R. J. Dennison

Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis

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.

The Mouse Retina as an Angiogenesis Model

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.

5-Lipoxygenase Metabolite 4-HDHA Is a Mediator of the Antiangiogenic Effect of ω-3 Polyunsaturated Fatty Acids

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. LOX and Vessels In a dreaded morning ritual, parents once force-fed cod liver oil—a source of vitamin D—to their children to prevent rickets. Today, fish oil has a new star: ω-3 polyunsaturated fatty acids (PUFAs). These molecules—eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)—can protect against the abnormal growth of blood vessels, a prime contributor to diabetes-related blindness and retinopathy associated with premature birth. Sapieha et al. have now enriched our understanding of the mechanism behind this protection by showing that the production of a DHA metabolite, 4-hydroxy-docosahexaenoic acid (4-HDHA), by the enzyme 5-lipoxygenase (5-LOX) is one of the main metabolic pathways required for the antiangiogenic effect. 4-HDHA acts via peroxisome proliferator–activated receptor γ (PPARγ) to directly inhibit the sprouting and proliferation of endothelial cells—those that line the inner surfaces of blood vessels. Mice that are exposed to high amounts of oxygen (75%) from day 7 to 12 after birth and then returned to ambient air develop abnormal revascularization of the retina. Using this system as an approximation of human retinal revascularization, the authors found that feeding mice a diet high in ω-3 PUFAs, but not ω-6 PUFAs, could prevent this event. By genetically removing, one by one, each of the four main enzymes that convert these ω-3 PUFAs to active metabolites, the authors found that protection against revascularization required 5-LOX, a non–heme iron dioxygenase that generates leukotrienes, lipoxins, and the recently identified ω-3 PUFA–derived resolvins and protectins. After identifying the critical 5-LOX as the one present in white blood cells, Sapieha et al. found that large amounts of DHA metabolites were generated under stress conditions such as those that produce retinopathy in premature newborns. One metabolite, 4-HDHA, was detected in the serum and white blood cells of mice and humans and in retinas of mice with oxygen-induced retinopathy. 4-HDHA is known to act through the PPARγ receptor, and pharmacological inhibition of PPARγ interfered with the ability of the ω-3 diet to alleviate oxygen-induced retinopathy in the mice. These results elucidate an important pathway through which ω-3 oils protect against retinopathy and perhaps exert some of their other beneficial effects: oxidation of ω-3 PUFAs by 5-LOX and subsequent inhibition of angiogenesis via PPARγ activation. One implication of this work is good news for those who take aspirin and ibuprofen: The cyclooxygenase inhibition by these agents will not interfere with the healthy effects of your daily dose of fish oil. 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 microglial-derived tumor necrosis factor–α. 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 lipid-processing 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 oxidation of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxy-docosahexaenoic 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 anti-inflammatory effects. Our study suggests that ω-3 PUFAs may be profitably 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.

Wnt signaling mediates pathological vascular growth in proliferative 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.

Postnatal Weight Gain Modifies Severity and Functional Outcome of Oxygen-Induced Proliferative Retinopathy

In clinical studies, postnatal weight gain is strongly associated with retinopathy of prematurity (ROP). However, animal studies are needed to investigate the pathophysiological mechanisms of how postnatal weight gain affects the severity of ROP. In the present study, we identify nutritional supply as one potent parameter that affects the extent of retinopathy in mice with identical birth weights and the same genetic background. Wild-type pups with poor postnatal nutrition and poor weight gain (PWG) exhibit a remarkably prolonged phase of retinopathy compared to medium weight gain or extensive weight gain pups. A high (r(2) = 0.83) parabolic association between postnatal weight gain and oxygen-induced retinopathy severity is observed, as is a significantly prolonged phase of proliferative retinopathy in PWG pups (20 days) compared with extensive weight gain pups (6 days). The extended retinopathy is concomitant with prolonged overexpression of retinal vascular endothelial growth factor in PWG pups. Importantly, PWG pups show low serum levels of nonfasting glucose, insulin, and insulin-like growth factor-1 as well as high levels of ghrelin in the early postoxygen-induced retinopathy phase, a combination indicative of poor metabolic supply. These differences translate into visual deficits in adult PWG mice, as demonstrated by impaired bipolar and proximal neuronal function. Together, these results provide evidence for a pathophysiological correlation between poor postnatal nutritional supply, slow weight gain, prolonged retinal vascular endothelial growth factor overexpression, protracted retinopathy, and reduced final visual outcome.

Resveratrol Inhibits Pathologic Retinal Neovascularization in Vldlr / Mice

PURPOSE Macular telangiectasia (MacTel) is a vision-threatening retinal disease with unknown pathogenesis and no approved treatment. Very low-density lipoprotein receptor mutant mice (Vldlr(-/-)) exhibit critical features of MacTel such as retinal neovascularization and photoreceptor degeneration. In this study, the authors evaluate the therapeutic potential of resveratrol, a plant polyphenol, in Vldlr(-/-) mice as a model for MacTel. METHODS Vldlr(-/-) and wild-type mice at postnatal day (P) 21 to P60 or P10 to P30 were treated orally with resveratrol. The number of neovascular lesions was evaluated on retinal flatmounts, and resveratrol effects on endothelial cells were assessed by Western blot for phosphorylated ERK1/2, aortic ring, and migration assays. Vegf and Gfap expression was evaluated in laser-capture microdissected retinal layers of angiogenic lesions and nonlesion areas from Vldlr(-/-) and wild-type retinas. RESULTS From P15 onward, Vldlr(-/-) retinas develop vascular lesions associated with the local upregulation of Vegf in photoreceptors and Gfap in the inner retina. Oral resveratrol reduces lesion formation when administered either before or after disease onset. The reduction of vascular lesions in resveratrol-treated Vldlr(-/-) mice is associated with the suppression of retinal Vegf transcription. Resveratrol also reduces endothelial ERK1/2 signaling as well as the migration and proliferation of endothelial cells. Furthermore, a trend toward increased rhodopsin mRNA in Vldlr(-/-) retinas is observed. CONCLUSIONS Oral administration of resveratrol is protective against retinal neovascular lesions in Vldlr(-/-) mice by inhibiting Vegf expression and angiogenic activation of retinal endothelial cells. These results suggest that resveratrol might be a safe and effective intervention for treating patients with MacTel.

Short communication: PPAR gamma mediates a direct antiangiogenic effect of omega 3-PUFAs in proliferative retinopathy.

Rationale Omega3 long-chain polyunsaturated fatty acids (&ohgr;3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing &ohgr;3-PUFA–dependent attenuation of angiogenesis. Objective This study aims to identify a major mechanism by which &ohgr;3-PUFAs attenuate retinal neovascularization. Methods and Results Administering &ohgr;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)&ggr; almost completely abrogates this effect. Inhibition of PPAR&ggr; also reverses the &ohgr;3-PUFA–induced reduction of retinal tumor necrosis factor-&agr;, 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&ggr;-mediated effect of &ohgr;3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

SOCS3 is an endogenous inhibitor of pathologic angiogenesis

Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.

Short Communication: PPARγ Mediates a Direct Antiangiogenic Effect of ω3-PUFAs in Proliferative RetinopathyNovelty and Significance

Rationale Omega3 long-chain polyunsaturated fatty acids (&ohgr;3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing &ohgr;3-PUFA–dependent attenuation of angiogenesis. Objective This study aims to identify a major mechanism by which &ohgr;3-PUFAs attenuate retinal neovascularization. Methods and Results Administering &ohgr;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)&ggr; almost completely abrogates this effect. Inhibition of PPAR&ggr; also reverses the &ohgr;3-PUFA–induced reduction of retinal tumor necrosis factor-&agr;, 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&ggr;-mediated effect of &ohgr;3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice

Objective:Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM).Design:Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks–26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet.Results:The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs.Conclusion:This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.