|Author:||Cheung, Ka Wai|
|Title:||A chick vitreous proteome database and differential vitreous protein expressions during myopia development|
|Advisors:||Lam, Chuen Thomas (SO)|
To, Chi Ho (SO)
Hong Kong Polytechnic University -- Dissertations
|Department:||School of Optometry|
|Pages:||xxvii, 231 pages : color illustrations|
|Abstract:||Myopia is the most common refractive error, estimated to affect half the world's population by 2050. High myopia increases the risk of more complicated ocular diseases, which could eventually lead to total blindness. Although multiple factors have been associated with myopia, the exact mechanism of myopia onset or progression is still yet to be identified. Vitreous humor (VH) is a transparent gelatin-like substance that takes up two-thirds of the eyeball and alters the most during eye elongation, covering normal growth and abnormal growth periods such as myopia. As myopia can be seen as an excess growth of the eyeball, quantitative proteomics on the normal ocular growth period and the myopia progression period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia.|
Specific characteristics of the VH, such as being highly hydrated (making the sample more diluted in terms of protein content), and the gel-like elasticity properties, hampered the advancement in ocular proteomics studies with the use of VH. Therefore, a series of optimization studies aiming at the tissue extraction process and mass spectrometry acquisition, data analysis, and processing were conducted in earlier chapters of this thesis. Vitreous homogenized in a 1:1 ratio [v/v, tissue protein extraction reagent (T-PER), lysis buffer: vol] homogenization method was found to yield the optimal protein concentration (0.2 μg/μl) while maintaining a reasonable sample volume for downstream analysis. Acetone precipitation (100%) was found to be the best precipitation for the in-solution protocol, with digested peptides cleaned up using the solid phase extraction (SPE) column. With the use of high-pH fractionation, a loading quantity of 1 μg VH peptides with a 90 mins liquid chromatography (LC) gradient was injected into the mass spectrometer for data analysis for optimal results.
Using a next-generation mass spectrometry label-free data-independent acquisition (DIA) method or termed Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), relative protein changes in vitreous during the normal growth period (7, 14, 21, and 28 days old) in the chick model were identified and quantified. This was followed by protein confirmation using a novel high-resolution multiple reaction monitoring (MRMHR) mass spectrometry using separate batches of animals. The average changes in the refraction showed a reduction in diopter (D), from a hyperopic state on Day 7 to a nearly emmetropic state on Day 28, where both eyes at each time point remained the same (≥ 0.05, paired T-test, n= 6 at each time point) and the vitreous chamber depth (VCD) were found to be significantly elongated (P< 0.05, paired T-test, n= 6 at each time point) during the growth period. Using a highly sensitive nanoLC-ESI-MS/MS system, this study was able to identify a total of 1576 non-redundant proteins (22987 distinct peptides) at 1% FDR without the need for fractionation or protein depletion, making it the most comprehensive chick vitreous protein library covering the emmetropization period (from day 7 across today 28), while 159 proteins were found as "core vitreous proteins". The top abundance proteins identified were Serum albumin (ALB), reelin (RELN), fibronectin, tenascin, and Ovotransferrin. With stringent filtering criteria set as fold change (FC) cut off threshold at ≥1.5 or ≤0.7 folds, differential expression in the same direction from both eyes and each identified protein must have at least 2 quantifiable peptides, 27 proteins were found up-regulated, and 37 proteins were found down-regulated across all time points compared to the baseline at day 7. Targeted MRMHR MS further confirmed proteins such as cadherins (CDH), neurocan (NCAM), and reelin (RELN), which are known to be related to structural and growth-related pathways for the first time, providing novel evidence on which might be key molecules involved in the overall ocular elongation mechanism in the chicks.
Next, myopia progression was studied using the monocular Lens-Induced Myopia (LIM) chick model. -10D lens was attached to a random eye of each chick for 3 days (LIM3) and 7 days (LIM7) to induce myopia progression., Significant LIM was successfully introduced for short-term (LIM3) and long-term (LIM7) old chicks (n= 7 for each time point). The refraction of LIM3 treated eye indicated that the eyes were not fully compensated to the -10D lens, but significant differences were found in Rx compared to the controlled eyes. The eyes further compensated and had an average change in refraction up to -11.61 ± 0.90 D in LIM7 eyes while the change in refraction in controlled eyes remained at -0.46 ± 0.96 D. Furthermore, the VCD changed significantly during this phase (for both LIIM3 and LIM7 with biometric parameters from A-Scan showing that the eyes were able to respond to hyperopic defocus and compensated to the lens introduced successfully. Using the high-pH fractionation technique for constructing a specific ion library for SWATH-MS acquisition data extraction, a total of 1242 proteins (15181 distinct peptides) were identified using the shorter 90 mins MS running gradient as the protein library. With an FC cut off at ≥1.5 or ≤0.7 and p≤ 0.05, unpaired T-test, >1 peptide per protein: For myopia LIM3 study using SWATH-MS analysis, a total of 8 down-regulated proteins were found differentially expressed. The extracellular matrix (ECM) proteins such as Inter-alpha-Trypsin inhibitor heavy chain 3 (ITIH3) indicated a possible breakdown in the VH structural integrity. Also, neuropeptides such as Vasoactive intestinal polypeptide (VIP) and Corticotropin-releasing factor-binding protein (CRHBP) suggested a transfer of molecules from the neurons for multiple functions including oxidative stress which might take part in myopia. For the LIM7 experimental group, a total of 23 proteins were found differentially expressed (10 up and 13 down-regulated), with several upregulated proteins found to be related to α2-macroglobulin (A2M) and were responsible for inflammation/ immune responses. These differential expressed proteins (DEPs) were again mostly neuroproteins that could be leaked from the neighboring tissues, indicating chances of biofluids exchange between the vitreous and its neighboring tissues.
VIP was found to be significantly down-regulated in both LIM groups (LIM3 FC: -0.67, P= 0.004 and LIM7 FC: -0.70, P= 0.0012, n= 7 at each time point). Its expression was previously found reduced in FDM animal myopia studies and its suggested to be involved in circadian rhythm. The down-regulation of VIP was further validated with the targeted multiple reaction monitoring (MRMHR) approach in LIM3 (n= 5) and LIM7 (n= 6) using another batch of animals. However, VIP was not detected in the VH in qPCR studies of LIM3 and LIM7, where else a down-regulation of VIP gene expression was found in both time points in the retina (LIM3 FC: 0.665 ± 0.267, P= 0.046, and LIM7 FC: 0.696 ± 0.117, P= 0.02, n= 4 at both time points). This further suggested that VIP could be transferred from neighboring tissues close to the VH during axial elongation in LIM and FDM.
Along with the other novel candidate proteins obtained using SWATH-MS from this study, a list of potential candidates from the VH under normal growth and myopia progression may serve as potential therapeutic targets to be tested in animal trials to further solidify the understanding of the mechanical of myopia.
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