Bent Honoré

Differences in the protein expression in limbal versus central human corneal epithelium - a search for stem cell markers.

In the search for potential limbal stem cell protein markers, the purpose of this study was to characterize differences in protein expression between human central and limbal corneal epithelium by a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry (LC-MS/MS). The results were subsequently confirmed by Western blotting and immunohistochemistry. We detected more than 1000 protein spots in each gel. Thirty-two spots were significantly over-expressed in the central part and 70 spots were significantly over-expressed in the limbal part. We identified 25 different proteins. Among these 11 proteins representing different cellular locations and functions were selected for further investigations. Most interestingly, superoxide dismutase 2 (SOD2), was expressed in clusters of cells in the basal limbal epithelium. Heat shock protein 70 protein 1 (HSP70.1) and annexin I were highly abundant in limbal epithelium, although they were also present in the central epithelium to a minor extent. Among the proteins primarily expressed in the limbal fraction we further identified cytokeratin (CK) 15, CK19 and alpha enolase, which have been reported previously to be related to the limbal basal epithelium. The basal limbal epithelium consists of clusters of slow cycling limbal stem cells and rapid cycling transient amplifying cells. Ideally, proteins exclusively expressed in the limbal part of the epithelium may serve as markers for the basal limbal cells. SOD2 and CK15 identify clusters of limbal basal cells and therefore they may serve as markers for limbal stem cells in conjunction with the earliest transient amplifying cells.

Dye-Free Porcine Model of Experimental Branch Retinal Vein Occlusion: A Suitable Approach for Retinal Proteomics

Branch retinal vein occlusion induces complex biological processes in the retina that are generated by a multitude of interacting proteins. These proteins and their posttranslational modifications can effectively be studied using modern proteomic techniques. However, no method for studying large-scale protein changes following branch retinal vein occlusion has been available until now. Obtainment of retinal tissue exposed to branch retinal vein occlusion is only available through experimental animal models. Traditional models of experimental branch retinal vein occlusion require the use of Rose Bengal dye combined with argon laser photocoagulation. The use of Rose Bengal dye is problematic in proteomic studies as the dye can induce multiple protein modifications when irradiated. This paper presents a novel technique for proteomic analysis of porcine retinal tissue with branch retinal vein occlusion combining a dye-free experimental model with label-free liquid chromatography mass spectrometry based proteomics.

Proteins Involved in Focal Adhesion Signaling Pathways are Differentially Regulated in Experimental Branch Retinal Vein Occlusion

Branch retinal vein occlusion (BRVO) is a common retinal vascular disease, but global protein changes following the condition remain largely unelucidated. To bring new insights into pathological processes and identify potential therapeutic targets, large-scale retinal protein changes following BRVO were studied by combining a porcine model of experimental BRVO with proteomic analysis by label-free liquid chromatography mass spectrometry. Among a total set of 1974 proteins, 52 significantly upregulated proteins and 10 significantly downregulated proteins were identified in retinas with BRVO after 15 days. Significantly upregulated proteins were involved in signaling pathways of focal adhesion via integrin and blood coagulation. Proteins involved in focal adhesion signaling included collagen α-2 chain, laminin subunit β-2, laminin subunit γ-1, lipocalin-7, nidogen-2, osteopontin, integrin-β, α-actinin-1, isoform 2 of α-actinin-1, talin-2 and filamin C. The identified proteins indicate that BRVO was associated with extracellular matrix remodeling processes. The present study identified focal adhesion signaling and ECM remodeling as important biological mechanisms to evaluate in the search for signaling pathways that promote neovascularisation and macular edema following BRVO.

Retinal proteome changes following experimental branch retinal vein occlusion and intervention with ranibizumab

Animal models of experimental branch retinal vein occlusion (BRVO) provide a unique opportunity to study protein changes directly in retinal tissue. Results from these experimental models suggest that experimental BRVO is associated with an upregulation of extracellular matrix remodeling and adhesion signaling processes. To study whether these processes could be blocked by inhibition of VEGF-A, a porcine model of experimental BRVO was combined with proteomic analyses. In six Danish Landrace pigs experimental BRVO was induced with argon laser in both eyes. After 24xa0h an injection of 0.05xa0mL ranibizumab was given in the right eyes of the animals while left eyes received an injection of 0.05xa0mL 9xa0mg/mL sodium chloride water. Retinas were dissected three days after BRVO and the retinal samples were analyzed with label-free quantification as well as tandem mass tag based proteomics. In retinas treated with ranibizumab five proteins exhibited statistically significant changes in content with both proteomic techniques. These five proteins, which were all decreased in content, included integrin β-1, peroxisomal 3-ketoacyl-CoA thiolase, OCIA domain-containing protein 1, calnexin and 40S ribosomal protein S5. As anti-integrin therapies are under development for inhibition of angiogenesis in retinal diseases it is interesting that inhibition of VEGF-A in itself resulted in a small decrease in the content of integrin β-1. The decreased content of integrin β-1 indicates that extracellular matrix remodeling and adhesion processes associated with BRVO are at least partly reversed through inhibition of VEGF-A.

Dexamethasone intravitreal implant downregulates PDGFR-α and upregulates caveolin-1 in experimental branch retinal vein occlusion

Abstract A dexamethasone (DEX) intravitreal implant (OZURDEX) provides an effective treatment of inflammation secondary to branch retinal vein occlusion (BRVO). Retinal proteome changes which mediate the beneficial effects of the implant remain poorly understood. To study retinal proteome changes in BRVO following an intervention with a DEX implant this study combined an experimental model of BRVO with proteomic techniques. In eight Danish Landrace pigs experimental BRVO was induced in both eyes using argon laser. After inducing BRVO a DEX implant was injected into the right eye of each animal while the left control eye was given an identical injection without an implant. Fifteen days after BRVO and DEX implant intervention the retinas were excised and analyzed with tandem mass tag based mass spectrometry. A total of 26 significantly changed proteins were identified. DEX intervention reduced the retinal levels of platelet‐derived growth factor receptor‐&agr; (PDGFR‐&agr;) and vascular endothelial growth factor receptor 2 (VEGFR‐2). DEX treatment resulted in increased levels of caveolin‐1, peptidyl‐prolyl cis‐trans isomerase FKBP5 and transgelin. Changes in PDGFR‐&agr; and caveolin‐1 were confirmed with immunohistochemistry. In BRVO treated with the DEX implant a strong reaction for caveolin‐1 was observed in the innermost retinal layers. DEX implant intervention may inhibit PDGF signaling by decreasing the retinal level of PDGFR‐&agr; while an increased content of caveolin‐1 may help maintain the integrity of the blood‐retinal barrier. Graphical abstract Figure. No Caption available. HighlightsProteome changes in experimental BRVO after dexamethasone intervention were studied.Dexamethasone intervention decreased the level of retinal PDGFR‐&agr;.A small decrease in VEGFR‐2 following dexamethasone treatment was observed.Caveolin‐1 was increased following dexamethasone treatment.The increase in caveolin‐1 was pronounced in the inner retinal layers.