Project description:Aqueous humor (AH) is the fluid in the anterior and posterior chambers of the eye that contains proteins regulating ocular homeostasis. Analysis of aqueous humor proteome is challenging, mainly due to low sample volume and protein concentration. In this study, by utilizing state of the art technology, we performed Liquid-Chromatography Mass spectrometry (LC-MS/MS) analysis of 88 aqueous humor samples from subjects undergoing cataract surgery. A total of 2263 unique proteins were identified, which were sub-divided into four categories that were based on their detection in the number of samples: High (n = 152), Medium (n = 91), Low (n = 128), and Rare (n = 1892). A total of 243 proteins detected in at least 50% of the samples were considered as the constitutive proteome of human aqueous humor. The biological processes and pathways enriched in the AH proteins mainly include vesicle mediated transport, acute phase response signaling, LXR/RXR activation, complement system, and secretion. The enriched molecular functions are endopeptidase activity, and various binding functions, such as protein binding, lipid binding, and ion binding. Additionally, this study provides a novel insight into race specific differences in the AH proteome. A total of six proteins were upregulated, and five proteins were downregulated in African American subjects as compared to Caucasians.
Project description:The purpose of this study is to discover genes that might increase aqueous humor outflow when human ciliary muscle or human trabecular meshwork cells are treated with the prostaglandin analogues latanoprost free acid or prostaglandin F2alpha. Five tissue donors were pooled on each chip. Keywords: other
Project description:A miRNA PCR array comprising 84 miRNAs was used to analyze the AH (glaucoma, n=3; control, n=3) and LC samples (glaucoma, n=3; control, n=4). Expression levels of 19 and 3 miRNAs were significantly upregulated in the AH and LC samples of the glaucoma group, respectively (p < 0.05).
Project description:Glaucoma is the leading cause of irreversible blindness worldwide. The proteome characterization of glaucoma is not clearly understood. A total of 175 subjects, including 57 primary acute angle-closure glaucoma (PAACG), 50 primary chronic angle-closure glaucoma (PCACG), 35 neovascular glaucoma (NVG), and 33 cataract patients, were enrolled and comparison proteomic analysis was provided. The samples were randomly divided into discovery group or validation group, whose aqueous humor proteome was analyzed by data-independent acquisition or by parallel reaction monitoring. The common proteome features of three types of glaucoma were immune response, lipid metabolism, and cell death. Three proteins, VTN, SERPIND1, and CD14, showed significant upregulation in glaucoma and could discriminate glaucoma from cataract. Mutual differential proteomic analysis of PAACG, PCACG, and NVG showed different proteome characterization of the three types of glaucoma. NVG was characterized with activated angiogenesis. PAACG was characterized with activation of inflammation response. SERPIND1 was discovered to play vital role in glaucoma occurrences, which is associated with eye transparency decrease and glucose metabolism. This study would provide insights in understanding proteome characterization of glaucoma and benefit the clinical application of AH proteome.
Project description:PurposeHuman aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH.MethodshAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified.ResultsA total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LC-ESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESI-MS/MS and antibody protein arrays.ConclusionsProteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.
Project description:In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving aqueous humor outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.
Project description:PurposeTrabecular meshwork (TM) bypass surgeries attempt to enhance aqueous humor outflow (AHO) to lower intraocular pressure (IOP). While TM bypass results are promising, inconsistent success is seen. One hypothesis for this variability rests upon segmental (non-360 degrees uniform) AHO. We describe aqueous angiography as a real-time and physiologic AHO imaging technique in model eyes as a way to simulate live AHO imaging.MethodsPig (n = 46) and human (n = 6) enucleated eyes were obtained, orientated based upon inferior oblique insertion, and pre-perfused with balanced salt solution via a Lewicky AC maintainer through a 1mm side-port. Fluorescein (2.5%) was introduced intracamerally at 10 or 30 mm Hg. With an angiographer, infrared and fluorescent (486 nm) images were acquired. Image processing allowed for collection of pixel information based on intensity or location for statistical analyses. Concurrent OCT was performed, and fixable fluorescent dextrans were introduced into the eye for histological analysis of angiographically active areas.ResultsAqueous angiography yielded high quality images with segmental patterns (p<0.0001; Kruskal-Wallis test). No single quadrant was consistently identified as the primary quadrant of angiographic signal (p = 0.06-0.86; Kruskal-Wallis test). Regions of high proximal signal did not necessarily correlate with regions of high distal signal. Angiographically positive but not negative areas demonstrated intrascleral lumens on OCT images. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways.ConclusionsAqueous angiography is a real-time and physiologic AHO imaging technique in model eyes.
Project description:The aqueous humor is a colourless, transparent fluid that fills the anterior chamber of the eye. It has an important role to play in maintaining the intraocular pressure of the eye and providing nourishment to avascular structures such as the lens and cornea. It is also essential for providing a clear path for the entry and exit of light from the eye and maintaining the refractive power of the eye. In this study, 250 samples of aqueous humor were collected from patients undergoing cataract surgery. The samples were pooled, divided into aliquots and subjected to ingel digestion, insolution digestion followed by strong cation exchange chromatography and basic reverse phase liquid chromatography fractionation. MS/MS analysis was carried out on a Fourier transform LTQ-Orbitrap velos mass spectrometer and the data were searched with the search algorithms Mascot, SEQUEST and X!Tandem against the NCBI RefSeq human protein database. We have identified 818 proteins of which 447 have been identified for the first time. Proteins such as sorbitol dehydrogenase, filensin and phakinin were some of the novel proteins identified in this study. Classification of proteins based on domain information yielded 304 proteins with signal peptides, 46 proteins with transmembrane domain and 75 proteins with both signal peptide and transmembrane domains. Our study provides a detailed profile of the human aqueous humor. The results from this study of aqueous proteins should help facilitate research into pathological conditions of the eye such as glaucoma, myopia, cataract and uveitis.
Project description:Primary open-angle glaucoma (POAG) is characterized by degeneration of retinal ganglion cells associated with an increase in intraocular pressure (IOP) due to hindered aqueous humor (AH) drainage through the trabecular meshwork and uveoscleral pathway. Polyunsaturated fatty acids and oxylipins are signaling lipids regulating neuroinflammation, neuronal survival and AH outflow. Among them, prostaglandins have been previously implicated in glaucoma and employed for its treatment. This study addressed the role of signaling lipids in glaucoma by determining their changes in AH accompanying IOP growth and progression of the disease. Eye liquids were collected from patients with POAG of different stages and cataract patients without glaucoma. Lipids were identified and quantified by UPLC-MS/MS. The compounds discriminating glaucoma groups were recognized using ANCOVA and PLS-DA statistic approaches and their biosynthetic pathways were predicted by bioinformatics. Among 22 signaling lipids identified in AH, stage/IOP-dependent alterations in glaucoma were provided by a small set of mediators, including 12,13-DiHOME, 9- and 13-HODE/KODE, arachidonic acid and lyso-PAF. These observations correlated with the expression of cytochromes P450 (CYPs) and phospholipases A2 in the ocular tissues. Interestingly, tear fluid exhibited similar lipidomic alterations in POAG. Overall, POAG may involve arachidonic acid/PAF-dependent pathways and oxidative stress as evidenced from an increase in its markers, KODEs and 12,13-DiHOME. The latter is a product of CYPs, one of which, CYP1B1, is known as POAG and primary congenital glaucoma-associated gene. These data provide novel targets for glaucoma treatment. Oxylipin content of tear fluid may have diagnostic value in POAG.