Diabetes is a lifelong progressive disease resulting from the body's inability to produce or utilize insulin to break down circulating glucose. It affects more than 230 million people worldwide, is the fourth leading cause of death by disease globally, and results in one death every 10 seconds from diabetes-related causes.1 The sustained hyperglycemia in diabetes attacks both microvessels and macrovessels throughout the body, including the eye. In the eye, chronic end organ damage is manifested as diabetic retinopathy (DR), the leading cause of blindness in adults of working age. In DR, there is pericyte loss followed by further damage to the microvasculature of the retina, leading to microaneurysms, telangiectatic vessels, and exudative leakage. Continued ischemia ultimately leads to development of neovascularization, vitreous hemorrhage, and/or tractional retinal detachment and blindness.2 Retinal neovascularization occurs in up to 20% of patients with diabetes (Figure).3 Current photocoagulation treatment for DR has changed little over the 50 years since its inception it is applied only after onset of neovascularization, reduces peripheral and night vision, and is uncomfortable, destructive, and expensive.4
INFLAMMATION IN DR
The pathogenesis of DR is characterized by many features typical of inflammation. These include increased blood flow and vascular permeability, tissue (macular) edema and neovascularization,5 microglial cell activation,6-8 increased leukocyte adhesion,9,10 complement activation,11 Fas ligand (FasL) upregulation,12 and the increased expression of inflammatory mediators, including VEGF,13,14 interleukin 1 beta (IL-1β),15 TNF-a,15 IL-6,16,17 and IL-8.17 These proinflammatory factors promote the pathogenesis of DR in various ways.
VEGF promotes ocular neovascularization,18 vascular permeability,19 and leukostasis.20 IL-1β, in addition to promoting inflammation, seems to directly induce retinal capillary cell degeneration and death through caspase 1 signaling.21 TNF-a induces upregulation of adhesion molecules, immune cell infiltration, and induction of apoptosis.22 The complement system is a part of the innate immune systems, and activation of complement results in increased inflammation and microthrombosis.23 This inflammation, which typically has beneficial effects as an acute phase response, has undesirable tissue damaging effects if it persists chronically.
CELLULAR INFILTRATE IN DR
In addition to soluble inflammatory mediators, there is significant infiltration of immune cells into the ocular environment following DR. Neutrophils have been reported to be elevated in both choroidal and retinal blood vessels of patients with diabetes.24 Elevated numbers of polymorphonuclear cells (PMCs) have been reported in the choriocapillaries of diabetic patients.25 CD4+ and CD8+ T cells have been found to be elevated in the vitreous humor of diabetics.26
Many studies have implicated immune cell infiltrate with promoting the pathogenesis of DR by mediating retinal vascular damage. For example, the increase in PMCs in the choriocapillaries of diabetics have been correlated with losses in viable endothelial cells, leukocyte queuing in capillaries, and capillary dropout.25 Also, similar correlations of elevated leukocyte numbers and capillary damage have been reported in animal models.27,28 In streptozotocin (STZ)-induced diabetic rats, an increase in retinal leukostasis correlated with increased leakage of retinal vasculature.9
Interestingly, infiltrating immune cells have also been shown to prevent the development of abnormal blood vessels in the eye. There is both experimental and clinical evidence to show that these infiltrating cells lead to injury and death of endothelial cells.29 Prevention of leukostasis with blocking antibodies to either CD18 or ICAM-1 resulted in significant reduction of endothelial cell apoptosis.30 Fas-FasL interaction may be important in these events, as FasL was found to be upregulated on neutrophils while Fas was upregulated in retinal blood vessels in STZ-induced rats.12 This was accompanied by an enhanced capacity of neutrophils to induce apoptosis of cultured endothelial cells.12 Also, in vivo administration of anti-FasL antibody resulted in decreased endothelial cell apoptosis as determined by TUNEL staining.12 T lymphocytes may also play a role in preventing angiogenesis. Systemic administration of an antibody against CD2, a key adhesion molecule for T lymphocytes, resulted in increased pathological neovascularization following oxygen-induced retinopathy (OIR).31
MACROPHAGES
In addition to PMNs and T lymphocytes, macro-phages also are involved in the pathogenesis of DR. Macrophages have been found to be elevated in the vitreous humor of diabetic patients.17 Also, macro-phages have been found in membranes surgically removed from diabetics.32 Macrophages produce many proinflammatory cytokines in DR. Macrophages constitute the principal cellular source of TNF-a during diabetes.22 Among the wide variety of biological functions attributed to macrophages, regulation of angiogenesis is a key role.33 Macrophages can exhibit both pro-angiogenic and anti-angiogenic functions.34,35 This dual function of macrophages seems to be largely dependent upon the polarization of macrophages. Polarization, in turn, seems to be regulated by the production of cytokines in the resident tissue micro-milieu. Macrophages stimulated to produce high levels IL-12, IL-23, IL-6, and TNF-a and low levels of IL-10 are termed "classically-activated" macrophages, or M1 macrophages. M1 macrophages display an antiangiogenic phenotype and play an important role in antbacterial and proinflammatory functions. Macrophages stimulated to produce high levels of IL-10 and VEGF, and low levels of pro-inflammatory cytokines such as IL-6 and TNF-a are termed "alternatively activated" macrophages, or M2 macrophages, and are proangiogenic.
The role of macrophages in ocular angiogenesis is complex. In murine models of choroidal neovascularization (CNV) and OIR, mice depleted of macrophages with intravitreous injections of clodronate liposomes had decreased ocular angiogenesis,31,36,37 suggesting that macrophages function in a proangiogenic role. Contrary to this, macrophages may also be functioning in an antiangiogenic role in the eye. For example, macrophages are responsible for the induction of apoptosis in the vascular endothelial cells of the temporary hyaloid vessels of the developing eye.38 Also, mice lacking a key macrophage recruitment chemokine (CCL-2) developed spontaneous CNV.39 Plus, the above mentioned study involving the work of macrophage depletion with clodronate liposomes resulting in increased CNV involved the use of senescent animals.36 As macrophages age, they tend to skew toward a proangiogenic phenotype.35 Therefore, depletion of these pro-angiogenic macrophages may explain the decrease in CNV observed.
IL-10 PROMOTES PROANGIOGENIC MACROPHAGES IN THE RETINA
Interleukin-10 is an antiinflammatory cytokine that inhibits T cell and macrophage functions. It is a potent inhibitor of cytokine and chemokine production, including a number of molecules known to attract monocytes and macrophages to sites of inflammation. The absence of IL-10 typically results in substantial increases in inflammation.40 In the eye, IL-10 seems to promote pathological angiogenesis. In the murine laser-induced injury model of CNV, neovascularization was increased in the presence of IL-10.34 This was due to the ability of IL-10 to prevent the infiltration of antiangiogenic macrophages to the choroid following injury, and due to the ability of IL-10 to polarize macrophages to an M2 phenotype.34,35
Recent studies in our lab have suggested that both IL-10 and the hypoxic environment of the retina seen in DR contribute to the proangiogenic function of macrophages and pathological angiogenesis. Using the OIR model, the presence of IL-10 resulted in increased retinal angiogenesis compared with mice lacking IL-10. Instead of preventing macrophage infiltration to the retina, IL-10 instead promoted nitric oxide and VEGF expression in macrophages. Also, hypoxia caused macrophages to produce IL-10 and undergo IL-10 mediated STAT-3 signaling. Taken as a whole, inhibition of IL-10 signaling in macrophages may be beneficial in preventing aberrant angiogenesis in the eye during DR.
Dru S. Dace, PhD, is a Postdoctoral Research Associate with the Department of Ophthalmology and Visual Sciences at Washington University School of Medicine in St. Louis, MO.
Rajendra S. Apte, MD, PhD, is Assistant Professor, Ophthalmology and Visual Sciences and the Developmental Biology Program at Washington University School of Medicine. Dr. Apte states that he is a consultant for Eyetech, Genentech, Allergan, Inc., Gerson Lehman Group, and Ophthotech. He is on the advisory boards of Genentech and Allergan, Inc., and he receives research grants from and serves on the speakers' bureau for Genentech. He may be reached at +1 314 747 5262; fax: +1 314 362 6793 or e-mail: apte@vision.wustl.edu.
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