Genome-wide Expression of Trabecular Meshwork in POAG
ABSTRACT: This study compared genome-wide expression profiles of individuals with and without Primary Open-Angle Glaucoma (POAG). One POAG case (case #6 with two replicates #10 and #11) carried a Q368X myocilin mutation. Overall design: This study compared the genome-wide expression in human trabecular meshwork tissue between 13 controls and 15 POAG cases. Six controls and one POAG cases had the expression performed from both left and right eyes. One technical replicate was done between two cases. The average from the biological replicates for each inidividual was used for analysis.
Project description:This study compared genome-wide expression profiles of individuals with and without Primary Open-Angle Glaucoma (POAG). One POAG case (case #6 with two replicates #10 and #11) carried a Q368X myocilin mutation. This study compared the genome-wide expression in human trabecular meshwork tissue between 13 controls and 15 POAG cases. Six controls and one POAG cases had the expression performed from both left and right eyes. One technical replicate was done between two cases. The average from the biological replicates for each inidividual was used for analysis.
Project description:To identify the specific genes in human trabecular meshwork (TM) related to POAG.Primary open-angle glaucoma TM specimens were obtained from routine trabeculectomy surgery. Nonglaucomatous control TM specimens were dissected from donor eyes using the same approach as a standard trabeculectomy. All cases were screened for myocilin (MYOC) mutations. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips. Expression data were normalized and analyzed using the R package limma in Bioconductor. Pathway analyses were performed using DAVID Bioinformatics Resources.Our study included surgical TM specimens from 15 cases and 13 controls. One case was identified with a heterozygous Q368X MYOC mutation. If TMs were available from both eyes in an individual, the expression data were combined for analysis. The following three comparisons were performed for differential analyses: (1) MYOC POAG case versus 14 non-MYOC POAG cases, (2) MYOC POAG case versus 13 controls, and (3) 14 non-MYOC POAG cases versus 13 controls. Limited by one MYOC case in comparisons 1 and 2, expression changes were reported comparing the fold changes but without P values. Comparison 3 identified 483 genes, including 36 components of TM exosomes. Gene ontology analysis identified several enriched functional clusters, including cell adhesion, extracellular matrix, and secretion.This is the largest TM expression study of POAG cases and controls performed to date and represents the first report of TM expression in a patient having POAG with a Q368X MYOC mutation. Our data suggest the potential role of endocytic and exosome pathways in the pathogenesis of POAG.
Project description:<h4>Background</h4>A traditional Chinese medicine, Tetramethylpyrazine (TMP), has been prescribed as a complementary treatment for glaucoma to improve patient prognosis. However, the pharmacological mechanism of action of TMP is poorly understood. In previous studies, we demonstrated that TMP exerts potent inhibitory effects on neovascularization, suppresses the tumorigenic behavior of glioma cells, and protects neural cells by regulating CXCR4 expression. Here, we further investigated whether the SDF-1/CXCR4 pathway is also involved in the TMP-mediated activity in trabecular meshwork cells.<h4>Methodology/principal findings</h4>CXCR4 expression was examined by quantitative real-time PCR in trabecular and iris specimens from 54 primary open-angle glaucoma (POAG) patients who required surgery and 19 non-glaucomatous donors. Our data revealed markedly elevated CXCR4 expression in the trabecular meshwork of POAG patients compared with that of controls. Consistently, CXCR4 expression was much higher in glaucomatous trabecular meshwork cells than in normal trabecular meshwork cells. Using RT-PCR and western blot assays, we determined that glaucoma-related cytokines and dexamethasone (DEX) also significantly up-regulated CXCR4 expression in primary human trabecular meshwork (PHTM) cells. Moreover, the TGF-?1-mediated induction of CXCR4 expression in PHTM cells was markedly down-regulated by TMP compared with control treatment (PBS) and the CXCR4 antagonist AMD3100. In addition, TMP could counteract the TGF-?1-induced effects on stress fiber accumulation and expansion of PHTM cells. TMP markedly suppressed the migration of PHTM cells stimulated by TGF-?1 in transwell and scratch wound assays. TMP also suppressed the extracellular matrix (ECM) accumulation induced by TGF-?2.<h4>Conclusions</h4>Our findings demonstrate that CXCR4 might be involved in the pathogenetic changes in the trabecular meshwork of patients with POAG. Additionally, TMP might exert its beneficial effects in POAG patients by down-regulating CXCR4 expression.
Project description:We investigated in vivo changes in Schlemm's canal and the trabecular meshwork in eyes with primary open angle glaucoma (POAG). Relationships between Schlemm's canal diameter, trabecular meshwork thickness, and intraocular pressure (IOP) were examined. Forty POAG patients and 40 normal individuals underwent 80-MHz Ultrasound Biomicroscopy examinations. The Schlemm's canal and trabecular meshwork were imaged in superior, inferior, nasal and temporal regions. Normal individuals had an observable Schlemm's canal in 80.3% of sections, a meridional canal diameter of 233.0±34.5 ?m, a coronal diameter of 44.5±12.6 ?m and a trabecular meshwork thickness of 103.9±11.1 ?m, in POAG patients, Schlemm's canal was observable in 53.1% of sections, a meridional canal diameter of 195.6±31.3 ?m, a coronal diameter of 35.7±8.0 ?m, and a trabecular meshwork thickness of 88.3±13.2 ?m, which significantly differed from normal (both p <0.001). Coronal canal diameter (r = -0.623, p < 0.001) and trabecular meshwork thickness (r = -0.663, p < 0.001) were negatively correlated with IOP, but meridional canal diameter was not (r = -0.160, p = 0.156). Schlemm's canal was observable in 50.5% and 56.6% of POAG patients with normal (<21 mmHg) and elevated (>21 mmHg) IOP, respectively (? = 1.159, p = 0.282). Coronal canal diameter was significantly lower in the elevated IOP group (32.6±4.9 ?m) than in the normal IOP group (35.7±8.0 ?m, p < 0.001). This was also true of trabecular meshwork thickness (81.9±10.0 ?m vs. 97.1±12.0 ?m, p < 0.001). In conclusion, eyes with POAG had fewer sections with an observable Schlemm's canal. Canal diameter and trabecular meshwork thickness were also lower than normal in POAG patients. Schlemm's canal coronal diameter and trabecular meshwork thickness were negatively correlated with IOP.
Project description:Long non-coding RNAs were associated with the development and progression of glaucoma. Our study aim to identify the potential genes in human trabecular meshwork related to primary open-angle glaucoma (POAG). Overall design: LncRNAs and mRNAs expression profiles in trabecular meshwork from 4 normal controls and 4 POAG patients were determined through microarray analysis
Project description:Objectives:Primary open-angle glaucoma (POAG) is a neurodegenerative disorder leading to a gradual vision loss caused by progressive damage to the optic nerve. Immunological processes are proposed to be involved in POAG pathogenesis. Altered serological autoantibody levels have been frequently reported, but complete analyses of the natural autoantibodies with respect to disease-related alterations are scarce. Here, we provide an explorative analysis of pathways and biological processes that may involve naturally immunogenic proteins and highlight POAG-specific alterations. Methods:Mass spectrometry-based antibody-mediated identification of autoantigens (MS-AMIDA) was carried out in healthy and glaucomatous trabecular meshwork (TM) cell lines, using antibody pools purified from serum samples of 30 POAG patients and 30 non-glaucomatous subjects. Selected antigens were validated by protein microarray (n = 120). Bioinformatic assessment of identified autoantigens, including Gene Ontology (GO) enrichment analysis and protein-protein interaction networks, was applied. Results:Overall, we identified 106 potential autoantigens [false discovery rate (FDR) < 0.01], from which we considered 66 as physiological targets of natural autoantibodies. Twenty-one autoantigens appeared to be related to POAG. Bioinformatic analysis revealed that the platelet-derived growth factor receptor beta (PDGFRB) pathway involved in TM fibrosis was particularly rich in POAG-related antigens. Antibodies to threonine-tRNA ligase (TARS), component 1 Q subcomponent-binding protein (C1QBP) and paraneoplastic antigen Ma2 (PNMA2) showed significantly (P < 0.05) higher levels in POAG patients as validated by protein microarray. Conclusion:This study provides new insights into autoimmunity in health and glaucoma. Bioinformatic analysis of POAG-related autoantigens showed a strong association with the PDGFRB pathway and also increased levels of PNMA2, TARS, and C1QBP autoantibodies in the serum of POAG patients as potential glaucoma biomarkers.
Project description:PURPOSE:To determine whether interleukin-20 receptors (IL-20R) are expressed in trabecular meshwork cells and the effect of a T104M mutation in IL-20R2 on downstream cellular functions. METHODS:Evaluation of signal transducer and activator of transcription (STAT)3 phosphorylation and generic matrix metalloproteinase (MMP) activity in primary open angle glaucoma (POAG) dermal fibroblasts (pHDF) with the T104M IL-20R2 mutation were compared with normal human dermal fibroblasts (HDF). Expression of IL-20R1 and IL-20R2 in human trabecular meshwork (HTM) cells was determined by immunohistochemistry and western immunoblotting. RESULTS:A T104M mutation in IL20-R2 was identified in a large POAG family in which the GLC1C locus was originally mapped. pHDFs harboring this mutation had significantly increased phosphorylated STAT3 (pSTAT3) activity compared with normal HDFs. However, stimulation with either IL-19 or IL-20 for 15?min resulted in significantly decreased levels of pSTAT3 in pHDFs compared with controls. Generic MMP activity was significantly decreased in pHDFs compared with controls after stimulation with IL-20 for 24?h. Both IL-20R1 and IL-20R2 receptors were expressed in HTM cells by western immunoblot and immunofluorescence, and they appeared to be up-regulated in response to cytokine treatment. CONCLUSIONS:A T104M mutation in IL-20R2 significantly impacts the function of this receptor as shown by decreased pSTAT3 levels and generic MMP activity. Reduced MMP activity may affect the ability of glaucoma patients to alter outflow resistance in response to elevated intraocular pressure.
Project description:We compared phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol) profiles of control and glaucomatous trabecular meshwork (TM) derived from human donors.Control TM and most primary open angle glaucoma (POAG) TM were collected from cadaver donors. A select subset of POAG surgical TM samples also were collected for analyses. Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford method, and for select samples confirmed with densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ quantum Access Max triple quadrupole mass spectrometer with precursor ion scan (PIS) or neutral ion loss scan (NLS), using appropriate class specific lipid standards.The comparative profiles of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and phosphatidylinositol between control and glaucomatous TM showed several species common between them. A number of unique lipids in all four phospholipid classes also were identified in control TM that were absent in glaucoma TM and vice versa.A number of phospholipids were found to be uniquely present in control but absent in glaucomatous TM and vice versa. Compared to a previous study of control and POAG blood, a number of these phospholipids are absent locally (TM), as well as systemically (in blood).
Project description:Transforming growth factor beta 2 (TGF??) is often elevated in the aqueous humor (AH) and trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) and appears to contribute to POAG pathogenesis. To better understand TGF?? signaling in the eye, TGF??-induced proteomic changes were identified in cells cultured from the TM, a tissue involved in intraocular pressure (IOP) elevation in glaucoma.Primary cultures of human TM cells from four donors were treated with or without TGF?? (5 ng/mL) for 48 hours; then cellular protein was analyzed by liquid chromatography-mass spectrometry iTRAQ (isobaric tags for relative and absolute quantitation) technology.A total of 853 proteins were quantified. TGF?? treatment significantly altered the abundance of 47 proteins, 40 of which have not previously been associated with TGF?? signaling in the eye. More than half the 30 elevated proteins support growing evidence that TGF?? induces extracellular matrix remodeling and abnormal cytoskeletal interactions in the TM. The levels of 17 proteins were reduced, including four cytoskeletal and six regulatory proteins. Both elevated and decreased regulatory proteins implicate TGF??-altered processes involving transcription, translation, and the glutamate/glutamine cycle. Altered levels of eight mitochondrial proteins support TGF??-induced mitochondrial dysfunction in the TM that in POAG could contribute to oxidative damage in the AH outflow pathway, TM senescence, and elevated IOP.The results expand the repertoire of proteins known to participate in TGF?? signaling, provide new molecular insight into POAG, and establish a quantitative proteomics database for the TM that includes candidate glaucoma biomarkers for future validation studies.
Project description:The receptor-associated prorenin system (RAPS) is associated with several pathologic conditions, including diabetic retinopathy, age-related macular degeneration, and uveitis. Here, we show the involvement of RAPS in the trabecular meshwork (TM) from patients with primary open-angle glaucoma (POAG) and neovascular glaucoma (NVG) due to proliferative diabetic retinopathy. Anterior chamber (AC) levels of prorenin significantly increased in both POAG and NVG, as did those of angiotensin II in NVG alone, compared to cataract. In surgically excised TM tissues, (pro)renin receptor ((P)RR) and angiotensin II type 1 receptor (AT1R) co-localized with prorenin and angiotensinogen, respectively. In screening for various genes related to glaucoma, prorenin stimulation to human TM cells exclusively upregulated cell junction constituents connexin 43 and zona occludens 1, while downregulating an extracellular matrix-degrading enzyme tissue plasminogen activator, all of which were reversed by (P)RR blockade. In contrast, angiotensin II application upregulated a pro-angiogenic factor placental growth factor alone, which was abolished by AT1R blockade. Consistently, (P)RR and AT1R co-localized with these corresponding proteins in patient TM tissues. Oxidative stress, a known etiology for glaucoma, induced the expression of prorenin and angiotensinogen in human TM cells. These data suggest the contribution of RAPS to the molecular pathogenesis of POAG and NVG through TM tissue remodeling and AC angle angiogenesis.