Thomas Chuen Lam

Application of proteomic technology in eye research: a mini review

Proteomics is a rapidly growing research area for the study of the protein cognate of genomic data. This review gives a brief overview of the modern proteomic technology. In addition to general applications of proteomics, we highlight its contribution to studying the physiology of different ocular tissues. We also summarise the published proteomic literature in the broad context of ophthalmic diseases, such as cataract, age‐related maculopathy, diabetic retinopathy, glaucoma and myopia. The proteomic technology is a useful research tool and it will continue to advance our understanding of a variety of molecular processes in ocular tissues and diseases.

Isotope-coded protein label based quantitative proteomic analysis reveals significant up-regulation of apolipoprotein A1 and ovotransferrin in the myopic chick vitreous

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching −10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing −10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

Proteomic analysis of chick retina during early recovery from lens‑induced myopia

Myopia development has been extensively studied from different perspectives. Myopia recovery is also considered important for understanding the development of myopia. However, despite several previous studies, retinal proteomics during recovery from myopia is still relatively unknown. Therefore, the aim of the present study was to investigate the changes in protein profiles of chicken retinas during early recovery from lens-induced myopia to evaluate the signals involved in the adjustment of this refractive disorder. Three-day old chickens wore glasses for 7 days (−10D lens over the right eye and a plano lens as control over the left eye), followed by 24 h without lenses. Protein expression in the retina was measured by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Pro-Q Diamond phosphoprotein staining 2D gel electrophoresis was used to analyze phosphoprotein profiles. Protein spots with significant differences (P<0.05) were analyzed by mass spectrometry. The minus lens-treated eye became myopic, however following 24 h recovery, less myopia was observed. 2D-DIGE proteomic analysis demonstrated that three identified protein spots were upregulated at least 1.2-fold in myopic recovery retinas compared with those of the controls, Ras related protein Rab-11B, S-antigen retina and pineal gland and 26S proteasome non-ATPase regulatory subunit 14. Pro-Q Diamond images further revealed three protein spots with significant changes (at least 1.8-fold): β-tubulin was downregulated, while peroxiredoxin 4 and ubiquitin carboxyl-terminal hydrolase-L1 were upregulated in the recovery retinas compared with the control eye retinas. The present study detected previously unreported protein changes in recovering eyes, therefore revealing their potential involvement in retinal remodeling during eye ball reforge.

Cyclic Adenosine Monophosphate Activates Retinal Apolipoprotein A1 Expression and Inhibits Myopic Eye Growth.

PURPOSE Apolipoprotein A1 (ApoA1) has been shown to inhibit myopia development in chicks, but the underlying biological mechanism remains unknown. Because ApoA1 interacts with cyclic adenosine monophosphate (cAMP) in many cellular systems, we examined whether this interaction is important in myopia development. METHODS The nonmetabolizable cAMP analogue 8-Bromo-cAMP (8-Br-cAMP) was administered intravitreally to the right eyes of 8-day old chicks for 4 consecutive days. Control eyes received vehicle. Chicks in group 1 received 8-Br-cAMP (0.1 mM or 1 mM) and were fitted with -10 diopter (D) lenses on both eyes, whereas chicks in group 2 (0.1 mM 8-Br-cAMP) wore plano lenses over both eyes. The levels of retinal cAMP and ApoA1 were examined in another two groups of chicks wearing -10 D (group 3) and +10 D lenses (group 4) over their right eyes for 3 days, respectively (plano over left eyes). RESULTS The 8-Br-cAMP significantly inhibited development of lens-induced myopia (group 1: 0.1 mM versus vehicle: +1.71 ± 1.22 D versus -8.00 ± 2.19 D; 1 mM versus vehicle: +1.38 ± 1.34 D versus -9.96 ± 1.14 D, mean ± SEM, P < 0.01 for both); 1 mM, but not 0.1 mM 8-Br-cAMP increased expression of retinal ApoA1 levels in right eyes (P < 0.01). The 8-Br-cAMP had minimal effect on normal eye growth. Both retinal cAMP and ApoA1 levels were significantly increased only in hyperopic eyes (group 4). CONCLUSIONS The 8-Br-cAMP robustly inhibited development of lens-induced myopia. The increase in retinal ApoA1 observed in cAMP-treated and hyperopic eyes suggested a possible interplay between ApoA1 and cAMP in regulating eye growth.

The Interactions Between Bright Light and Competing Defocus During Emmetropization in Chicks

Purpose The environment comprises multiple optical signals that affect eye growth. We aimed to determine if the inhibitory effects of myopic defocus and bright light (BL) against myopia are additive in the presence of the myopia-genic hyperopic defocus. Methods In experiment 1, three groups of 24 chicks each were fitted with the following multizone dual-power lenses (pl): pl/-10 D (50:50 area), +10/-10 D (50:50 area), and +10/-10 D (33:67 area) monocularly for 6 days. Half of each group were raised under normal illumination of 500 lux, 12/12-hour light/dark cycle, whereas the remainder were exposed to 6-hour BL of 40 klx and 6-hour 500 lux during the light cycle. In experiment 2, 38 chicks wore +10/-10 D (33:67 area) lenses monocularly for 8 days and were exposed to one of four light intensities for 6 hours per day-500 lux, 10 klx, 20 klx, or 40 klx-and received 500 lux for the remainder of the light cycle. Results In experiment 1, interocular difference in refractions after 6 days for the three groups were -3.6 D, +2.0 D, and -4.2 D, respectively, under normal light and were -0.9 D, +4.2 D, and +0.67 D under BL, manifesting as a shorter anterior segment and vitreous chamber. In experiment 2, the effect of BL increased with light intensity in the +10/-10 D (33:67) group, with a significant difference in refraction between the 10 klx and 20 klx groups (interocular difference -2.75 ± 2.76 D vs. 1.70 ± 2.40 D, P < 0.01), but plateaued between 20 klx and 40 klx (1.70 ± 2.40 D vs. 1.70 ± 0.35 D, P > 0.05). Conclusions The protective effects of myopic defocus and BL against experimental myopia were additive. The inhibitory effect of BL against myopia was dose dependent at 10 klx and above but plateaued at 20 klx.

The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation

Purpose Biomechanical properties changes and α-smooth muscle actin (α-SMA) overexpression are involved in myopia scleral remodeling. However, interactions between altered tissue biomechanics and cellular signaling that sustain scleral remodeling have not been well defined. We determine the mechanisms of mechanotransduction in the regulation of α-SMA expression during myopia scleral remodeling. Methods Guinea pigs were used to establish a form-deprivation myopia (FDM) model. Protein profiles in myopic sclera were examined using tandem mass spectrometry. Ras homolog gene family member A (RhoA) and α-SMA expressions were confirmed using quantitative (q) RT-PCR and Western blotting. Scleral fibroblasts were cultured and subjected to 4% cyclic strain. Levels of RhoA, rho-associated protein kinase-2 (ROCK2), myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), and α-SMA were determined by qRT-PCR and Western blotting in groups with or without the RhoA siRNA or ROCK inhibitor Y27632. MRTF-A and α-SMA were evaluated by confocal immunofluorescent microscopy and myofibroblasts were enumerated using flow cytometry. Results mRNA and protein levels of RhoA and α-SMA were significantly increased in the FDM eyes after 4 weeks of form-deprivation treatment. The 4% static strain increased expressions of RhoA, ROCK2, MRTF-A, SRF, and α-SMA as well as nuclear translocalization of MRTF-A in scleral fibroblasts compared to those without strain stimulation. Additionally, the percentage of myofibroblasts increased after strain stimulation. Conversely, inhibition of RhoA or ROCK2 reversed the strain-induced α-SMA expression and myofibroblast ratio. Conclusions Mechanical strain activated RhoA signaling and scleral myofibroblast differentiation. Strain also mediated myofibroblast differentiation via the RhoA/ROCK2-MRTF-A/SRF pathway. These findings provided evidence for a mechanical strain-induced RhoA/ROCK2 pathway that may contribute to myopia scleral remodeling.

Data on differentially expressed proteins in retinal emmetropization process in guinea pig using integrated SWATH-based and targeted-based proteomics

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. Retinal protein profile changes using integrated SWATH and MRM-HR MS were studied in guinea pigs at 3- and 21-days of age, where the axial elongation was significantly detected. Differential proteins expressions were identified, and related to pathways which are important in postnatal development in retina, proliferation, breakdown of glycogen-energy and visual phototransduction. These results are significant as key retinal protein players and pathways that underlying emmetropization can be discovered. All raw data generated from IDA and SWATH acquisitions were accepted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS00746). A more comprehensive analysis of this data can be obtained in the article “Integrated SWATH-based and targeted-based proteomics provide insights into the retinal emmetropization process in guinea pig” in Journal of Proteomics (Shan et al., 2018) [1].

Post-translational modifications and their applications in eye research (Review)

Gene expression is the process by which genetic information is used for the synthesis of a functional gene product, and ultimately regulates cell function. The increase of biological complexity from genome to proteome is vast, and the post-translational modification (PTM) of proteins contribute to this complexity. The study of protein expression and PTMs has attracted attention in the post‑genomic era. Due to the limited capability of conventional biochemical techniques in the past, large‑scale PTM studies were technically challenging. The introduction of effective protein separation methods, specific PTM purification strategies and advanced mass spectrometers has enabled the global profiling of PTMs and the identification of a targeted PTM within the proteome. The present review provides an overview of current proteomic technologies being applied in eye research, with a particular focus on studies of PTMs in ocular tissues and ocular diseases.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

Astigmatic shift and conjunctival epithelial ingrowths following late-onset iatrogenic LASIK flap dehiscence during scleral buckling surgery.

Quan Liu* MD PhD Chi Ho To* PhD Jian Ge* MD PhD Chu Yan Chan PhD Lin Lv* MD PhD Thomas Chuen Lam PhD Andrew W Siu PhD * State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China † Centre for Myopia Research and § Laboratory of Experimental Optometry, School of Optometry, The Hong Kong Polytechnic University, Hong Kong, China || Department of Anatomy, The Chinese University of Hong Kong, Shatin, Hong Kong, China E-mail: sochto@inet.polyu.edu.hk