Centre for Eye Research Australia (CERA) - Research Publications

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    Alterations in Retinal Microvascular Geometry in Young Type 1 Diabetes
    Sasongko, MB ; Wang, JJ ; Donaghue, KC ; Cheung, N ; Benitez-Aguirre, P ; Jenkins, A ; Hsu, W ; Lee, M-L ; Wong, TY (AMER DIABETES ASSOC, 2010-06-01)
    OBJECTIVE: To describe retinal microvascular geometric parameters in young patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Patients with type 1 diabetes (aged 12-20 years) had clinical assessments and retinal photography following standardized protocol at a tertiary-care hospital in Sydney. Retinal microvascular geometry, including arteriolar and venular tortuosity, branching angles, optimality deviation, and length-to-diameter ratio (LDR), were measured from digitized photographs. Associations of these geometric characteristics with diabetes duration, A1C level, systolic blood pressure (SBP), and other risk factors were assessed. RESULTS: Of 1,159 patients enrolled, 944 (81.4%) had gradable photographs and 170 (14.7%) had retinopathy. Older age was associated with decreased arteriolar (P = 0.024) and venular (P = 0.002) tortuosity, and female subjects had larger arteriolar branching angle than male subjects (P = 0.03). After adjusting for age and sex, longer diabetes duration was associated with larger arteriolar branching angle (P 8.5 vs.
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    Quantitative Assessment of Early Diabetic Retinopathy Using Fractal Analysis
    Cheung, N ; Donaghue, KC ; Liew, G ; Rogers, SL ; Wang, JJ ; Lim, S-W ; Jenkins, AJ ; Hsu, W ; Lee, ML ; Wong, TY (AMER DIABETES ASSOC, 2009-01-01)
    OBJECTIVE: Fractal analysis can quantify the geometric complexity of the retinal vascular branching pattern and may therefore offer a new method to quantify early diabetic microvascular damage. In this study, we examined the relationship between retinal fractal dimension and retinopathy in young individuals with type 1 diabetes. RESEARCH DESIGN AND METHODS: We conducted a cross-sectional study of 729 patients with type 1 diabetes (aged 12-20 years) who had seven-field stereoscopic retinal photographs taken of both eyes. From these photographs, retinopathy was graded according to the modified Airlie House classification, and fractal dimension was quantified using a computer-based program following a standardized protocol. RESULTS: In this study, 137 patients (18.8%) had diabetic retinopathy signs; of these, 105 had mild retinopathy. Median (interquartile range) retinal fractal dimension was 1.46214 (1.45023-1.47217). After adjustment for age, sex, diabetes duration, A1C, blood pressure, and total cholesterol, increasing retinal vascular fractal dimension was significantly associated with increasing odds of retinopathy (odds ratio 3.92 [95% CI 2.02-7.61] for fourth versus first quartile of fractal dimension). In multivariate analysis, each 0.01 increase in retinal vascular fractal dimension was associated with a nearly 40% increased odds of retinopathy (1.37 [1.21-1.56]). This association remained after additional adjustment for retinal vascular caliber. CONCLUSIONS: Greater retinal fractal dimension, representing increased geometric complexity of the retinal vasculature, is independently associated with early diabetic retinopathy signs in type 1 diabetes. Fractal analysis of fundus photographs may allow quantitative measurement of early diabetic microvascular damage.
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    Modelling the potential role of saliva use during masturbation in the transmission of Neisseria gonorrhoeae at multiple anatomical sites
    Xu, X ; Chow, EPF ; Ong, JJ ; Shen, M ; Wang, C ; Hocking, JS ; Fairley, CK ; Zhang, L ; Hogben, M (CSIRO PUBLISHING, 2021-12-17)
    Background Neisseria gonorrhoeae can be cultured from saliva in men with pharyngeal gonorrhoea and could theoretically be transmitted from the pharynx to the urethra when saliva is used as a lubricant for masturbation. In this work, we proposed that saliva use during masturbation may be a potential transmission route of gonorrhoea. Methods We analysed the transmission of Neisseria gonorrhoeae at the oropharynx, urethra and anorectum with mathematical models among men who have sex with men using data from six different studies. Model 1 included transmission routes (oral sex, anal sex, rimming, kissing, and three sequential sex practices). In Model 2, we added saliva use during solo masturbation and mutual masturbation to model 1. Results Model 2 could replicate single site infection at the oropharynx, urethra and anorectum and multi-site infection across six different datasets. However, the calibration of Model 2 was not significantly different from Model 1 across four datasets. Model 2 generated an incidence of gonorrhoea from masturbation of between 5.2% (95% CI: 3.2-10.1) to 10.6% (95% CI: 5.8-17.3) across six data sets. Model 2 also estimated that about one in four cases of urethral gonorrhoea might arise from solo masturbation and mutual masturbation. Conclusions Our models raise the possibility that saliva use during masturbation may play a role in transmitting gonorrhoea. This is an important area to explore because it contributes to the knowledge base about gonorrhoea transmission.
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    Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly
    Cipullo, M ; Pearce, SF ; Sanchez, IGL ; Gopalakrishna, S ; Kruger, A ; Schober, F ; Busch, JD ; Li, X ; Wredenberg, A ; Atanassov, I ; Rorbach, J (OXFORD UNIV PRESS, 2021-01-11)
    Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.
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    AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo
    Hung, SSC ; Chrysostomou, V ; Li, F ; Lim, JKH ; Wang, J-H ; Powell, JE ; Tu, L ; Daniszewski, M ; Lo, C ; Wong, RC ; Crowston, JG ; Pebay, A ; King, AE ; Bui, BV ; Liu, G-S ; Hewitt, AW (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06-01)
    PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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    Editorial: Somatic Cell Gene Editing for Treating Diseases
    Wong, RC-B ; Huang, J ; Li, D ; Amaral, O (FRONTIERS MEDIA SA, 2021-12-23)
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    Bulk Gene Expression Deconvolution Reveals Infiltration of M2 Macrophages in Retinal Neovascularization
    Wang, J-H ; Kumar, S ; Liu, G-S (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2021-11-01)
    Purpose: This study interrogated the transcriptional features and immune cellular landscape of the retinae of rats subjected to oxygen-induced retinopathy (OIR). Methods: Bulk RNA sequencing was performed with retinal RNA isolated from control and OIR rats. Gene set enrichment analysis (GSEA) was undertaken to identify gene sets associated with immune responses in retinal neovascularization. Bulk gene expression deconvolution analysis by CIBERSORTx was performed to identify immune cell types involved in retinal neovascularization, followed by functional enrichment analysis of differentially expressed genes (DEGs). Protein-protein interaction analysis was performed to predict the hub genes relevant to identified immune cell types. CIBERSORTx was applied to profile immune cell types in the macula of patients with both proliferative diabetic retinopathy (PDR) and diabetic macular edema using a public RNA-seq dataset. Results: Transcriptome analysis by GSEA revealed that the retina of OIR rats and patients with PDR is characterized by increased immunoregulatory interactions and complement cascade. Deconvolution analysis demonstrated that M2 macrophages infiltrate the retinae of OIR rats and patients with PDR. Functional enrichment analysis of DEGs in OIR rats showed that the dysregulated genes are related to leukocyte-mediated immunity and myeloid leukocyte activation. Downstream protein-protein interaction analysis revealed that several potential hub genes, including Ccl2, Itgam, and Tlr2, contribute to M2 macrophage infiltration in the ischemic retina. Conclusions: This study highlights application of the gene expression deconvolution tool to identify immune cell types in inflammatory ocular diseases with transcriptomes, providing a new approach to assess changes in immune cell types in diseased ocular tissues.
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    Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice
    Cao, M ; Zhang, L ; Wang, J-H ; Zeng, H ; Peng, Y ; Zou, J ; Shi, J ; Zhang, L ; Li, Y ; Yoshida, S ; Tang, L ; Zhou, Y (IVYSPRING INT PUBL, 2019-01-01)
    Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.
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    Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid
    Frisca, F ; Crombie, DE ; Dottori, M ; Goldshmit, Y ; Pebay, A (ELSEVIER, 2013-05-01)
    We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.
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    TAK1 blockade as a therapy for retinal neovascularization
    Lin, F-L ; Wang, J-H ; Chen, J ; Zhu, L ; Chuang, Y-F ; Tu, L ; Ma, C ; Lama, S ; Ling, D ; Wong, RC-B ; Hewitt, A ; Tseng, C-L ; Bui, B ; van Wijngaarden, P ; Dusting, G ; Wang, P-Y ; Liu, G-S ( 2021-01-29)
    Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-β-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-β1 and other pro-inflammatory cytokines. TAK1 is also a key mediator of inflammation, innate immune responses, apoptosis and tissue homeostasis and plays an important role in physiological angiogenesis. Its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. We investigated the regulatory role of TAK1 in pathological angiogenesis in the retina. Transcriptome analysis of human retina featuring retinal neovascularization revealed enrichment of known TAK1-mediated signaling pathways. Selective inhibition of TAK1 activation by 5Z-7-oxozeaenol attenuated aberrant retinal angiogenesis in rats following oxygen-induced retinopathy. Transcriptome profiling revealed that TAK1 activation in human microvascular endothelial cells under TNFα stimulation led to increase the gene expression related to cytokines and leukocyte-endothelial interaction, mainly through nuclear factor kappa B (NFκB) signaling pathways. These results reveal that inhibition of TAK1 signaling may have therapeutic value for the treatment of pathological angiogenesis in the retina.