Leukocytes in Diabetic Retinopathy - Semantic Scholar€¦ · Rakesh Chibber*, Bahaedin M. Ben-Mahmud, Surina Chibber and Eva M. Kohner Cardiovascular Division, GKT School of Biomedical

Current Diabetes Reviews, 2007, 3, 3-14 3

1573-3998/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Leukocytes in Diabetic Retinopathy

Rakesh Chibber*, Bahaedin M. Ben-Mahmud, Surina Chibber and Eva M. Kohner

Cardiovascular Division, GKT School of Biomedical & Health Sciences, King's College London, Guy’s Campus,

London, UK

Abstract: 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.

Keywords: Diabetes, Retinopathy, Leukocytes.

1. INTRODUCTION

Diabetic retinopathy is a leading cause of blindness in working age people [1]. Research and advances in treatment have greatly reduced the risk of blindness from retinopathy, but because of the growing diabetic population, retinopathy remains an important problem. During the

first two decades

of disease, nearly all patients with type 1 diabetes and >60%

of patients with type 2 diabetes have retinopathy. Duration of

diabetes, pregnancy, and onset of puberty, blood glucose

control, hypertension, serum lipid levels, and cataract sur-gery are risk factors that may contribute to the progression of diabetic retinopathy [2-3]. The present strategies of care for diabetic retinopathy as supported by large, prospective, randomised clinical trials include good metabolic control, tight control of blood pressure (BP) and laser photoco-agulation for retinal neovascularization and clinically signi-ficant macular edema. Vitrectomy, a microsurgical pro-cedure, clears media opacities; relieve retinal traction, and makes adequate laser treatment of the retina possible.

2. CLASSIFICATION AND NATURAL PROG-RESSION

Damage to blood vessels of the retina is the cause of dia-betic retinopathy. According to the International Clinical Diabetic Retinopathy Disease Severity Scale [4], the classi-fication includes no retinopathy, nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). It progresses from mild NPDR, to moderate and

*Address correspondence to this author at the Cardiovascular Division, GKT School of Biomedical & Health Sciences, 2nd Floor, New Hunt's House, King's College London, London, SE1 1UL, UK; Tel: 0044-(020) 7848-6213; Fax: 0044-(020) 7848-6220; E-mail: [email protected]

severe NPDR, and finally to PDR, characterized by the growth of new blood vessels [5-6] which are fragile and tend to leak (Fig 1B). NPDR is graded as mild, moderate and severe. In mild NPDR, at least one microaneurysm, and dot, blot or flame-shaped haemorrhages are present in one of the fundus quadrants. In moderate NPDR, numerous micro-aneurysms, retinal haemorrhages, and dot and blot haemorr-hages of greater severity than mild NPDR (Fig 1A), are present in one to three quadrants. Cotton wool spots, venous beading, and intraretinal microvascular abnormalities (IRMAs) if present are mild. Patients with moderate NPDR have a risk of 12 to 27% for progression to PDR within 1 year. Severe NPDR is characterized by any one of the following “4-2-1 rule” a) ‘severe’ haemorrhages and micro-aneurysms in all four quadrants of the fundus, b) venous beading, which is more marked in at least two quadrants, and c) IRMAs, which are more severe in at least one quadrant.

Diabetic maculopathy (exudative, edematous, or ischemic) (Fig 1C) may be associated with NPDR and PDR. The uncontrolled growth of new blood vessels and macular edema are the main causes of visual loss in type 1 and type 2 diabetes [7].

3. PATHOGENIC MECHANISMS

The pathogenesis of diabetic retinopathy remains unclear, but results from the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have confirmed that hyperglycaemia

is the

major factor in its development [8-9]. The prevailing bio-chemical theories on how high glucose leads to retinopathy include increased polyol (sorbitol/aldose reductase) pathway flux, hexosamine pathway, accelerated formation of ad-vanced glycation end-products (AGEs), haemodynamics

4 Current Diabetes Reviews, 2007, Vol. 3, No. 1 Chibber et al.

changes, oxidative stress, and activation of diacylglycerol and protein kinase C beta (PKC ) isoforms [10-12]. To date, clinical testing of these pathogenic mechanisms has not established an effective drug based therapy for the treatment of diabetic retinopathy. The most recent interest has been with the PKC 1/2 inhibitor, ruboxistaurin. However, results from a large randomised, placebo-controlled, multi-centre clinical trial suggested no significant beneficial effect of ruboxistaurin on the progression of retinopathy or the need for laser photocoagulation, but treatment did appear to reduce the relative risk of moderate visual [13]. A recent study has also underscored the importance of the hexosamine pathway, AGEs, and PKC in diabetic retinopathy. The lipid-soluble thiamine derivative benfotiamine, which acts by activating the pentose phosphate pathway enzyme trans-ketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars, has been reported to prevent the development of retinopathy in diabetic rats [14]. However, it is difficult to extrapolate these initial early encouraging results to humans without further research to determine effectiveness, long-term safety and pharmacokinetics.

Inflammation and the activation of microglia, which are resident macrophages and immune cells in the central ner-

vous system (CNS) and the retina, has also been implicated in the pathogenesis of diabetic retinopathy [15]. Early evi-dence suggests that minocycline, an antibiotic with anti-in-flammatory properties has the potential to block activation of microglia, and prevent diabetic retinopathy [15]. Adminis-tration of minocycline reduced diabetes-induced inflam-matory

cytokine production, reduced the release of cyto-

toxins from activated microglia, and significantly reduced

caspase-3 activity within the retina. The results from this

novel study suggested that minocycline could be a strong candidate for further consideration as a therapeutic drug in reducing the retinal complications of diabetes. Further studies are still necessary to test the prediction that minocycine will reduce damage to the retina. In addition to these and other mechanisms [16], there is now growing evidence that increased leukocyte adhesion to the endothelial wall and entrapment (leukostasis) is a key early mechanism in the development of diabetic retinopathy. This review will sum-marise evidence for this mechanism with special attention to the role of leukocyte carbohydrates surface changes in the pathogenesis of diabetic retinopathy.

4. LEUKOCYTES AND RETINOPATHY

As shown by fluorescein angiography (Fig. 2), an early clinical feature of diabetic retinopathy is the occlusion of

Fig. (1). Natural progression of diabetic retinopathy. (A) Superior temporal region of left eye shows severe nonproliferative

(preproliferative) diabetic retinopathy with venous beading, IRMA, cotton wool spots and haemorrhages. (B) Same as in (A) but 10 months

later showing new vessels and a large haemorrhage arising from the new vessels. (C) Left macular region showing multiple hard exudates

(exudative maculopathy).

Fig. (2). Capillary occlusion in early diabetic retinopathy. (A) Colour photograph of right macular region of moderate nonproliferative

diabetic retinopathy. There are multiple microaneurysms, haemorrhages and a few soft and hard exudates. (B) Fluorescein angiogram of area

shown in (A). Note large areas of capillary nonperfusion, around and lateral to macula.

Leukocytes in Diabetic Retinopathy Current Diabetes Reviews, 2007, Vol. 3, No. 1 5

capillaries [17-18], although the pathogenic mechanism whereby this arises remains unclear. Changes in retinal blood flow [19]; blood cell components (erythrocytes, platelets) and blood viscosity [20-23] could play some role. Vascular endothelial growth factors (VEGF)-induced endot-helial cell hypertrophy and resulting narrowing of the retinal

capillary has also suggested as a possible cause of capillary occlusion [24]. Studies in the last decade have also suggested that leukocytes may play an important role in the develop-ment of diabetic retinopathy [25-31] (Fig. 3). There is now general acceptance that retinopathy is a low-grade chronic

A

Fig. (3). Increased leukocyte entrapment (leukostasis) in human diabetic retinopathy. (A) Schematic diagram of increased leukocyte-

endothelial cell adhesion and entrapment (leukostasis) in diabetic retinopathy. Diabetes-induced expression of adhesion molecules, such as

intracellular adhesion molecule-1 (ICAM-1), and P-selectin causes increased leukocyte-endothelial cell adhesion, localized production of

reactive oxygen species (ROS), capillary nonperfusion, and the vascular damage. (B) Evidence showing two neutrophils (arrows) entrapped

on either end of a vascular segment that lacks ADPase activity from a spontaneously diabetic monkey. Arrowhead shows a branch of the

capillary devoid of ADPase activity. (C) A neutrophil is present in an aneurysm that has formed at a branch point in a capillary of a

spontaneously diabetic monkey. (D) Brightfield illumination showing neutrophils (arrows) in a capillary segment adjacent to a nonperfused

area (arrowhead) in spontaneously diabetic monkey. Retinal artery, and vein are represented by a, and v, respectively. (E) Darkfield

illumination of the same area in (D) demonstrating reduction in ADPase activity in some of the capillaries in this area of capillary dropout.

(F) Leukocytes stained with lectin in the arteriole of a diabetic rat. (G) Areas of capillary nonperfusion occur downstream from adherent

leukocytes in retinal capillaries of diabetic rats. Photographs B, C, D & E are adapted from Kim et al. [36] with kind permission of Dr Gerald

Lutty (Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, MD 21287-9115, USA). Photographs F & G are adapted from Joussen

et al. [30] with kind permission of Dr. AP Adamis (Eyetech Research Center, Woburn, Massachusetts, USA).


Fig. (4). Leukostasis in human diabetic retinopathy. Leukocytes (arrows) tracked by scanning laser ophthalmoscope (SLO) normal (A) and

diabetic patients (B). Photographs (unpublished data) are adapted with kind permission of Dr John Forrester (Department of Bio-Medical

Physics and Bio-Engineering, Forresterhill, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK).

inflammatory condition associated with increased leukocyte

entrapment in retinal capillaries, and areas of capillary non-perfusion and endothelial cell damage [32]. The recently reported Hoorn Study [33] has provided clinical evidence for the close association between retinopathy and inflammation. The study measured the levels of C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), von Willebrand factor, and soluble vascular adhesion molecule-1 (sVCAM-1), together with the urinary albumin: creatinine ratio in subjects with and without type 2 diabetes. Results revealed that the inflammatory activity and endothelial dys-function are strongly associated with retinopathy, suggesting their involvement in the onset and progression of retinopathy [33]. This has been further supported by Meleth et al. [34] showing a close association between serum inflammatory markers and cell adhesion molecules with severity of diabetic retinopathy.

Evidence from animal studies suggests that increased leukocyte-endothelial cell adhesion and entrapment (leuko-stasis) is an early event in diabetic retinopathy [21-28] leading to the breakdown of the blood-retinal barrier (BRB), endothelial cell damage, and capillary non-perfusion [32-33]. In a rat model of diabetic retinopathy, areas of capillary occlusion, endothelial cell damage, and capillary loss was closely with associated entrapped leukocytes [34].

There is also compelling evidence for the involvement of leukocytes in diabetic retinopathy from studies using human tissue that demonstrate a strong relationship between leukocyte-endothelial cell adhesion and choroidal capillary damage in diabetes [35]. The adhesion and accumulation of leukocytes in the retinal microvessels is rapid and happens within a week from the onset of diabetes [28-29]. More recently, Kim et al. [36] have provided further strong evidence for the role of leukocytes in the development of diabetic retinopathy. The authors demonstrated a close association between the severity of diabetes and the development

of retinopathy with increased numbers of

leukocytes in the retina of spontaneously diabetic monkeys

[36] (Fig. 3D). The number and site of neutrophil accu-mulation was

associated with loss in capillary viability, as

evidenced by the loss in ADPase, capillary dropout, and the

formation of microaneurysms [36]. The leukocyte population appears to involve the monocytes, neutrophils, and T-lymphocytes [25, 37]. Consistent with the observations in the animal models of diabetes, there is also some evidence of increased number of leukocytes in the human diabetic retina (Fig. 4; unpublished data kindly provided by Dr. John Forrester, Aberdeen, Scotland).

Leukocyte-endothelial cell interaction appears to be the underlying mechanism in active role for leukocytes in tight junction disruption and BRB breakdown during retinal inflammation [38]. A recent study showing that argatroban, a direct thrombin inhibitor, suppresses both leukocyte-endo-thelial cell interactions and BRB breakdown after scatter laser photocoagulation, supports this association [39]. Furthermore, injection of corticosteroids was recently shown to prevent diabetes-induced leukocyte accumulation in the retina, BRB breakdown, and the expression of intracellular adhesion molecule-1 (ICAM-1) [40]. High dose of aspirin also potentially prevents some clinical features of retino-pathy (formation of acellular capillaries, retinal haemorr-hages) in diabetic dogs. Although not significant, aspirin also prevented the loss of pericytes and formation of micro-aneurysms [41]. These findings are consistent with results from a recent study testing the potential of non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin to prevent the development of diabetic retinopathy [42]. High dose of aspirin, meloxicam

(cyclo-oxygenase-2 inhibitor),

and etanercept (soluble tumour necrosis factor- receptor)

significantly prevented the expression of retinal ICAM-1, leucocyte adhesion, and BRB breakdown in diabetic rats [42].

The pathogenic mechanisms mediating abnormal leuko-cyte-endothelial cell adhesion include increased expression of cell adhesion molecules on the surface of endothelial cells, and changes on the leukocyte surface. Leukocyte


adhesion to the vascular endothelium, a key step in inflam-mation, involves three steps in leukocyte sequestration: rolling, firm adhesion and transmigration. The adhesion of leukocytes to the vascular endothelium is largely dependent upon interactions between endothelial cell and leukocyte expressed adhesion molecules [43]. An initial and key step in leukocyte recruitment is the “low-affinity” adhesion medi-ated by three members of the selectin family - E-, P-, and L-selectins. E-selectin is expressed on endothelial cells, P-selectin is stored in preformed cytosolic storage granules which are called Weibel–Palade bodies, and is expressed on both platelets and endothelial cells surface, and L-selectin is expressed on leukocyte surface is located on the tips of microvilli [44-45]. This low-affinity adhesion between the selectins and their counter-receptors initially captures and tethers the leukocyte to the vessel wall and, in the context of vascular shear flow, cause the tethered leukocytes to roll along the vascular endothelium. Selectin-dependent tethering and rolling brings the leukocyte into close physical proxi-mity to the vessel wall. This process facilitates activation of leukocytes, via chemokines, which is elaborated by the endo-thelium, the vessel wall and peri-vascular structures [46-47], and which engage leukocyte-borne chemokine receptors.

4.1. Endothelial Cell Changes

Endothelial cell surface glycoproteins such as, intra-cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) facilitate the attachment of leukocytes to the endothelium and are key mediators of the low-grade inflammation [44-45]. Support for diabetes-induced changes in the adhesion molecules on the endo-thelial cells comes from studies using cells derived from large vessels. However, most of the studies do suggest that exposure to high glucose promotes leukocyte-endothelial cell adhesion by upregulating cell surface adhesion molecules, such ICAM-1, E-selectin, P-selectin, and CAM-1, ELAM-1 [48-52]. Recently, Hirata et al. [53] confirmed that high glucose also enhances leukocyte-endothelial adhesion on cultured human retinal capillary endothelial cells (HREC) through increased ICAM-1 expression. In addition to glucose, insulin also appears to increase neutrophil–endothelial cell adhesion via ICAM-1 expression on human umbilical endothelial cells (HUVEC) [54].

The close associations between increased numbers of T lymphocytes and high expression of ICAM-1 in ocular tissues, suggests an important role of these factors in development of diabetic retinopathy [55]. Intermittent high glucose seems to enhance the ICAM-1, VCAM-1, E-selectin expression in endothelial cells [56]. This is particularly important since fluctuations in blood glucose are thought to play a direct

and significant role in the pathogenesis of

vascular diabetic complications [57-58].

The underlying mechanisms in glucose-mediated leuko-cyte-endothelial cell adhesion remain to be elucidated, but according to Morgi et al. [48], it seems to involve the nuclear factor- B (NF- B) and protein kinase C (PKC) intracellular signalling pathways. The role of PKC in the glucose-induced expression of adhesion molecules (ICAM-1, P-selectin, E-selectin) has also been supported by studies showing that inhibition of PKC activity reduces leukocyte-endothelium interactions by suppressing surface expression

of endothelial cell adhesion molecules in response to increased oxidative stress [59-60]. The activation of PKC and increased ICAM-1 expression also appears to be the underlying mechanism in the glucose-induced neutrophil adhesion to human retinal endothelial cells [53].

Phosphorylation of Ser-660 by PKC 2 seems to represent a selective regulatory mechanism for glucose-induced up regulation of VCAM-1 [61]. This finding suggests that PKC 2-selective inhibitors may be promising drugs for treatment of endothelial dysfunction during acute hyperglycaemia and possibly in diabetes. Interestingly, glica-zide (sulfonylurea), epalrestat (aldose reductase inhibitor), pravastatina and fluvastatin (statins) prevent glucose-induced leukocyte-endothelial cell adhesion by inhibiting the expression of endothelial adhesion molecules [62-64].

It is also conceivable that instead of glucose, higher levels of TNF- may up-regulate expression of adhesion molecules, such as E-selectin, ICAM-1 and VCAM-1 [65-67], on the surface of endothelial cells and cause leukocyte adhesion to the diabetic retinal vasculature. Its actions include the translocation of selectins and integrins on leukocytes and protein synthesis for the expression of E-selectin, ICAM-1, or VCAM-1 on endothelial cells [68-69]. The contribution of TNF- to the pathogenesis of diabetic retinopathy is clearly supported by a number of reports [70-76], and significantly higher levels of TNF- are found in the plasma of patients affected by either type 1 or type 2 diabetes versus age-matched healthy control subjects [77-80].

Further support for the abnormalities on the endothelial cell surface comes from the immunohistochemical demon-stration of increased expression of ICAM-1 in response to hyperglycaemia

in both human [81] and experimental models

[28]. Additional support comes from the observation that a monoclonal antibody against ICAM-1 partially reverses the increased leukocyte adhesion (leukostasis) in experimental diabetic animals [28]. Moreover,

ICAM-1 knockout mice

made diabetic do not develop the expected retinal vascular

changes evident in their wild type counterparts [82]. How-

ever, the recent study by Hughes et al. [83] appears to question the involvement of ICAM-1 in diabetic retinopathy. The authors carried out an immunohistochemical study

of

human specimens, and found that in contrast with previous

studies, that there was little difference in the expression of

ICAM-1 between normal and diabetic retinas. However, their studies did appear to demonstrate diffuse ICAM-1 staining of neural retina that was increased in the diabetic specimens and appeared to correlate

with local breakdown of

the blood-retinal barrier.

Interestingly, higher serum concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1, and sELAM-1) have been detected in diabetic patients [84-85], and correlated with hyperglycaemia and augmented oxidative stress [86-87].

4.2. Leukocyte Changes

The leukocytes are relatively large with high cytoplasmic rigidity [88-89], and often completely fill the capillary lumen; thus, significantly higher forces are needed to deform them during their passage through capillaries. Early studies


suggested that leucocytes, particularly polymorphonuclear (PMN) leukocytes in diabetes have decreased deformability and thus the potential to damage retinal capillaries [88-89]. This decreased deformability along with small lumen dia-meter of retinal capillaries (3.5 - 6 m), and increased ex-pression of ICAM-1 [28] may contribute to the sustained leukocyte entrapment in diabetes. In addition, there is a suggestion of a link between elevated leukocyte count that is within the

normal range, and the development of micro and

macrovascular complication in diabetes [90].

Leukocytes from diabetic patients also release enhanced amounts of reactive oxygen species (ROS) when compared with normal cells [91-95] possibly via PKC-mediated acti-vation of NADPH oxidase [96]. NADPH oxidase is a multi-component,

membrane-associated, enzyme that catalyses the

one electron reduction of oxygen to superoxide anion (O2-)

using NADPH as the

electron donor [97]. Subunits of NADPH oxidase components include gp91phox,

p22phox,

p40phox, p47phox, and p67phox. While gp91phox

and p22phox are present in the plasma membrane and bind the components

of the electron transport chain heme and FAD,

forming cytochrome b558 [98-99], p40phox, p47phox,

and

p67phox are cytosolic and are involved in activation of the

enzyme complex. Exposure to high glucose increases the expression of p47phox [100] and/or PKC-mediated phos-phorylation of p47phox [101]. There is evidence that neutro-phils from diabetic subjects contain elevated diglycerides [95] and increased activation of PKC in vivo [102] and in vitro [103], particularly PKC 2 [96]. The localized release of ROS, particularly O2- by entrapped leukocytes could potentially damage endothelial cells and pericytes (Fig. 3). This would support the role of oxidative stress in diabetic complications, including retinopathy [104].

Studies in vitro have also shown that monocytes isolated from diabetic patients are more adhesive to cultured human endothelial cells than those from healthy control subjects and that this leukocyte-endothelial cell adhesion is CDII-CD18 dependent [105]. The observation that direct inhibition of C18 bioactivity prevents retinal

leukocyte adhesion in

diabetic animals, not only suggests the importance of leukocyte surface changes in diabetic retinopathy [29]. Circulating lymphocytes are also changed in the diabetic patients with retinopathy showing increased adhesion to human endothelial cells [37].

To explain why leukocytes in diabetic patients are more adhesive to endothelial cells, we proposed modification of cell surface carbohydrates (O-glycans) expressed on leuko-cyte surface and control cell adhesion events. The O-glycans that have been shown to play an important role in the initial recruitment of leukocytes to the site of inflammation [106-108], serve as ligands for the selectins that mediate tethering and rolling

of leukocytes on activated endothelial cells [109-

110]. In cells there are variety of O-glycans [111] and those with a 1,6-GlcNAc branch, namely the

core 2-type, are

extended with poly (N-acetyllactosamine) and capped with

the LewisX antigen, an 2–3-sialylated,

1–3-fucosylated tetrasaccharide that represents the minimal

carbohydrate

epitope recognized by P-, E-, and L-selectins. The Golgi enzyme UDP-GlcNAc synthesizes these core 2 sugars: Gal 1, 3GalNAc-R (GlcNAc to GalNAc) 1, 6-N-acetyl-

glucosaminyltransferase (i.e., core 2 GlcNAc-T, EC 2.4.1.102) [112]. The enzyme converts core 1 (i.e. Gal ( 1-3) GalNAc ( 1-O)) to core 2 structures (i.e. Gal 1, 3 [GlcNAc

1, 6] GalNAc -O) expressed on serine or threonine residues

[113-114] (Fig. 5). These O-linked glycans synthesized by

core 2 GlcNAc-T are associated with cellular adhesion, immune responses, and

disease states, such as malignant

transformation, T-cell activation, inflammation, myocardial

dysfunction,

capillary morphogenesis, and myeloblastic leukemia [115-122].

Initial studies suggested that core 2 GlcNAc-T is possibly

involved in the increased adhesion [123] of PMN leukocytes

to retinal endothelial cells. The activity of core 2 GlcNAc-T

was significantly higher in the leukocytes isolated from type

1 and type 2 diabetic patients compared with those from age-

matched healthy control subjects. Enzyme activity was also

significantly higher in leukocytes isolated from diabetic

patients with retinopathy than those isolated from patients

without retinopathy. Interestingly, worsening of retinopathy

was associated with increasing activity of core 2 GlcNAc-T

in leukocytes of diabetic patients, and there was a significant

correlation between enzyme activity and the extent to which

leukocytes adhere to cultured retinal capillary endothelial

cells. This relationship between enzyme and leukocyte-

endothelial cell binding was also supported by transfection

experiments showing that overexpression of core 2 GlcNAc-

T DNA into a human myelocytic cell line (U937) increased

their binding to endothelial cells. The activity of core 2

GlcNAc-T in diabetic leukocytes appears to be raised

through glucose- and TNF- mediated serine/threonine

PKC 2-dependent phosphorylation of the enzyme [124-

125]. This regulatory mechanism, involving phosphorylation

of core 2 GlcNAc-T, is also present in leukocytes isolated

from type 1 and type 2 diabetic patients. This functional link

between PKC 2 and core 2 GlcNAc-T is further supported

by the study of Nonaka et al. [126] in which they

demonstrate that treatment with the specific PKC -inhibitor,

LY333531 attenuates the increase of leukocyte entrapment in the retinal micro-circulation of diabetic rats [126].

The raised activity of core 2 GlcNAc-T may lead to posttranslational modification of O-linked glycans

on leuko-

cyte surface thus leading to their increased adhesion to endothelial cells. In support of this, there appears to be increased O-linked glycosylation of P-selectin glycoprotein ligand-1 (PSGL-1)

on the surface of leukocytes of diabetic

patients compared with

those from age-matched control subjects [124]. Previous work has already

demonstrated a

crucial role for core 2 GlcNAc-T in the binding of PSGL-1 to

P-selectin [127-128]. PSGL-1, a major ligand for E- and P-selectin is expressed as homodimeric sialomucin on all leucocytes [129]. It preferentially binds to P-selectin and with lower affinity to E-selectin. Glycosylation and dimeri-zation of PSGL-1 appears to increase the rate and strength of tethering to P-selectin

under flow [130]. On the basis of its

sequence, PSGL-1 is thought to contain more than 70 sites for O-linked glycosylation and 3 sites for N-linked glycosylation in each monomer [131-132]. Since platelets also express P-selectin, changes in PSGL-1 could lead to the increased number of circulating heterophilic plateletleuko-


Fig. (5). Core 2 GlcNac-T reaction. UDP-Glucose ( ) is converted to UDP-Galactose ( ), and finally to UDP-Galactosamine ( ). O-

glycosylation occurs in the Golgi complex with the addition of an N-acetylgalactosamine (GalNAc, ) from uridine diphosphate (UDP)-

GalNAc residue to serine (Ser) or threonine (Thr) residues by a family of polypeptide N-acetylgalactosaminyltransferase (ppGalNAcT). The

addition of galactose ( ) to GalNAc, in a ß1, 3 linkage by the enzyme core-1 1-3 galactosyltransferase ( 3GalT) 3-beta-galactosyl-

transferase, forms the core 1 structure (Gal ( 1-3)GalNAc ( 1-O). Core 1 structure is then converted to the core 2 structure (Gal 1-

3(GlcNAc 1-6) GalNAc -O) by the addition of GlcNAc ( ) to GalNAc, a reaction catalyzed by core 2 GlcNAc-T.

cyte aggregates that have been associated with the pathogenesis of retinopathy [133].

Our findings so far are summarised in Fig. 6. In diabetes, glucose and/or plasma TNF- possibly activates NADPH oxidase, leading to increased generation of reactive oxygen species (ROS) (Chibber et al. unpublished results). ROS then acts as intracellular second messengers increasing the activity of core 2 GlcNAc-T in leukocytes via PKC 2-dependent phosphorylation (Chibber et al. unpublished results). The raised activity of core 2 GlcNAc-T leads to posttranslational modification (glycosylation) of glyco-proteins, such as PSGL-1 on the surface of diabetic leukocytes, increased rolling/adhesion to activated and/or damaged endothelial cells overexpressing adhesion mole-cules (ICAM-1, P-selectin), and finally to entrapment in diabetic retinal capillaries. Since platelets express P-selectin, leukocytes may also form aggregates through PSGL-1. Platelet-leukocyte interaction may also lead to the formation of microthrombi detected in human and animal diabetic retinal capillaries [134-135]. This accumulation of platelets to sites of retinal cell endothelial cell damage occurs very early in experimental diabetes [136].

5. SUMMARY

Over the years, a number of mechanisms have been tested as potential link between hyperglycemia, and the development of diabetic retinopathy, but so far their clinical targeting has not led an effective pharmacological based therapy. Increasing evidence now suggests that retinopathy is a low-grade, chronic inflammatory condition associated with increased leukocyte adhesion to the diabetic retinal vascu-lature, resulting in blood-retinal barrier breakdown, capillary non-perfusion, and endothelial cell damage. The increased leukocyte-endothelial cell adhesion and entrapment (leuko-stasis) is the result of glucose-mediated changes on endo-thelial cells (adhesion molecules) and leukocytes (deform-ability, upregulation of integrins which are the counter-receptors for vascular adhesion molecules). Recent results also suggest that raised activity of the Golgi enzyme, UDP-GlcNAc: Gal 1, 3GalNAc-R (GlcNAc to GalNAc) 1, 6-N-acetylglucosaminyltransferase (i.e., core 2 GlcNAc-T, EC 2.4.1.102) in diabetic leukocytes increases their adhesion to retinal endothelial cells. Core 2 GlcNAc-T is an enzyme located in the cell's O-linked glycosylation pathway that creates a cell-surface carbohydrate structure (O-glycans)


involved in the recruitment of leukocytes to the site of the inflammation. Based on the results with animal models of diabetes, drug-based therapy (e.g. non-steroidal anti-inflam-matory drugs) with potential to inhibit leukostasis in the retinal microcirculation is a promising target in the treatment of diabetic retinopathy. Our recent work further suggests a novel rationale toward a specific treatment of diabetic retinopathy, based on normalising raised activity of core 2 GlcNAc-T in diabetic leukocytes.

ACKNOWLEDGEMENTS

We gratefully acknowledge Guy’s & St Thomas’ Charitable Foundation, Charities Advisory Trust (London, UK), Juvenile Diabetes Foundation International (JDRFI) and European Foundation of Diabetes Study/Servier for supporting our past and present work on the role of core 2 GlcNAc-T in diabetic retinopathy. The authors thank Dr Gerald Lutty (Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, MD 21287-9115, USA), Dr AP Adamis (Eyetech Research Center, Woburn, Massachusetts, USA), and Professor John Forrester & Dr Mackinon (Department of Bio-Medical Physics and Bio-Engineering, Forresterhill, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK) for kind permission to include their published images of leukocyte entrapment (leukostasis) in diabetic retinopathy. There is no conflict of interest for the authors. We apologise to any investigators whose work, owing to space limitation, has not been discussed in the present review.

NON-STANDARD ABBREVIATIONS

Core 2 GlcNAc-T = Core 2 -1,6-N-acetyl-glucosaminyltransferase

PMN = Polymorphonuclear

ICAM-1 = Intracellular adhesion molecule-1

PKC = Protein kinase C

ETDRS = Early Treatment Diabetic Retinopathy Study

TNF- = Tumour necrosis factor-alpha

PDR = Proliferative diabetic retinopathy

NPDR = Nonproliferative diabetic retino-pathy

VCAM-1 = Vascular cell adhesion molecule-1

sICAM-1 = Soluble intracellular adhesion molecule-1

sVCAM-1 = Soluble vascular cell adhesion molecule-1

AGEs = Advanced glycation end products

DCCT = Diabetes Control and Compli-cations Trial

Fig. (6). Schematic diagram of core 2 GlcNAc-T in diabetic retinopathy. Glucose and/or plasma TNF- levels raise the activity of core 2

GlcNAc-T via diacylglycerol (DAG) and/or NADPH oxidase-derived reactive oxygen species (ROS) activation of PKC 2-dependent

phosphorylation leading to posttranslational modification of surface glycoproteins such as, P-selectin glycoprotein ligand-1 (PSGL-1) and

increased rolling/adhesion to activated and/or damaged endothelial cells which over express adhesion molecules (P-selectin, ICAM-1). There

is also possibility that leukocytes bind directly to platelets that also express P-selectin forming aggregates. Platelet-leukocyte interaction may

also lead to the formation of microthrombi detected in diabetic retinal capillaries [132-135]. Core 2 structures can be further extended with

polylactosamine and Lewis antigens (e.g. sialyl-LewisX), the latter of which have been shown to participate in leucocyte adhesion and homing when expressed on various surface glycoproteins such as PSGL-1.


UKPDS = United Kingdom Prospective Study

PMN = Polymorphonuclear

HREC = Human retinal capillary endo-thelial cells

HUVEC = Human umbilical endothelial cells

CRP = C-reactive protein

TJs = Tight junctions

BRB = Blood retinal barrier

IRMA = Intraretinal microvascular abnor-malities

ELAM-1 = Endothelial-leukocyte adhesion molecule 1

VEGF = Vascular endothelial growth factors

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Received: 09 May, 2006 Revised: 12 July, 2006 Accepted: 01 October, 2006

Leukocytes in Diabetic Retinopathy - Semantic Scholar€¦ · Rakesh Chibber*, Bahaedin M. Ben-Mahmud, Surina Chibber and Eva M. Kohner Cardiovascular Division, GKT School of Biomedical

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