Diabetes Upregulates P-Selectin Glycoprotein Ligand-1 (PSGL-1) Function

Abstract

Purpose: : Capillary occlusion due to leukocyte-endothelial interaction is considered a critical early event in diabetic retinopathy (DR). However, little is known about changes in leukocytes surface molecules during disease. Here, we investigate the expression and function of PSGL-1, the principal rolling ligand of endothelial P-selectin, in experimental diabetes.

Methods: : Diabetes was induced in 150-175mg male Long Evans rats by intraperitoneal injection of 60mg/kg streptozotocin. To investigate leukocyte PSGL-1 on a functional level during diabetes, we utilized our autoperfused micro flow chamber (0.4x0.04mm), coated with 5µg/ml recombinant P-selectin. Blood flow through the chamber allows live microscopy of leukocyte interaction with the immobilized P-selectin. The inlet blood pressure was kept at 40mmHg (18.7dyne/cm2). Leukocyte rolling velocity in the chambers was quantified in 4wks diabetic rats and controls. Furthermore, PSGL-1 expression in peripheral blood leukocytes (PBLs) was analyzed in flow cytometry, using soluble recombinant P-selectin Fc fusion protein (1µg/10⁶) and a secondary FITC-labeled anti-human Fc mAb for staining.

Results: : Leukocyte rolling velocity on immobilized P-selectin was significantly reduced by 51.7% in 4wks diabetic animals (1.4±0.02µm/s) compared to normal controls (2.9±0.42µm/s, n=4 each, p<0.05). Flow cytometry revealed an 11% increase in mean fluorescence intensity (MFI) of PSGL-1 on PBLs of diabetic rats (89.9, n=5) compared to controls (79, n=4).

Conclusions: : Diabetes causes reduced PSGL-1/P-selectin mediated leukocyte-rolling velocity. Our data provide direct evidence for functional PSGL-1 upregulation in experimental diabetes. This novel finding advances our understanding of molecular and cellular events that precede leukostasis and capillary occlusion in DR.

Keywords: diabetes • inflammation • vascular occlusion/vascular occlusive disease