Abstract
Purpose: :
Increasing evidence implicates inflammation in diabetic complications, in particular, retinopathy. Vascular endothelial cells are protected from deposition of autologous C3b on their surfaces by the cell surface regulator DAF (or CD55). Once deposited, C3b not only can serve as the site for further propagation of the complement cascade, but also can function as a ligand for C3b receptors (CR1) and iC3b receptors (CR3) on monocytes/macrophages and other phagocytic cells. To determine whether alterations in DAF–dependent protection against C3 deposition are involved in diabetic retinopathy, we studied 1) whether DAF protein is susceptible to functional inactivation by non–enzymatic glycation, 2) whether diabetic retinopathy develops more rapidly in Daf1–/– mice, and 3) whether modification of DAF protein by glycation adducts can be documented in poorly controlled diabetic patients.
Methods: :
For studying whether DAF is susceptible to glycation, purified DAF protein was incubated with glucose or ribose and the product tested for function in hemolytic assays. For the in vivo studies in Daf1–/– mice, Daf1–/– mice and their Daf1+/+ littermate controls were rendered diabetic with streptozopocin. Enucleated eyes at 6 mo and 12 mo were examined for 1) retinal vascular endothelial changes and 2) caspase 3. For assaying DAF proteins in poorly controlled diabetics, erythrocytes from blood samples and retinal tissue from pathologic specimens were purified by anti–DAF affinity chromatography and examined in Western blots by antibodies specific for different AGEs.
Results: :
Exposure of purified DAF protein to both glucose and ribose inactivated its function. Mass spectrometry analyses and in–gel trypsin digests of the protein showed that the modifications were localized at the junction of CCPs 2–3 and elsewhere in CCP 3, sites shown by alanine substitution mutagenesis to be essential for DAF function. Retinal vascular endothelium of Daf1–/– mice showed marginal adherent leukocytes; whereas, littermate controls demonstrated little change. In preliminary studies, caspase 3 levels were elevated in the Daf1–/– mice relative to controls. Affinity purification of DAF protein from erythrocytes and retinal tissue in poorly controlled diabetic patients showed reactivity with several AGE–specific Abs, in particular, argpyrimidine.
Conclusions: :
These findings indicate that circumvention of C3b deposition on retinal vascular endothelium by DAF plays an important role in protecting against diabetic retinopathy.
Keywords: diabetic retinopathy • immunomodulation/immunoregulation • inflammation