Samuel Burnim

Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy–consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr−/− mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr−/− photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr−/− retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.

Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis

Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases.

Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization

Objective—Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of &ohgr;-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but &ohgr;-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of &ohgr;-3 LCPUFA on neovascular eye diseases. Approach and Results—The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of &ohgr;-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and &ohgr;-3 LCPUFA–derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of &ohgr;-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a &ohgr;-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C &ohgr;-3 LCPUFA–derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected &ohgr;-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. Conclusions—Inhibition of CYP2C activity adds to the protective effects of &ohgr;-3 LCPUFA on pathological retinal neovascularization and CNV.

Corrigendum: Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Jean-Sébastien Joyal, Ye Sun, Marin L Gantner, Zhuo Shao, Lucy P Evans, Nicholas Saba, Thomas Fredrick, Samuel Burnim, Jin Sung Kim, Gauri Patel, Aimee M Juan, Christian G Hurst, Colman J Hatton, Zhenghao Cui, Kerry A Pierce, Patrick Bherer, Edith Aguilar, Michael B Powner, Kristis Vevis, Michel Boisvert, Zhongjie Fu, Emile Levy, Marcus Fruttiger, Alan Packard, Flavio A Rezende, Bruno Maranda, Przemyslaw Sapieha, Jing Chen, Martin Friedlander, Clary B Clish & Lois E H Smith Nat. Med.; doi:10.1038/nm.4059; corrected 24 March 2016

Inflammatory signals from photoreceptor modulate pathological retinal angiogenesis via c-Fos

Pathological neovessels growing into the normally avascular photoreceptors cause vision loss in many eye diseases, such as age-related macular degeneration and macular telangiectasia. Ocular neovascularization is strongly associated with inflammation, but the source of inflammatory signals and the mechanisms by which these signals regulate the disruption of avascular privilege in photoreceptors are unknown. In this study, we found that c-Fos, a master inflammatory regulator, was increased in photoreceptors in a model of pathological blood vessels invading photoreceptors: the very low-density lipoprotein receptor–deficient (Vldlr−/−) mouse. Increased c-Fos induced inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor (TNF), leading to activation of signal transducer and activator of transcription 3 (STAT3) and increased TNF&agr;–induced protein 3 (TNFAIP3) in Vldlr−/− photoreceptors. IL-6 activated the STAT3/vascular endothelial growth factor A (VEGFA) pathway directly, and elevated TNFAIP3 suppressed SOCS3 (suppressor of cytokine signaling 3)–activated STAT3/VEGFA indirectly. Inhibition of c-Fos using photoreceptor-specific AAV (adeno-associated virus)-hRK (human rhodopsin kinase)–sh_c-fos or a chemical inhibitor substantially reduced the pathological neovascularization and rescued visual function in Vldlr−/− mice. These findings suggested that the photoreceptor c-Fos controls blood vessel growth into the normally avascular photoreceptor layer through the inflammatory signal–induced STAT3/VEGFA pathway.

Sema3f Protects Against Subretinal Neovascularization In Vivo

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr−/−) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr−/− model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.

FGF21 Administration Suppresses Retinal and Choroidal Neovascularization in Mice

Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration: very-low-density lipoprotein-receptor-deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited tumor necrosis α (TNF-α) expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration.

Photoreceptor glucose metabolism determines normal retinal vascular growth

The neural cells and factors determining normal vascular growth are not well defined even though vision‐threatening neovessel growth, a major cause of blindness in retinopathy of prematurity (ROP) (and diabetic retinopathy), is driven by delayed normal vascular growth. We here examined whether hyperglycemia and low adiponectin (APN) levels delayed normal retinal vascularization, driven primarily by dysregulated photoreceptor metabolism. In premature infants, low APN levels correlated with hyperglycemia and delayed retinal vascular formation. Experimentally in a neonatal mouse model of postnatal hyperglycemia modeling early ROP, hyperglycemia caused photoreceptor dysfunction and delayed neurovascular maturation associated with changes in the APN pathway; recombinant mouse APN or APN receptor agonist AdipoRon treatment normalized vascular growth. APN deficiency decreased retinal mitochondrial metabolic enzyme levels particularly in photoreceptors, suppressed retinal vascular development, and decreased photoreceptor platelet‐derived growth factor (Pdgfb). APN pathway activation reversed these effects. Blockade of mitochondrial respiration abolished AdipoRon‐induced Pdgfb increase in photoreceptors. Photoreceptor knockdown of Pdgfb delayed retinal vascular formation. Stimulation of the APN pathway might prevent hyperglycemia‐associated retinal abnormalities and suppress phase I ROP in premature infants.

Fibroblast Growth Factor 21 Protects Photoreceptor Function in Type 1 Diabetic Mice

Retinal neuronal abnormalities occur before vascular changes in diabetic retinopathy. Accumulating experimental evidence suggests that neurons control vascular pathology in diabetic and other neovascular retinal diseases. Therefore, normalizing neuronal activity in diabetes may prevent vascular pathology. We investigated whether fibroblast growth factor 21 (FGF21) prevented retinal neuronal dysfunction in insulin-deficient diabetic mice. We found that in diabetic neural retina, photoreceptor rather than inner retinal function was most affected and administration of the long-acting FGF21 analog PF-05231023 restored the retinal neuronal functional deficits detected by electroretinography. PF-05231023 administration protected against diabetes-induced disorganization of photoreceptor segments seen in retinal cross section with immunohistochemistry and attenuated the reduction in the thickness of photoreceptor segments measured by optical coherence tomography. PF-05231023, independent of its downstream metabolic modulator adiponectin, reduced inflammatory marker interleukin-1β (IL-1β) mRNA levels. PF-05231023 activated the AKT-nuclear factor erythroid 2–related factor 2 pathway and reduced IL-1β expression in stressed photoreceptors. PF-05231023 administration did not change retinal expression of vascular endothelial growth factor A, suggesting a novel therapeutic approach for the prevention of early diabetic retinopathy by protecting photoreceptor function in diabetes.

RORα modulates semaphorin 3E transcription and neurovascular interaction in pathological retinal angiogenesis

Pathological proliferation of retinal blood vessels commonly causes vision impairment in proliferative retinopathies, including retinopathy of prematurity. Dysregulated crosstalk between the vasculature and retinal neurons is increasingly recognized as a major factor contributing to the pathogenesis of vascular diseases. Class 3 semaphorins (SEMA3s), a group of neuron‐secreted axonal and vascular guidance factors, suppress pathological vascular growth in retinopathy. However, the upstream transcriptional regulators that mediate the function of SEMA3s in vascular growth are poorly understood. Here we showed that retinoic acid receptor–related orphan receptor α (RORα), a nuclear receptor and transcription factor, is a novel transcriptional regulator of SEMA3E‐mediated neurovascular coupling in a mouse model of oxygen‐induced proliferative retinopathy. We found that genetic deficiency of RORα substantially induced Sema3e expression in retinopathy. Both RORα and SEMA3E were expressed in retinal ganglion cells. RORα directly bound to a specific ROR response element on the promoter of Sema3e and negatively regulated Sema3e promoter–driven luciferase expression. Suppression of Sema3e using adeno‐associated virus 2 carrying short hairpin RNA targeting Sema3e promoted disoriented pathological neovascularization and partially abolished the inhibitory vascular effects of RORα deficiency in retinopathy. Our findings suggest that RORα is a novel transcriptional regulator of SEMA3E‐mediated neurovascular coupling in pathological retinal angiogenesis.—Sun, Y., Liu, C.‐H., Wang, Z., Meng, S. S., Burnim, S. B., SanGiovanni, J. P., Kamenecka, T. M., Solt, L. A., Chen, J. RORα modulates semaphorin 3E transcription and neurovascular interaction in pathological retinal angiogenesis. FASEB J. 31, 4492–4502 (2017). www.fasebj.org