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Wei Li, Nora Blanca Caberoy, Gabriela Alvarado, Hui Wang, Feng Wang, Rui Chen; Systematic identification of diabetic retinopathy-associated endothelial ligands. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4920.
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Endothelial cells (ECs) with direct contact to diabetic blood play critical roles in the initiation and progression of diabetic retinopathy (DR), and are extrinsically regulated by serological ligands. DR-related ligands are the key to delineating disease mechanisms and therapeutic targets but are traditionally identified on a case-by-case basis with technical challenging. This study is to systematically identify DR-related EC ligands by a new approach.
We performed three rounds of in vivo binding selection by intravenously injecting a cDNA library of open reading frame (ORF) phage display into diabetic or control mice to enrich EC-binding clones. After circulating for 20 min, unbound phages were removed by intracardiac perfusion. The retinas were isolated with EC-bound phages, which were released by homogenization, amplified in bacteria and used as input for the next round of selection. After 3 rounds of in vivo selection, the cDNA inserts of enriched phage clones were amplified by PCR and identified by DNA sequencing. The copy number of phage clones as the equivalent of EC-binding activity for the displayed ligands was quantitatively compared between control and diabetic retina to identify DR-related EC ligands.
Sequencing analysis identified hundreds putative endothelial ligands. Quantitative comparison of enriched phage clones for diabetic vs. control retina identified ~660 DR-related EC ligands, including 529 ligands with increased binding activity to diabetic endothelium and 131 with decreased binding (p<0.0001). Among them are fibronectin 1 (Fn1), collagen type IV alpha 3 (Col4a3) and cadherin 1 (Cdh1). All these three molecules are adhesion molecules involved in cell-cell interactions. The copy numbers of these three phage clones identified on diabetic and control retinal endothelium were 419:0, 409:2 and 132:0, respectively. These results are consistent with the upregulation of adhesion molecules in diabetic retinopathy and increased leukocyte adhesion.
This study identified Fn1, Col4a3 and Cdh1 as DR-related endothelial ligands, suggesting that our ORF phage display is a valid approach to systematically identify DR-related EC ligands. This approach will substantially improve our technical capability to delineate the role of EC ligands in DR pathogenesis and exploit their potentials for DR therapy and diagnosis.
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