiment 4 studied the indirect effect of apoA1 on normal growth, myopic and hyperopic progression by oral administration of nicotinic acid. Proteomic analysis was applied to reveal the biological mechanism of eye growth in the presence of apoA1 and nicotinic acid. The spherical equivalent refraction (spherical power + half of cylindrical power) was measured by steak retinoscopy. Ocular parameters were measured by high frequency A-scan ultrasound system (30MHz probe sampled at 100MHz). Sealing Foiland LightCycler® 480 system from Roche Company and LightCycler® 480 SYBR Green I Master kit were used to perform qPCR analysis. Western blot and liquid chromatography coupled tandem mass spectrometry (LC MS/MS) were used to identify and analyze differential protein expressions. SPSS software (version 23, SPSS, Chicago, Illinois, USA) was used for statistical analysis. Results: ApoA1 protein differential expression in LIM, LIH and control chick eyes: Positive optical lenses of +10D could effectively induce hyperopic eye growth after 4 days of LIH (plano vs. LIH, mean±SD; VCD: 0.283±0.073mm vs. -0.121±0.044mm, P<0.001, n=5; AXL: 0.496±0.062mm vs. 0.120±0.058mm, P<0.001, n=5). Whereas, -10D lenses led to myopic eye growth after 4 days (Len Induced Myopia, LIM) (plano vs. LIM, mean±SD; VCD: 0.283±0.073mm vs. 0.713±0.084mm, P<0.001, n=6; AXL: 0.496±0.062mm vs. 0.983±0.093mm, P<0.001, n=6). Retinal apoA1 protein expression was found to increase in LIH (by MS analysis, LIH/plano=2.174, P=0.026, n=5). The results were consistent with previous studies (Bertrand et al., 2006, Chun et al., 2015, Summers et al., 2016). However, the retinal apoA1 expression did not show any down-regulation in LIM when compared to the control (by MS analysis, LIM/plano=0.935, P=1.000, n=6). According to pathway analysis by differential expression proteins, insulin signaling pathway, regulation of actin cytoskeleton, endocytosis and hippo signaling pathways were identified. ApoA1 mRNA differential expression in LIM, LIH and control chick eyes: LIH induced significant changes in the mRNA expression of apoA1 as early as after one day of lens wear and the changes remained significant at 4 days (after 1 day LIH, LIH/plano=1.195, P=0.030, n=7; after 4 days LIH, LIH/plano=1.430, P=0.049, n=8; recovery, + 10D/plano=1.198, P=0.200, n=7). Similarly, the LIM eyes demonstrated a significant differential mRNA expression of apoA1 after 1 day (LIM/plano=1.460, P=0.023, n=8), 4 days of lens wear (LIM/plano=1.549, P=0.033, n=8) and after the removal of lens (LIM/plano=1.303, P=0.062, n=8). Direct effect of apoA1 on myopic progression by apoA1 protein intravitreal injection: Intravitreal injection of apoA1 protein could retard myopia in LIM eyes of different stages of myopia development. In this experiment, 10µl of total 2µg apoA1 protein was randomly injected into the treatment eye while 10µl of control mixture (1×PBS and 0.1% sodium lauroyl sarcosine, PH 7.4) was injected to the control eye. The results showed that apoA1 retarded the myopia development in LIM chicks if injected daily from PN4 to PN6 (changes between PN4 to PN7 were compared; Treatment vs. control, mean±SD; VCD: 0.008±0.101mm vs. 0.154±0.144mm, P=0.013; AXL: 0.115±0.090mm vs. 0.321±0.128mm, P=0.002, n=8). It was also affected in LIM chicks (lens wear from PN4 to PN12) if injected daily from PN9 to PN11 (changes between PN9 to PN12; Treatment vs. control, mean±SD; VCD: 0.079±0.130mm vs. 0.224±0.099mm, P=0.001; AXL: 0.196±0.145mm vs. 0.363±0.111mm, P=0.016, n=7). ApoA1 could even reverse the myopia development in LIM chicks (lens wear from PN4 to PN22) if injected daily from PN19 to PN21 (changes between PN19 to PN22; Treatment vs. control, mean±SD; VCD: -0.057±0.024mm vs. 0.070±0.076mm, P=0.049; AXL: 0.064±0.045mm vs. 0.253±0.056mm, P=0.003, n=4). Effect of nicotinic acid on normal growth, myopic and hyperopic progression: Nicotinic acid is known to increase apoA1 expression in the blood plasma. The effects of oral administration of nicotinic acid on LIM, LIH and normal eye growth were examined in this study. Nicotinic acid significantly retarded eyes growth in both the LIH (NA LIH vs. LIH, mean±SD; VCD: 0.223±0.046mm vs. -0.121±0.044mm, P=0.012; AXL: -0.021±0.097mm vs. 0.120±0.058mm, P=0.030, n=4) and LIM eyes (NA LIM vs. LIM, mean±SD; VCD: 0.548±0.146mm vs. 0.713±0.084mm, P=0.015; AXL: 0.801±0.172mm vs. 0.983±0.093mm, P=0.046, n=8). However, nicotinic acid did not affect normal eye growth. In terms of pathway analysis based on differential expression proteins, insulin signaling pathway, regulation of actin cytoskeleton and endocytosis pathways were identified in this experiment. Conclusion: The present study profiled the retinal differential expression proteins in LIM and LIH in chicks. ApoA1 was found to be highly expressed in hyperopic eye. Increasing the retinal apoA1 by direct intravitreal injection of apoA1 or by oral intake of nicotinic acid led to retardation in eye growth in chicks. Therefore, the results strongly suggested that apoA1 is a "stop" signal to eye growth. Modulating the retinal apoA1 expression with new therapeutics could be a novel way to control human myopia in the future. |
| Rights: | All rights reserved |
| Access: | open access |
Copyright Undertaking
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item:
https://theses.lib.polyu.edu.hk/handle/200/10578