Karen I. Guerin

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.

SIRT1 Is Essential for Normal Cognitive Function and Synaptic Plasticity

Conservation of normal cognitive functions relies on the proper performance of the nervous system at the cellular and molecular level. The mammalian nicotinamide-adenine dinucleotide-dependent deacetylase SIRT1 impacts different processes potentially involved in the maintenance of brain integrity, such as chromatin remodeling, DNA repair, cell survival, and neurogenesis. Here we show that SIRT1 is expressed in neurons of the hippocampus, a key structure in learning and memory. Using a combination of behavioral and electrophysiological paradigms, we analyzed the effects of SIRT1 deficiency and overexpression on mouse learning and memory as well as on synaptic plasticity. We demonstrated that the absence of SIRT1 impaired cognitive abilities, including immediate memory, classical conditioning, and spatial learning. In addition, we found that the cognitive deficits in SIRT1 knock-out (KO) mice were associated with defects in synaptic plasticity without alterations in basal synaptic transmission or NMDA receptor function. Brains of SIRT1-KO mice exhibited normal morphology and dendritic spine structure but displayed a decrease in dendritic branching, branch length, and complexity of neuronal dendritic arbors. Also, a decrease in extracellular signal-regulated kinase 1/2 phosphorylation and altered expression of hippocampal genes involved in synaptic function, lipid metabolism, and myelination were detected in SIRT1-KO mice. In contrast, mice with high levels of SIRT1 expression in brain exhibited regular synaptic plasticity and memory. We conclude that SIRT1 is indispensable for normal learning, memory, and synaptic plasticity in mice.

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.

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.

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.

PPARγ Mediates a Direct Antiangiogenic Effect of Ω3-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.

Short Communication: PPARγ Mediates a Direct Antiangiogenic Effect of ω3-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.