Assessment of Endothelial Progenitor Cells in the Peripheral Blood of Patients with Diabetic Retinopathy

Abstract

Abstract: : Purpose: Previous studies have demonstrated that specific subsets of mononuclear cells which express the CD34 marker appear to be enriched for endothelial cell progenitors or angioblasts. Others have shown that fluorescence-labeled human CD34⁺ cells, when injected into ischemic limbs of mice will incorporate into the neovasculature. If CD34⁺ leukocytes are indeed involved as endothelial progenitors cells (EPC) in patients undergoing active retinal proliferation, an increased amount of circulating CD34⁺ leukocytes may be demonstrated. Cancer researchers have also demonstrated that monocytes/macrophages are involved in inducing endothelial cell proliferation and promoting angiogenic activity. The purpose of this study was to estimate the CD14⁺ and CD34⁺ leukocytes in peripheral blood of diabetic patients, as well as the circulating EPC. We wished to determine if a correlation exists between these cell types and the severity of diabetic retinopathy to devise a simple blood test for diagnosis and/or prognosis of the disease. Methods: Peripheral blood was collected from 28 diabetic patients with varying degrees of retinopathy including: nonproliferative diabetic retinopathy, preproliferative diabetic retinopathy, proliferative diabetic retinopathy (PDR), and involutional PDR post panretinal photocoagulation. Blood was also collected from 7 control patients. 100 µl of peripheral blood was labeled with FITC labeled anti-CD14 antibodies and anti-CD34 antibodies. Samples were then quantified using flow cytometry. Mononuclear cells were also separated from peripheral blood by density centrifugation on Histopaque 1077 and plated into culture wells coated with fibronectin. Cells were labeled to confirm endothelial phenotype by acetylated LDL-DiI uptake and with FITC-Ulex europeus lectin. Positively staining cells were photographed and counted in three microsopic fields. Results: Our analysis did not find a significant difference in the number of circulating monocytes, CD34+ cells and of EPCs between control and diabetic patients. Also, there was no significant difference among diabetics with varying degrees of retinopathy. Conclusion: Despite the negative result, this study is informative regarding the contribution of circulating EPC to diabetic retinopathy, and for the prospects of possible blood tests for its assessment. It remains that more sensitive blood cell assays are to be discovered, the number of patients may need to be increased, and more localized studies both in vivo on diabetic retinas and/or in vitro with longer incubations should be conducted.

Keywords: flow cytometry • diabetic retinopathy • retinal neovascularization