Hypoxia up-regulates CD36 expression and function via hypoxia-inducible factor-1- and phosphatidylinositol 3-kinase-dependent mechanisms.

Neovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1alpha down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway.

Activation of CD36 inhibits and induces regression of inflammatory corneal neovascularization.

PURPOSE: This study was undertaken to investigate the role of the antiangiogenic receptor CD36 during inflammatory corneal neovascularization (CNV). METHODS: In a murine model of inflammatory CNV, CD36 expression was evaluated by RT-PCR and immunofluorescence. Mice subjected to CNV were treated topically (thrice daily) with CD36 functionally neutralizing antibodies against the oxidized low-density lipoprotein (oxLDL) and thrombospondin (TSP)-1 sites (clones JC63.1 and FA6-152, respectively). Neovascularization was analyzed by CD31-immunostained corneal flatmounts. The role of the less characterized oxLDL site during angiogenesis was elucidated by using the CD36 ligand 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine (POVPC; 50, 100 microg/mL) 24 hours after corneal injury for 7 days, whereas in angioregressive studies, POVPC treatments were initiated 10 days after induction of CNV. In this process, VEGF expression was also studied. Effects of CD36 activation were further examined ex vivo using the mouse aortic ring assay. RESULTS: CD36 expression was upregulated after corneal injury; CD36 was expressed in corneal epithelium, limbus, invading microvessels, and stromal macrophages. Blocking CD36 activity with FA6-152 significantly increased CNV (P <0.001). Conversely, activating CD36 with POVPC dose dependently inhibited CNV (P = 0.003); this effect was blocked by JC61.3. POVPC also significantly regressed preformed blood vessels (P < 0.001). Ex vivo experiments on aortic rings confirmed the angioinhibitory and -regressive effects of POVPC. Because corneal macrophages express CD36 and may partake in angiogenesis via VEGF-A secretion, we surmised that VEGF-A could be modulated by CD36. Indeed, POVPC downregulated VEGF-A expression in a time-dependent fashion (P < 0.001), whereas FA6-152 induced its expression (P < 0.05). CONCLUSIONS: CD36 is involved both physiologically and pharmacologically in inhibition and regression of CNV, by direct effect on endothelial cells and partly by negatively regulating VEGF expression in macrophages.

New insights into the retinal circulation: inflammatory lipid mediators in ischemic retinopathy.

Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Consequently, the retina constitutes an excellent model for testing the functional consequences of membrane lipid peroxidation. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane (TXA2) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA2 and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.

The role of lysophosphatidic acid receptor (LPA1) in the oxygen-induced retinal ganglion cell degeneration.

PURPOSE: Although previous studies have demonstrated that hypoxia induces retinal ganglion cell (RGC) apoptosis and that transient retinal ischemia upregulates the expression of lysophosphatidic acid (LPA) receptors, it remains to be determined whether LPA(1) receptor mediates RGC degeneration during retinopathy of prematurity (ROP). By using an immortalized RGC line (RGC-5), primary neonatal RGC cultures, and oxygen-induced retinopathy (OIR) to model ROP, the authors explored whether LPA(1) receptor induces RGC degeneration and the potential mechanisms thereof. METHODS: OIR was induced by exposing rat pups to alternating cycles of hyperoxia/hypoxia from postnatal day (P) 0 to P14. RGC viability was evaluated by Fluorogold labeling. Effects of hyperoxia or hypoxia on LPA(1) expression were determined in the RGC-5 line by Western blot. Roles of hypoxia, LPA(1) receptor (with agonist, stearoyl-LPA; antagonist, THG1603; LPA(1) knock-down, shRNA-LPA(1)), and Rho kinase (with inhibitor Y-27632) in mediating RGC survival and neurite outgrowth were assessed by MTT assay and phase-contrast microscopy, respectively. Expression of GFP-LPA(1) in RGC-5 under hypoxia was examined by confocal microscopy. RESULTS: OIR caused pronounced RGC loss in the retina. LPA(1) receptor was expressed by RGCs in retinal tissue, whereas oxygen stress induced its expression in RGC-5. Exposure to stearoyl-LPA or hypoxia substantially reduced the viability of RGCs; this was abrogated by THG1603 and shRNA-LPA(1). THG1603 and Y-27632 treatment also attenuated the adverse effects of hypoxia on RGC-5 neurite outgrowth, and their intravitreal administration prevented OIR-induced RGC loss. Interestingly, overexpression of LPA(1) increased RGC-5 susceptibility to hypoxia-induced cell loss. CONCLUSIONS: Current data strongly support a critical role for LPA(1) receptor in mediating RGC degeneration during OIR.

Genetic ablation of CD36 induces age-related corneal neovascularization.

PURPOSE: Corneal avascularity is tightly regulated by a balance between angiogenic and antiangiogenic factors (angiogenic privilege). In the current study, we tested the hypothesis that the CD36+/+ antiangiogenic receptor contributes toward the maintenance of corneal avascularity. METHODS: Corneas of CD36 wild-type (CD36) and knockout (CD36) mice aged 4, 16, 52, and 78 weeks were histologically evaluated for corneal haze and neovascularization (NV). Quantitative real-time polymerase chain reaction was performed on corneal tissue from CD36+/+ and CD36-/- mice aged 4 and 52 weeks to examine the effect of CD36 deficiency on expression of relevant angiogenic factors. RESULTS: Corneal haze and NV were absent in CD36+/+ mice at all ages. Conversely, corneal haze and NV were evident at 52 and 78 weeks in CD36-/- mice, and the latter demonstrated a significant increase in vessel density at 52 and 78 weeks. Interestingly, compared with CD36+/+ mice, in the corneas of 52-week-old CD36-/- mice, thrombospondin-1 messenger RNA was repressed, and vascular endothelial growth factor A, c-Jun N-terminal kinase-1, and c-Jun levels were robustly upregulated. CONCLUSIONS: CD36-/- mice develop corneal NV that increases in severity with age, thus accentuating the role of CD36 in preserving corneal avascularity.

Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.

Recent studies have demonstrated that lymphocyte-derived microparticles (LMPs) impair endothelial cell function. However, no data currently exist regarding the contribution of LMPs in the regulation of angiogenesis. In the present study, we investigated the effects of LMPs on angiogenesis in vivo and in vitro and demonstrated that LMPs strongly suppressed aortic ring microvessel sprouting and in vivo corneal neovascularization. In vitro, LMPs considerably diminished human umbilical vein endothelial cell survival and proliferation in a concentration-dependent manner. Mechanistically, the antioxidants U-74389G and U-83836E were partially protective against the antiproliferative effects of LMPs, whereas the NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium significantly abrogated these effects. Moreover, LMPs increased not only the expression of the NOX subunits gp91(phox), p22(phox), and p47(phox), but also the production of ROS and NOX-derived superoxide (O(2)(-)). Importantly, LMPs caused a pronounced augmentation in the protein expression of the CD36 antiangiogenic receptor while significantly downregulating the protein levels of VEGF receptor type 2 and its downstream signaling mediator, phosphorylated ERK1/2. In summary, LMPs potently suppress neovascularization in vivo and in vitro by augmenting ROS generation via NOX and interfering with the VEGF signaling pathway.

Inflammatory lipid mediators in ischemic retinopathy.

Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane A(2) (TXA(2)) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA(2) and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.