Diabetic retinopathy is one of the most common diabetic complications, and is a major cause of new blindness in the working-age population of developed countries. Progression of vascular abnormalities, including the selective loss of pericytes, formation of acellular capillaries, thickening of the basement membrane, and increased vascular permeability characterizes early nonproliferative diabetic retinopathy (NPDR). Capillary occlusion, as shown on fluorescein angiograms, is also one of the earliest clinically recognizable lesion of NPDR. In response to capillary non-perfusion, there is dilation of neighbouring capillaries, leading to early blood-retinal barrier breakdown, capillary non-perfusion, and endothelial cell injury and death. The resulting ischemia leads to increased production of growth factors, and the development of proliferative diabetic retinopathy (PDR), which is characterized by growth of new vessels and potential severe and irreversible visual loss. The exact pathogenic mechanism by which capillary non-perfusion occurs is still unclear but growing evidence now suggests that increased leukocyte-endothelial cell adhesion and entrapment (retinal leukostasis) in retinal capillaries is an early event associated with areas of vascular non-perfusion and the development of diabetic retinopathy. The leukocytes in diabetic patients are less deformable more activated, and demonstrate increased adhesion to the vascular endothelium. This review summarizes the current literature on the role of leukocytes in the pathogenesis of capillary occlusion, and discusses the potential of leukostasis as a new promising target in the treatment of diabetic retinopathy.
Increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy and atherogenesis in diabetic patients. We recently reported that raised activity of glycosylating enzyme []1,6 acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. Here, we demonstrate that elevated glucose increases the activity of core 2 GlcNAc-T and adhesion of human leukocytes to retinal capillary endothelial cells, in a dose-dependent manner, through diabetes-activated serine/threonine protein kinase C 2 (PKC2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients. Inhibition of PKC2 activation with the specific inhibitor, LY379196, attenuated serine phosphorylation of core 2 GlcNAc-T and prevented increased leukocyte-endothelial cell adhesion. Raised activity of core 2 GlcNAc-T was associated with a threefold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 on the surface of leukocytes of diabetic patients compared with age-matched control subjects. PKC2-dependent phosphorylation of core 2 GlcNAc-T may thus represent a novel regulatory mechanism for activation of this key enzyme in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy. Diabetes 52: 1519 -1527, 2003 D iabetic retinopathy, a leading cause of severe visual loss in type 1 and type 2 diabetic patients (1) is characterized in its early stage by areas of capillary nonperfusion and microvascular damage (2,3). As in atherogenesis (4), increased leukocyteendothelial cell adhesion is a key early event in the development of capillary occlusion in retinopathy (5-10). Leukocytes from diabetic patients are more adhesive to endothelial cells (11), and in experimental diabetes, their increased entrapment in retinal capillaries leads to areas of capillary nonperfusion and endothelial cell damage (12). Increased adhesion of leukocytes in diabetic patients may result from an increased expression of intracellular adhesion molecule (ICAM)-1 on endothelial cells and/or expression of integrins (CD11a, CD11b, and CD18b) on leukocytes (13-16).We recently reported that raised activity of the glycosylating enzyme []1,6-acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy (17). This Golgi enzyme plays a crucial role in the biosynthesis of O-linked glycans by converting core 1 (i.e., Gal (21), T-cell activation (22), inflammation (23), myocardial dysfunction (24,25), capillary morphogenesis (26), and myeloblastic leukemia (27). On the basis that O-linked sugars are also involved in cell-cell interactions (28), we proposed that their modification by raised activity of core 2 GlcNAc-T, together with glucose-induced expression of adhesion molecules on endoth...
The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyteendothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Gal1-3GalNAc␣R-N-acetylglucoaminyltransferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with agematched control subjects (1,638 ± 91 [n = 42] vs. 249 ± 35 pmol · h -1 · mg -1 protein [n = 24], P = 0.00013; 1,459 ± 194 [n = 58] vs. 334 ± 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA 1c (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyteendothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy. Diabetes
A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme core 2  1,6-N-acetylglucosaminyltransferase (GlcNAc-T) through protein kinase C (PKC)2-dependent phosphorylation plays a fundamental role in increased leukocyteendothelial cell adhesion and capillary occlusion in retinopathy. In the present study, we demonstrate that following exposure to plasma from diabetic patients, the human promonocytic cell line U937 exhibits a significant elevation in core 2 GlcNAc-T activity and increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by PKC2-dependent phosphorylation of the core 2 GlcNAc-T protein, were found to be triggered by increased plasma levels of tumor necrosis factor (TNF)-␣. Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of diabetic retinopathy, with the highest values observed upon treatment with plasma of patients affected by proliferative retinopathy. Furthermore, we noted much higher correlation, as compared with control subjects, between increased values of core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-␣ in the development of diabetic retinopathy, these observations further validate the significance of core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.
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