Lipid signaling is dysregulated in many diseases with vascular pathology, including cancer, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. We have previously demonstrated that diets enriched in ω-3 polyunsaturated fatty acids (PUFAs) effectively reduce pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy, in part through metabolic products that suppress microglialderived tumor necrosis factor–a. To better understand the protective effects of ω-3 PUFAs, we examined the relative importance of major lipid metabolic pathways and their products in contributing to this effect. ω-3 PUFA diets were fed to four lines of mice deficient in each key lipidprocessing enzyme (cyclooxygenase 1 or 2, or lipoxygenase 5 or 12/15), retinopathy was induced by oxygen exposure; only loss of 5-lipoxygenase (5-LOX) abrogated the protection against retinopathy of dietary ω-3 PUFAs. This protective effect was due to 5-LOX oxida-tion of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxydocosahexaenoic acid (4-HDHA). 4-HDHA directly inhibited endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator–activated receptor γ (PPARγ), independent of 4-HDHA’s antiinflammatory effects. Our study suggests that ω-3 PUFAs may be profit-ably used as an alternative or supplement to current anti–vascular endothelial growth factor (VEGF) treatment for proliferative retinopathy and points to the therapeutic potential of ω-3 PUFAs and metabolites in other diseases of vasoproliferation. It also suggests that cyclooxygenase inhibitors such as aspirin and ibuprofen (but not lipoxygenase inhibitors such as zileuton) might be used without losing the beneficial effect of dietary ω-3 PUFA. Source Science Translational Medicine, Vol. 3, Issue 69, Pages 1-12: “5-Lipoxygenase Metabolite 4-HDHA Is a Mediator of the Antiangiogenic Effect of w-3 Polyunsaturated Fatty Acids.” February 09, 2011 Effect of Omega-3 Fatty Acid Supplementation on Omega-3 Index and Red Blood Cell (RBC) Membrane Fatty Acid Composition ABSTRACT Omega-3 fatty acids (n-3 FA) have been shown from epidemiological studies and clinical trials to reduce the incidence of cardiovascular disease (CVD) in patients with the pre-existing CVD as well as in healthy individuals. In randomized secondary prevention trials fish or fish oil have been shown to reduce total and coronary heart disease (CHD) mortality at intakes of about 1 g/day. The Omega-3 Index (EPA + DHA expressed as % of total fatty acid) has been proposed as a physiologically relevant, modifiable, independent and graded risk factor for death from CHD. RBC membrane fatty acid composition correlates well with biomarkers of n-3 FA including serum EPA and DHA, whole blood EPA, DPA and DHA and fatty acid composition of cardiac tissue. The present dietary intervention study investigates the effect of a fish-oil based, n-3 FA supplement on Omega-3 Index and RBC fatty acid composition. Thirty healthy men and women consumed a supplement providing 1070 mg total n-3 FA (460 mg DHA, 480 mg EPA, and 80 mg other n-3 FA) daily for 4 wks. At the end of this period there was a significant 23% increase (p<0.01) in the Omega-3 Index (Fig.1). While there were no significantOmega-3 fatty acids (n-3 FA) have been shown from epidemiological studies and clinical trials to reduce the incidence of cardiovascular disease (CVD) in patients with the pre-existing CVD as well as in healthy individuals. In randomized secondary prevention trials fish or fish oil have been shown to reduce total and coronary heart disease (CHD) mortality at intakes of about 1 g/day. The Omega-3 Index (EPA + DHA expressed as % of total fatty acid) has been proposed as a physiologically relevant, modifiable, independent and graded risk factor for death from CHD. RBC membrane fatty acid composition correlates well with biomarkers of n-3 FA including serum EPA and DHA, whole blood EPA, DPA and DHA and fatty acid composition of cardiac tissue. The present dietary intervention study investigates the effect of a fish-oil based, n-3 FA supplement on Omega-3 Index and RBC fatty acid composition. Thirty healthy men and women consumed a supplement providing 1070 mg total n-3 FA (460 mg DHA, 480 mg EPA, and 80 mg other n-3 FA) daily for 4 wks. At the end of this period there was a significant 23% increase (p<0.01) in the Omega-3 Index (Fig.1). While there were no significant changes in % linoleic, gamma linoleic, and alpha linolenic acid; EPA, DPA and DHA Omega III Salmon Oil Plus Published in FASEB online Journal April 2008

Background Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and use of peroxisome proliferator activator receptor (PPAR)-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG) co-activator 1 alpha (PPARGC1A), a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR) transcription complex, may influence neovascularization (NV) in age-related macular degeneration (AMD). Methods We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A. Results A DNA coding variant (rs3736265) and a 3'UTR-resident regulatory variant (rs3774923) in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs). SNP-SNP interactions existed for NV AMD (P<0.005) with rs3736265 and a AMD-associated variant in complement factor B (CFB, rs512559). PPARGC1A influences activation of the AMD-associated complement component 3 (C3) promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P≤0.003) of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033), a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678) showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P≤0.003). C3 expression was down-regulated 2-fold in retinas of ω-3 LCPUFA-fed mice – these animals also showed 70% reduction in retinal NV (P≤0.001). Conclusion Ligands and co-activators of the ω-3 LCPUFA sensing PPAR-RXR axis may influence retinal angiogenesis in NV AMD via the complement and VEGF signaling systems. We have linked the co-activator of a lipid-sensing transcription factor (PPARG co-activator 1 alpha, PPARGC1A) to age-related macular degeneration (AMD) and AMD-associated genes.

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5(-/-)) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5(-/-) mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5(-/-) and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5(-/-) retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5(-/-) retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5(-/-) retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

In mice, ω-3 fatty acids in the diet are converted by 5-lipoxygenase to a metabolite that inhibits destructive vessel growth similar to diabetic retinopathy.

Background— Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. Methods and Results— In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor–related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5 −/− vessels. Blocking claudin5 significantly suppresses Wnt pathway–driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. Conclusions— These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

Rationale: Omega3 long-chain polyunsaturated fatty acids (ω3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing ω3-PUFA–dependent attenuation of angiogenesis. Objective: This study aims to identify a major mechanism by which ω3-PUFAs attenuate retinal neovascularization. Methods and Results: Administering ω3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)γ almost completely abrogates this effect. Inhibition of PPARγ also reverses the ω3-PUFA–induced reduction of retinal tumor necrosis factor-α, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor. Conclusions: These results identify a direct, PPARγ-mediated effect of ω3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

The mouse retina has been used extensively over the past decades to study both physiologic and pathologic angiogenesis. Over time, various mouse retina models have evolved into well-characterized and robust tools for in vivo angiogenesis research. This article is a review of the angiogenic development of the mouse retina and a discussion of some of the most widely used vascular disease models. From the multitude of studies performed in the mouse retina, a selection of representative works is discussed in more detail regarding their role in advancing the understanding of both the ocular and general mechanisms of angiogenesis.

Rationale: Omega3 long-chain polyunsaturated fatty acids (omega 3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing omega 3-PUFA-dependent attenuation of angiogenesis.Objective: This study aims to identify a major mechanism by which omega 3-PUFAs attenuate retinal neovascularization.Methods and Results: Administering omega 3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)gamma almost completely abrogates this effect. Inhibition of PPAR gamma also reverses the omega 3-PUFA-induced reduction of retinal tumor necrosis factor-alpha, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor.Conclusions: These results identify a direct, PPAR gamma -mediated effect of omega 3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor. (Circ Res. 2010; 107: 495-500.)

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

PURPOSE:Retinopathy is a result of pathologic angiogenesis influenced by insulinlike growth factor (IGF)-1. The authors examined the local expression of the IGF/insulin family.METHODS:In retinas with and without oxygen-induced retinopathy, the authors assessed with real-time RT-PCR mRNA expression of the IGF-1 receptor (IGF-1R), insulin receptor (IR), IGF-1, IGF-2, insulin (Ins2), and IGF-binding protein 1 (IGFBP1) to IGFBP6 in total retina from postnatal day (P) 7 to P33 to examine changes over time with the induction of retinopathy and at P17 on laser-captured retinal components to quantitatively localize mRNA expression in the ganglion cell layer, the outer nuclear layer, the inner nuclear layer, normal blood vessels, and neovascular tufts.RESULTS:IGF-1R and IR are expressed predominantly in photoreceptors and in vessels, with scant expression in the rest of the neural retina. IGF-1R expression is more than 100-fold greater than IR. The major local growth factor (expressed in photoreceptors and in blood vessels) is IGF-2 (approximately 1000-fold greater than IGF-1). IGF-1 (approximately 600 copies/10(6) cyclophilin) is expressed throughout the retina. IGFBP2, IGFBP4, and IGFBP5 expression is unchanged with increasing retinal development and with the induction of retinopathy. In contrast, IGFBP3 expression increased more than 5-fold with hypoxia, found in neovascular tufts.CONCLUSIONS:IGF-1R, IR, and the ligand IGF-2 are expressed almost exclusively in photoreceptors and blood vessels. IGFBP3 and IGFBP5 expression increases in neovascular tufts compared with normal vessels. IGF-1 is expressed throughout the retina at much lower levels. These results suggest cross-talk between vessels and photoreceptors in the development of retinopathy and retinal vasculature.

PURPOSE:Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy. METHODS:The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17. RESULTS:Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%. CONCLUSIONS:Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

The mouse model of oxygen-induced retinopathy (OIR) has been widely used in studies related to retinopathy of prematurity, proliferative diabetic retinopathy and in studies evaluating the efficacy of antiangiogenic compounds. In this model, 7-d-old (P7) mouse pups with nursing mothers are subjected to hyperoxia (75% oxygen) for 5 d, which inhibits retinal vessel growth and causes significant vessel loss. On P12, mice are returned to room air and the hypoxic avascular retina triggers both normal vessel regrowth and retinal neovascularization (NV), which is maximal at P17. Neovascularization spontaneously regresses between P17 and P25. Although the OIR model has been the cornerstone of studies investigating proliferative retinopathies, there is currently no harmonized protocol to assess aspects of angiogenesis and treatment outcome. In this protocol we describe standards for mouse size, sample size, retinal preparation, quantification of vascular loss, vascular regrowth, NV and neovascular regression.