In experimental studies employing rodent models of diabetes, diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are temporally and spatially correlated with a low-level leukocyte influx and persistent retinal leukostasis. This leukostasis is mediated by retinal upregulation of intercellular adhesion molecule-1 (ICAM-1), together with an increased expression of its cognate integrin ligands on neutrophils. Subsequently, endothelial cell injury and death result from Fas/FasL-mediated apoptosis.

In response to this injury, the endothelium maintains a sustained high rate of cell division, which is believed to result in exhaustion of its regenerative capacity. This stress is further exacerbated by a diabetes-induced defect in the ability of endothelial precursor cells to repair the damaged vasculature. While the vascular damage is primarily a function of infiltrating leukocytes, DR also is associated with ischemic neovascularization, a process that is amplified by the influx of macrophages.

Numerous cytokines are upregulated in DR, and two of these, vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), are believed to play important roles in the inflammation-linked retinal damage. Upregulation of VEGF causes much of the increase in retinal ICAM-1 and the resultant leukostasis, with the VEGF165 isoform being especially important in promoting inflammatory responses. TNF-α also is involved in the upregulation of ICAM-1, and preclinical studies have established that inhibitors of both VEGF and TNF-α are able to reduce the DR-associated pathology.

The concept that DR is a low-grade inflammatory condition has proved useful in the clinic. Inhibitors of VEGF and TNF-α have shown efficacy in reducing the DR-related vision loss while high-dose aspirin has proved effective in reducing the number of DR-associated microaneurysms. There is thus reason for hope that further elucidation of the underlying cellular and molecular mechanisms of DR-associated inflammation will lead to the development of new molecularly targeted therapies and to the rational use of approaches employing more than one therapeutic agent.

Key Words: Bevacizumab; Inflammation; Leukostasis; Pegaptanib; Ranibizumab; Tumor necrosis factor-alpha; Vascular endothelial growth factor.

INTRODUCTION

The concept that diabetic retinopathy (DR) is inflammatory in nature has been under investigation since the 1960s following the results of a study demonstrating a lower incidence of DR in patients treated with salicylates for rheumatoid arthritis (1). Subsequent work has provided further support for this linkage and has identified many characteristic inflammatory features that accompany the progress of DR in patients and in animal models. These include increased blood flow and vascular permeability, edema, infiltration of inflammatory cells such as macrophages, and the development of neovascularization (Table 1) (2–27). Numerous physiologic derangements are believed to act in mediating the linkage between hyperglycemia and retinal damage, including the accumulation of polyols, reactive oxygen intermediates, and advanced glycation end products, in addition to the activation of protein kinase-C (reviewed by Brownlee (28), Sheetz and King (29), and Caldwell et al. (30) ). These topics are discussed elsewhere in this book, and will be referred to here when they have a bearing on inflammatory processes.

Table 1

Inflammatory features that characterize diabetic retinopathy (adapted from Antonetti et al. (2) )

Increased blood flow and vascular permeability (2)

Tissue (macular) edema (2)

Neovascularization (2)

Increased expression of inflammatory mediators (3–16)

Accelerated retinal neural (17) and microvascular (18) cell death

Macrophage infiltration (8,19)

Microglial cell activation (20–22)

Increased leukocyte adhesion (23,24)

Complement activation (25)

Fas ligand upregulation (26)

Acute-phase response protein expression (27)

The retinal abnormalities in DR involve neurons, glial elements, and the retinal microvasculature (31). While some of the earliest detectable defects in DR include alterations in neuronal function (2, 31), for which there is some evidence for an inflammatory contribution (27, 32), most research into inflammatory changes concerns DR-associated damage to the retinal vasculature. As described in accompanying chapters, the processes leading to the DR-associated vasculopathy include leukocyte entrapment, formation of acellular capillaries, capillary dropout, and local hypoxia. The focus of this chapter is on the inflammatory nature of many of the molecular and cellular processes leading to this vascular damage, as well as on the pathologic neovascularization that often accompanies it. Finally, clinical findings validating the role of inflammation in DR are described.

PATHOPHYSIOLOGY OF DIABETIC RETINAL VASCULAR

INJURY: THE ROLE OF LEUKOSTASIS

Several lines of correlative preclinical and clinical evidence have demonstrated that DR is associated with increased levels of leukocytes. In a key early clinical study, McLeod et al. (33) reported that neutrophil numbers were significantly elevated in the choroidal and retinal vasculature of patients with diabetes mellitus and that this elevation was accompanied by an increased expression of intercellular adhesion molecule-1 (ICAM-1) in both tissues. Subsequently, it was determined that vitreous levels of soluble ICAM-1 were significantly higher in the eyes of diabetic patients compared to nondiabetic controls (34). Macrophages also have been detected in surgically removed membranes (19) and vitreous samples of (8) patients with proliferative DR. In other studies, the choriocapillaris of eyes from diabetic patients had approximately twice the number of polymorphonuclear leukocytes as in nondiabetic controls, together with an elevated level of nonviable endothelial cells, leading to choriocapillary dropout (35). Increased numbers of polymorphonuclear cells also have been observed in the retinal

Fig. 3. Leukocytes induce capillary occlusion. Serial studies were completed 1 week (A, B), 2 weeks (C, D), and 4 weeks (E, F) after diabetes induction in rats by using both acridine orange leukocyte fluorography (A, C, E) and fluorescein angiography (B, D, F). The arrows show a patent capillary that is not blocked by a leukocyte (A, B) that subsequently becomes occluded downstream from a static leukocyte (C, D), and then subsequently opens up when the leukocyte disappears (E, F). The arrowheads show a patent capillary not blocked by a leukocyte (A, B) that becomes occluded downstream from a static leukocyte (C, D) and then remains closed after the leukocyte has disappeared (E, F). Scale bars: 100 m. (Reproduced from Miyamoto et al. 1999 (23) with permission from Proc Natl Acad Sci USA. Copyright 1999 National Academy of Sciences, USA.)

Possible mechanisms linking leukostasis with increased permeability may include a direct action on tight junction disruption (41, 42) as well as local upregulation of vascular endothelial growth factor (VEGF), either from the hypoxia induced by nonperfusion or from release by the leukocytes themselves (43–46). A major factor, however, appears to be the concomitant increase in endothelial cell injury and death (24, 26, 47), also reported in an earlier clinical study (18). Prevention of the interaction between leukocytes and the retinal vascular endothelium by the administration of antibodies to CD18 or ICAM-1 resulted in inhibition of leukostasis and endothelial cell apoptosis (47). Similarly, the genetic ablation of CD18 or ICAM-1 resulted in markedly reduced

45

PI–positive cells

Wild-type Wild-type CD18-KO CD18-KO ICAM-KO ICAM-KO

Wild-type Wild-type CD18-KO CD18-KO ICAM-KO ICAM-KO

Fig. 6. Genetic ablation of either CD18 or intercellular adhesion molecule-1 (ICAM-1) dramatically reduces leukocyte adhesion, retinal endothelial damage, and blood–retinal barrier (BRB) breakdown in diabetic mice. (A) Adherent leukocytes were quantified in the retinal vasculature following in situ labeling with Concanavalin A (open bars). After 11 months of diabetes, the number of adherent leukocytes in the wild-type mice was 3.7-fold greater than in the age-matched, nondiabetic, wild-type controls (P < 0.001). In contrast, the number of adherent leukocytes in both the diabetic CD18 and ICAM-1 knockout (KO) mice did not differ significantly from that of the nondiabetic, wild-type controls or the nondiabetic, CD18, and ICAM-1 KO controls (P > 0.05 for all). Endothelial cell injury was assessed with propidium iodide (PI), which labels nonviable cells (filled bars). While 11-month diabetic, wild-type mice had a marked increase in PI-positive cells (P < 0.001), diabetic CD18, and ICAM-1 KO mice had significantly fewer PI-positive cells than diabetic wild-type mice (P < 0.001). Nondiabetic, wild-type mice and the nondiabetic, CD18, and ICAM-1 KO mice also had few PI-positive retinal endothelial cells. (B) BRB breakdown was assessed with the use of Evans Blue dye, which binds covalently to albumin. In wild-type mice, long-term diabetes led to a 27.7-fold increase in vascular leakage whereas in both CD18 and ICAM-1 KOs the diabetes-associated increase was much less (1.6- and 2.75-fold, respectively). Data represent mean ± standard deviation. (Reproduced from Joussen et al. 2004 (24) with permission from Fedn of Am Societies for Experimental Bio [FASEB] Copyright 2004 by Fedn of Am Societies for Experimental Bio (FASEB).)

Fig. 7. FasL inhibition suppresses apoptosis and blood–retinal barrier (BRB) breakdown. (A) Retinal cell death was quantified 2 weeks after induction of diabetes in rats using a DNA fragmentation enzyme-linked immunosorbant assay 48 h after systemic administration of an anti-FasL antibody or a control antibody. Fragmented retinal DNA levels increased by 13.84 ± 0.41-fold in diabetic animals. Systemic administration of the anti-FasL antibody reduced this to nearly nondiabetic levels (1.22 ± 0.82-fold), whereas no attenuating effect was seen after treatment with a control IgG antibody. (B) BRB breakdown was determined by Evans Blue performed 48 h after treatment with the anti-FasL antibody or a control antibody. Retinal permeability in the 2-week diabetic rats increased to 2.6 times the value in nondiabetic animals; treatment with the anti-FasL antibody reduced this to a factor of 1.33. Data represent mean ± standard deviation. (Reproduced from Joussen et al. 2003 (26) with permission from Fedn of Am Societies for Experimental Bio [FASEB] Copyright 2003 by Fedn of Am Societies for Experimental Bio (FASEB)).

the DR is accompanied by an enhanced rate of endothelial cell proliferation and death in experimental (18, 51) and clinical (18) disease, suggesting that the replicative capacity of endothelial cells may eventually become exhausted and lead to capillary dropout (18). Moreover, subsequent studies have also demonstrated that endothelial precursor cells from diabetic patients are impaired in several essential functions, including migration in