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  • Recently we suggested that KYNA an endogenous GPR agonist co

    2022-05-13

    Recently, we suggested that KYNA – an endogenous GPR35 agonist could be produced in a human cornea since we showed the presence of the enzymes that catalyze the synthesis of KYNA – kynurenine aminotransferases I-III in corneal epithelium, stroma and endothelium (). Therefore, these findings provided plausible ground to suspect the presence of KYNA's target GPR35 in eye structures. We applied three-step approach to study the expression of GPR35 in human cornea; (a) gene expression data mining, (b) immunodetection by means of western blotting and (c) immunohistochemistry. Data on gene expression in human eye layers were extracted from Gene Expression Omnibus (GEO) database using Genevestigator v5.11.05 software by Nebion AG () as described previously (, ). Absolute expression values were plotted using Prism 6 (GraphPad Software). Querying gene expression repositories revealed that gene is expressed at medium levels in the cornea, retina, conjunctiva and uvea of the human eye (A). No data were available on the expression of in other eye structures. Since GPR35 is predominantly expressed in the gastrointestinal tract and immune system, but only minute amounts are observed in the nervous system, microarray data on GPR35 expression in intestine, peripheral blood mononuclear cells (PBMCs) and subcortical tissue were plotted for comparison (A). In order to confirm the expression of the GPR35 on the protein level we carried out an immunoblotting experiment using human cornea and the adjacent sclera tissue. The methods used in this study followed the tenets of the Declaration of Helsinki and the study protocol was approved by Local Bioethics Committee in Lublin, Poland (KE-0254/140/2018). Normal cornea and sclera samples were collected from three individuals (independently for left and right eyes; six corneas and six sclerae in total; all from men; aged 62–74 years). Tissues were obtained from Lublin Eye Bank, Poland. The corneas and scleras were surgically separated. The tissue samples were thinly sliced and homogenized as described previously (), and subsequently subjected to SDS-PAGE and western blotting according to standard protocols (). GPR35 expression was tracked using rabbit primary polyclonal Ethacrynic Acid from Thermo Fisher Scientific (#PA5-23237). The immunoreactive bands were visualized using ChemiDoc Gel Imaging Systems (BioRad). The anti-GPR35 antibody detected two protein species of approximately 34 and 68 kDa, presumably corresponding to the monomeric and dimeric form of the receptor (B). The densitometric analysis revealed that GPR35 exists predominantly as dimer in corneal tissue (C). Moreover, corneas were significantly richer in GPR35 dimers compared to scleras (C). We observed no differences in the expression of the monomeric receptor form between the two tissues (C). This experiment demonstrated that GPR35 is expressed at the protein level within the human cornea as well as within the adjacent sclera. The sclera and cornea are composed of similar structural proteins. The fibroblasts and macrophages are resident cells within the sclera (), while in the cornea there are also epithelium and endothelium. It may be speculated, that a higher cell count in the cornea is responsible for a higher GPR35 expression relative to the sclera. Finally, an immunohistochemical study was performed to gain more insight into the spatial localization of GPR35 within the microanatomical structures of the human cornea. Two human eyes from two patients enucleated due to choroidal malignant melanoma, two corneas with KC and two corneas with FECD were used (total n = 6; three females, three males; age range: 27–85 years). No other ocular diseases were diagnosed in the patients. The study protocol was approved by the Human Ethics Committee of University of Erlangen-Nuernberg, Germany. Following the, surgery all corneas were fixed and processed as described previously (; ). Staining was performed at least twice, using the same antibody as the one for western blotting, and streptavidin-biotin approach described previously (). The final sections were photographed with a microscope (Axiophot, Carl Zeiss) using colour film (Ektachrome 64 T, Eastman Kodak). Immunohistochemistry showed the presence of GPR35 immunoreactivity in all studied corneal sections, including the normal corneas, KC corneas and corneas with FECD (). The corneal endothelium and epithelium exhibited the strongest GPR35 reactivity. Only a slight staining of stroma was detected. Normal corneas and KC corneas showed a similar distribution of GPR35. However, in the sections of corneas with FECD, less intensive immunoreactivity for GPR35 was revealed in the endothelium. Thus, our experiments provide the first evidence for GPR35 expression in the human cornea.