Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes |
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Authors: | Paul Canning Bridget-Ann Kenny Vivien Prise Josephine Glenn Mosharraf H. Sarker Natalie Hudson Martin Brandt Francisco J. Lopez David Gale Philip J. Luthert Peter Adamson Patric Turowski Alan W. Stitt |
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Abstract: | Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.Breakdown of the blood–retinal barrier (BRB) is a significant pathophysiological event during diabetic retinopathy (DR). Leakage of plasma proteins and lipids into the neuropile form exudates, whereas excessive vasopermeability can lead to diabetic macular edema (DME), which is a major cause of vision loss (1). Both DME in patients and BRB dysfunction in animal models have been linked to inflammatory processes, endothelial dysfunction, and compromise of normal neuroglial–vascular interactions (2). A number of therapeutic options are available to treat DME. Laser photocoagulation is effective in slowing the progression of DME; however, it carries risk of serious adverse side effects including visual field defects, retinal scarring, and foveal burns. Intravitreal injection of antiinflammatory corticosteroids has proven value, but this intervention also carries significant side effects including cataract formation and elevated intraocular pressure. More recently, neutralization of vascular endothelial growth factor (VEGF) in the retina has become a mainstream treatment of DME (3) although this is not effective for all DME patients (4). Moreover, repeated intravitreal injections can result in some patients becoming refractory to VEGF blockade (5), which suggests that other mechanisms also drive vascular permeability during DME. Concerns have also been raised that targeting the VEGF pathway could inadvertently compromise retinal neuroglial and microvascular survival (6) although, thus far, no evidence of these outcomes have been observed in clinical trials of anti-VEGF for DME. Nevertheless there is a need for additional therapeutic approaches to treat DME, either alone or adjunctively with currently available therapeutics.An alternative permeability-inducing agent in diabetic retina could be lysophosphatidylcholine (LPC), which is elevated in plasma of diabetic patients (7) and has demonstrated permeability-enhancing activity in cultured nonneural endothelial cells (ECs) (8). The principal enzyme responsible for the production of LPC is a calcium-independent phospholipase A2 called lipoprotein-associated phospholipase A2 (Lp-PLA2) (also known as platelet-activating factor acetylhydrolase or type VIIA PLA2). Lp-PLA2 can elicit a broad range of proinflammatory and proapoptotic effects in blood vessels related to its hydrolysis of modified polyunsaturated fatty acids within oxidized low-density lipoprotein (oxLDL) into LPC and oxidized nonesterified fatty acids (OxNEFA) (9). Elevated Lp-PLA2 has been proposed as a predictive biomarker in stroke (10), atherosclerosis (11), and coronary heart disease (12). Macrophages and other proinflammatory cells are a primary source of Lp-PLA2 in the systemic circulation (13), although endothelial cells also express this enzyme (14).Darapladib, a specific inhibitor of Lp-PLA2, has been shown to reduce atherosclerosis in both diabetic/hypercholesterolemic pigs (15) and ApoE-deficient mice (16). In diabetic/hypercholesterolemic pigs, darapladib protected against blood–brain barrier (BBB) dysfunction and vascular permeability (17). Darapladib has been studied in nearly 16,000 patients with coronary heart disease, and approximately one-third of this study population had diabetes mellitus (18). In a further study, a 3-month daily treatment with 160 mg of darapladib orally showed reduction of DME and an improvement in visual acuity in patients (19). These observations suggest that Lp-PLA2 and its mechanism of action warrant further investigation in the context of DR. In this study, we have studied the inhibition of Lp-PLA2 in diabetic rats with regards to BRB function and assessed the effect of LPC on neural microvascular endothelial cells. We show inhibition of Lp-PLA2 was effective in reducing diabetes-induced retinal vasopermeability and that the effect of this inhibition was additive with VEGF neutralization, suggesting this enzyme could be an efficacious therapeutic target for DME, either alone or in combination with anti-VEGF therapeutics. |
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Keywords: | diabetic retinopathy, VEGF signaling, lysophosphatidylcholine, blood– retinal barrier, lipoprotein-associated phospholipase A2 |
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