Diabetic retinopathy (DR), a well-known consequence of long-standing and poorly controlled diabetes mellitus (DM), causes significant vision loss and blindness in the human population. DR currently affects approximately 150 million people worldwide, and the World Health Organization projects that the number of people affected will double by the year 2025 [1].

In patients with DM, the prevalence of any form of diabetic retinopathy is approximately 24% [2]. Diabetic macular edema (DME) is the major vision-threatening complication of DR. The Wisconsin Epidemiologic Study has reported that the prevalence of DME in diabetics of 15-year duration is approximately 20% in patients with Type 1 DM and 25% in patients with Type II DM that are on treatment [3]. Patients without macular edema can still maintain good vision even if they have advanced stages of the disease. Therefore, management of DME has become crucial for ophthalmologists in preventing vision loss to their diabetic patients.

Macular edema is diagnosed clinically by documenting retinal thickening and/or hard exudates using biomicroscopic examination. The Early Treatment Diabetic Retinopathy Study (ETDRS) has described a specific type of DME called “Clinically Significant Macular Edema” (CSME) for which laser treatment is indicated [4]. Based on angiographic findings, diabetic maculopathy has been classified as either focal or diffuse, and ischemic or non-ischemic. Diabetic maculopathy caused by leaking microaneurysms is classified as focal, while leakage at the level of the capillary beds leads to diffuse. The visual prognosis for patients with the ischemic type is very poor as there is deprivation of blood to the retina in ischemic zones.

Diabetic retinopathy is a disease of the retina, but similar vascular and ischemic changes can be found in other parts of the eye in the more advanced stages. Profound ischemia of the retina leads to neovascularization (growth of new vessels) of the disc (NVD), the retina (NVE), the iris (NVI) and the angle (NVA). NVA can lead to neovascular glaucoma (secondary to closure of the angle). For many years these conditions have been treated by panretinal photocoagulation (PRP) of the retina, which causes regression of the abnormal new vessels on the iris and angle [5]. In advanced cases of neovascular glaucoma where there is synechial closure of the angle, a surgical shunt such as a Baerveldt tube shunt or Ahmed glaucoma valve must be placed to facilitate aqueous drainage. Currently, anti-VEGF drugs are being investigated for the treatment of rubeosis. The RAVE (Rubeosis Anti-VEGF) clinical trial for Ischemic Central Retinal Vein Occlusion (CRVO) utilizes monthly ranibizumab injections for nine months to determine if this treatment can prevent the development of neovascular glaucoma in eyes with CRVO.

Intensive glycemic control is one of the two important ways to reduce the risk of vision loss from DME. The Diabetes Control and Complications Trial [6] and UK Prospective Diabetes Study [7] showed that intensive blood glucose control reduced the risk of development of DME by 23%.

Laser photocoagulation is the second most important course of action to prevent vision loss from DME. Clinical trials such as the Diabetic Retinopathy Study (DRS) and ETDRS have shown that timely laser intervention can reduce the loss of vision by 90% [8, 9]. For proliferative diabetic retinopathy, PRP laser is the treatment of choice as demonstrated in several clinical trials. Unfortunately, the areas of retina treated with PRP laser exhibit scarring and loss of function.

In eyes of patients with DME, corticosteroids inhibit the expression of VEGF and decrease angiogenesis [10]. Platelet-derived growth factor (PDGF) is a proinflammatory cytokine that induces the expression of the VEGF gene, an effect that is downregulated by corticosteroids [11]. Intravitreal injections of triamcinolone acetonide (IVTA) are now commonplace and widely used for treatment of retinal disorders. In spite of being a fairly safe procedure, intravitreal steroid injections carry an inherent risk of major complications such as cataract, glaucoma, endophthalmitis (both infective and sterile), vitreous hemorrhage, and retinal detachment.

Visual outcomes are generally poor in advanced cases of DR with complications such as long-standing vitreous hemorrhage (VH), tractional retinal detachments (TRD), and fibrovascular proliferation. In these instances, the surgical approach is the procedure of choice [12]. The Diabetic Retinopathy Vitrectomy Study (DRVS) was a National Eye Institute sponsored, multicenter clinical trial program that established treatment guidelines for patients with advanced proliferative diabetic retinopathy. After four years of follow-up, the conclusions were that an early vitrectomy provided a greater chance for prompt visual recovery in eyes with recent severe vitreous hemorrhage.

Despite the presence of varied therapeutic modalities, vision loss occurs at an alarming rate due to DME. The ETDRS showed that despite laser treatment, 12% of subjects continued to have moderate visual loss. In addition, laser treatment is a destructive procedure that does not take into account the pathophysiology of DR and can cause tissue damage itself.

Recently, interest has developed regarding use of anti-VEGF in cases of DME. Although adverse effects such as nonfatal cerebrovascular accidents and myocardial infarction have been described [13], anti-VEGF agents are proving to be far superior to the laser treatments which are associated with extensive resultant tissue damage. The incidence of complications such as glaucoma and cataract encountered with the intravitreal injections of corticosteroids can be avoided with the use of anti-VEGF. For these reasons, they are becoming the preferred choice of treatment in cases of DME with coexisting glaucoma and in cases recalcitrant to lasers and intravitreal steroids.

Diabetic patients with concomitant hypertension are at a higher risk of developing DME and DR. Moreover, the DR in these patients tends to progress much more rapidly [14, 15]. The renin-angiotensin-aldosterone system (RAAS), which regulates blood pressure and maintains water balance, has a direct connection to the pathologic microvascular changes occurring in DR [16]. Angiotensin II, the major bioactive product of the RAAS, acts as an endocrine, paracrine, and intracrine hormone, and is a powerful vasoconstrictor of arterioles. In vitro studies have shown that angiotensin II directly stimulates the secretion of VEGF in cultured smooth muscle cells [17] and in cardiac endothelial cells [18]. Therefore, constriction of the retinal microvasculature could result in local increased VEGF production within the retina, which in turn could lead to angiogenesis [17, 19]. The detailed pathway linking angiotensin II to increased synthesis of VEGF is yet to be elucidated, but hypoxia-inducible factor (HIF-1α) has been shown to be involved at the intracellular signaling pathway level.

In 1948, Isaac Michaelson proposed that in the development of pathological angiogenesis, a key event was the synthesis and release of a diffusible angiogenic factor called “Factor X” by the ischemic retina [20]. Later in 1971, Judah Folkman presented the idea of inhibiting angiogenesis as a means to treat cancer, which then led to the discovery of anti-angiogenic factors [21]. Nearly forty years passed before the originally proposed “Factor X” could be isolated and identified. In 1983, Senger, Dvorak and colleagues discovered a key mediator involved in angiogenesis to be a protein secreted from a guinea pig tumor cell line. It had a potent capacity to induce vascular leakage and was named vascular permeability factor (VPF) [22]. In 1989, Napoleone Ferrara and colleagues identified a molecule present in bovine pituitary follicular cells that promoted the proliferation of endothelial cells and called it vascular endothelial growth factor (VEGF) [23]. Subsequently, the cloning of VPF by Daniel Connelly et al. and VEGF by Ferrara et al. confirmed that the two factors were the same protein [24, 25]. In 1992, two independent studies demonstrated that hypoxia could upregulate VEGF expression [26, 27]. Evidence from clinical studies supported the critical role of VEGF in ocular neovascularization. It was demonstrated that vitreous samples of patients with active proliferative retinopathies associated with diabetes and venous occlusive disorders had elevated VEGF levels [28].

Vascular endothelial growth factors (VEGF) are a subfamily of growth factors that function as signaling proteins for both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF is secreted primarily from retinal pigmented epithelial cells, pericytes, astrocytes, müller cells, glial cells, and endothelial cells. VEGF has several members including VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PGF). VEGF-A is a 36-46 kDa glycosylated protein derived by alternate splicing of mRNA from a single, 8-exon VEGF gene [29]. VEGF-A is expressed as five mRNA splice variants – isoforms 121, 145, 165, 189, and 206, depending upon the number of amino acids present. Larger isoforms (isoforms 189 and 206) have increased heparin binding ability, which reflects their ability to bind to cell surfaces and basement membranes. Unfortunately, the larger isoforms also have limited or no diffusibility. Smaller isoforms (isoform 121 and 145) are readily diffusible but have limited heparin binding ability. Isoform 165 has intermediate levels of diffusibility and heparin binding. VEGF 165 is the principle isoform secreted, and is critical for both development and pathological angiogenesis [30]. The other VEGF members probably have a greater role to play in tumors and cancers than in DR [31].

VEGF production is induced in response to ischemia or hypoxia (Fig. 1). Tissue hypoxia leads to the production of a DNA binding protein called hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric, basic helix-loop-helix protein that consists of two subunits: the constitutively-expressed HIF-1 beta subunit and the growth factor regulated HIF-1 alpha subunit [32]. In addition to playing a crucial role in VEGF production, HIF-1 is also responsible for the upregulation of the human erythropoietin gene in hypoxic cells, which is another adaptation to tissue hypoxia [33]. HIF-1 binds to the VEGF gene and initiates the transcription process. This leads to the generation and accumulation of VEGF mRNA by both increased mRNA transcription and decreased mRNA degradation [34], eventually leading to the intracellular accumulation of VEGF molecules. A chaperone protein called ORP 150 (oxygen regulated protein) is responsible for the transfer of VEGF from the endoplasmic reticulum to Golgi bodies. The expression of ORP150 mRNA is also induced by hypoxia [35].

Fig. (1). Flowchart of the VEGF cascade showing the molecular chain of events that occur after retinal tissue hypoxia culminating in angiogenesis.

All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors on the endothelial cell surface, causing them to dimerize and become activated through transphosphorylation. VEGF-A has two types of receptors: VEGF receptor 1 (VEGFR-1 or Flt-1) and 2 (VEGFR-2 or KDR). In humans, VEGFR-1 is a protein that is encoded by the Flt-1 gene [35] and VEGFR-2 is a kinase insert domain receptor (KDR, a type III receptor tyrosine kinase) that is encoded by the KDR gene [36]. VEGFR-2 seems to be the primary receptor that mediates almost all of the cellular responses to VEGF-A. The VEGF receptors have three parts: an extracellular portion consisting of seven similar immunoglobulin-like domains, a single transmembrane hydrophobic spanning region, and an intracellular portion containing a split tyrosine kinase domain [37]. When the VEGF molecule binds to the extracellular portion of the VEGF receptors, the intracellular portion causes phosphorylation of the tyrosine amino acid residues and a cellular signal is transduced. This transduction in turn induces a cascade of intracellular signaling pathways. One of the pathways induced within the cells by VEGF is the protein kinase C (PKC) pathway [38]. The other pathways contribute to angiogenesis by synthesis of nitric oxide and induction of cellular proliferation.

VEGF is an important factor in the development of both proliferative DR and DME [39], altering retinal capillary permeability by increasing the phosphorylation of proteins involved with tight-junctions such as zonula occludens [40]. The VEGF induction activates mitogen-activated protein (MAP), resulting in endothelial cell proliferation. This cascade coincides with the activation of the phophatidylinositol 3-kinase (PI3)/Akt pathway after VEGFR-2 induction [33]. VEGF-A stimulates endothelial cells to release matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator, resulting in the degradation of basement membranes and making cell migration possible. The activated endothelial cells express integrins such as αvβ3 and αvβ5, which aid in the migration through the degraded matrix [41]. Proliferation and migration of endothelial cells is followed by synthesis of basement membranes for the newly formed capillaries. The stability of these capillaries is achieved by recruitment of pericytes and smooth muscle cells that are regulated by platelet-derived growth factor (PDGF) [42].

Much work has been done to study the mechanisms of VEGF in hopes of reducing neovascularization and vessel leakage by blocking its effects. Fig. (1) shows the sites of action of the drugs that target abnormal neovascularization.

Laser photocoagulation still remains the gold standard treatment for proliferative DR and DME, but it is not effective in many patients. Recently, retina specialists are inclined to use anti-VEGF agents for the management of DME. Although only some of these drugs are FDA approved for intraocular use (i.e., ranibizumab, pegaptanib), many are currently being used off label in clinical practices (i.e., bevacizumab). These anti-VEGF drugs are used as adjunct therapies to laser treatment, but there is an increasing trend towards using them as primary treatment for diabetic macular edema.