Short Communication: PPARγ Mediates a Direct Antiangiogenic Effect of ω3-PUFAs in Proliferative RetinopathyNovelty and Significance
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.
Proliferative retinopathy is a major cause of blindness that occurs in 2 stages. Loss of functional vasculature in the early phase leads to retinal hypoxia that in turn drives a compensatory but destructive growth of pathological neovessels in the later phase. We previously found that dietary ω3-PUFAs given before the onset of neovascularization in the oxygen-induced retinopathy (OIR) mouse model stimulate the regrowth of normal retinal vasculature,1 thus limiting the hypoxic stimulus for subsequent proliferative retinopathy. It is, however, not known if ω3-PUFAs also have direct effects on retinal neovascularization (NV) formation and what mechanisms might be involved in mediating this effect.
In this study, we expose mice to ω3-PUFAs exclusively during the peak proliferative stage (phase II) of retinopathy. With this approach ω3-PUFAs selectively target retinal NV formation, without affecting vasoobliteration or regrowth of normal vessels. Our results demonstrate that ω3-PUFAs given after the onset of active retinopathy exert a beneficial effect on the inflammatory and angiogenic activation state of the retina that, mediated via the endogenous ω3-PUFA receptor peroxisome proliferator-activated receptor (PPAR)γ, leads to attenuated retinal NV.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
O2-Induced Retinopathy, ω3-PUFA Intervention, and PPARγ Inhibition
O2-induced retinopathy has been described in detail.2,–,4 C57BL/6 mothers with pups were fed standard chow until postnatal day (P)14 and then switched to a defined diet with 10% (wt/wt) safflower oil containing either 2% ω3-PUFAs (docosahexaenoic acid and eicosapentaenoic acid) or 2% ω6-PUFAs (arachidonic acid).1 The lipid composition of the mother's milk reflects the mother's diet,5 and changes in the mother's diet are efficiently transferred to the pups.1 GW9662, a specific PPARγ-antagonist with a nanomolar inhibitory concentration6 that effectively blocks PPARγ in vivo7 was injected IP daily into mouse pups from P14 to P17 (1 mg/kg in 20 μL). Vehicle control was PBS/DMSO 1:1.
Results and Discussion
ω3-PUFA Intervention Directly Attenuates Retinal NV
In the OIR mouse model, vascular loss develops during hyperoxic incubation from P7 to P12. After return to room air, the avascular areas of the retina become hypoxic and respond with upregulation of angiogenic growth factors like vascular endothelial growth factor (VEGF) (Figure 1a). VEGF upregulation is followed by retinal neovessel formation seen morphologically from P15 onwards and displaying maximal severity at P17 (Figure 1b). Because dietary ω3-PUFAs are transferred from nursing mothers to the pups with some delay, ω3-PUFA treatment was started at P14 to specifically target the active phase of NV formation from P15 onwards (Figure 1c). At P17, ω3-PUFA–fed mice show significantly less retinal NV compared to ω6 controls (5.2±0.5 versus 9.1±0.5% NV; P=10−5) (Figure 1d). Vasoobliteration (VO) at P17 is not affected (ω3 versus ω6: 22.5±1.2 versus 21.5±1.3% VO; P=0.6) (Figure 1e). Enlarged retinal quadrants are shown in Online Figure I (a and b). The ω6-diet with arachidonic acid or other ω6-PUFAs did not significantly exacerbate retinal NV compared to standard rodent chow, indicating that the observed differences in NV formation are mainly attributable to a beneficial effect of ω3-PUFAs (Online Figure I, c and d).
These results demonstrate, for the first time, that ω3-PUFAs can attenuate retinal NV formation directly, even when retinal hypoxia is already established and pathological neovascularization is in its active stage.
PPARγ Activation Is Required for ω3-PUFA—Mediated Attenuation of NV
The rapidity by which ω3-PUFA intervention attenuates NV suggests a receptor-based mechanism. We therefore investigated the involvement of the endogenous ω3-PUFA receptor PPARγ.8 Pharmacological inhibition of PPARγ from P14 to P17 with the specific inhibitor GW96626,7 significantly reduces the inhibitory effects of ω3-PUFAs on retinal NV (4.7±0.4 versus 7.2±0.4% NV; P=0.0008) without altering retinal NV in ω6 groups (7.4±0.5 versus 6.9±0.6% NV; P=0.5) (Figure 2a and 2c). VO at P17 was not altered in either group (21.8±1.5% versus 22.1±1.5% VO; P=0.9 and 20.5±2% versus 20.8±1.5% VO; P=0.9) (Figure 2b). Original retinal images and enlarged quadrants are shown in Online Figure II. Notably, pharmacological inhibition of PPARγ also reduces the expression of PPARγ in circulating leukocytes from ω3-fed mice to levels seen in the ω6 control group (Online Figure III).
These results identify PPARγ as a major pathway through which ω3-PUFAs exert their beneficial effects on retinal neovascularization. The swiftness of the ω3-PUFA effects and the near-complete abrogation with PPARγ inhibition indicate a central role for this transcriptional regulator in conveying the ω3-PUFA effects. Importantly, pharmacological PPARγ activation with thiazolidinediones, used to control hyperglycemia in type II diabetes, similarly attenuates proliferative retinopathy both in animal models and in diabetic patients,9,10 raising the possibility that ω3-PUFAs might have clinical effects similar to pharmacological PPARγ activators.
ω3-PUFA Intervention Reduces Retinal Inflammatory Activity via PPARγ
Given that retinal VEGF levels were not altered by ω3-PUFA intervention (Figure 3a and 3b) and given the known involvement of PPARγ in inflammatory modulation,11 we hypothesized that ω3-PUFAs might limit NV by modulating retinal inflammatory pathways. Tumor necrosis factor (TNF)α is a major inflammatory mediator whose expression is upregulated during the proliferative stage of OIR.12 In P17 OIR retinas, TNFα localizes to retinal neovessels (arrows in Figure 3c), as well as to macrophages associated with these neovessels (arrowheads in Figure 3c). ω3-PUFA treatment reduces TNFα protein both in retina and serum in a PPARγ-dependent fashion (Figure 3d and 3e). Similarly, murine macrophages incubated with ω3-PUFAs (docosahexaenoic acid) in vitro express significantly less TNFα than those incubated with ω6-PUFAs (arachidonic acid); a difference that is also dependent on PPARγ (Figure 3f). Analogous to the retina, macrophage expression of VEGF is not modulated by ω3 or ω6 treatment in vitro (Figure 3g).
These results indicate that ω3-PUFAs attenuate NV formation without altering the hypoxia-driven VEGF axis but by modulating the inflammatory state of the retina via PPARγ. These findings are in line with data obtained with pharmacological PPARγ activators that reduce retinal TNFα and NV formation in the OIR model without changing VEGF overexpression9,13 and reduce TNFα levels in diabetic patients.14
ω3-PUFA Intervention Reduces Endothelial Cell Activation via PPARγ
Besides modulating inflammatory mediators, ω3-PUFAs can directly attenuate activation of endothelial cells (ECs).15 EC activation is in part characterized by upregulation of adhesion molecules16 and angiopoietin (Ang)-217 and renders ECs more responsive to angiogenic stimulation. Ang-2 in particular has been found to be expressed only weakly in resting endothelium but robustly following endothelial activation.18 At P17, both Ang-2 and the adhesion molecule intercellular adhesion molecule (ICAM)-1 are expressed in activated ECs from retinal neovessels but not in quiescent vessels nearby (Figure 4a and 4b). When neovessels are isolated using laser-capture microdissection (Figure 4c), NV from ω3 animals show a PPARγ-dependent reduction of ICAM-1, VCAM-1 (vascular cell adhesion molecule-1), E-Selectin (endothelial selectin), and Ang-2 compared to ω6 mice (Figure 4d through 4g). In line with the mRNA data, ω3-PUFAs reduce retinal Ang-2 protein, mediated in part via PPARγ (Figure 4h). Ang-1 and ICAM-2 are not significantly regulated between ω3- and ω6 groups (Online Figure IV, a and b). Importantly, other nonendothelial cells closely associated with neovessels may also contribute to the inflammatory profile presented. Particularly neovessel-associated macrophages (Figure 3c) may influence the local inflammatory milieu. This is illustrated by higher levels of local TNFα expression in laser-captured neovessels from ω6 mice compared to ω3 mice (Online Figure IV, c).
In summary, this study demonstrates that ω3-PUFA intervention during the proliferative phase of retinopathy decreases the neovascular activity of the retina via PPARγ-dependent reduction of inflammatory mediators and attenuation of EC activation. The PPARγ-dependent effect of ω3-PUFAs on NV formation is large and comparable to anti-VEGF treatment in the OIR model.4 This is the first study to identify a direct inhibitory effect of ω3-PUFAs on retinal neovascularization and the first to identify PPARγ as one of the major pathways mediating this effect. Because retinal VEGF levels are unaffected, ω3-PUFA–dependent activation of PPARγ might be considered as an additive treatment to the current anti-VEGF approaches for pathological retinal angiogenesis.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (to A.S.); Canadian Institutes of Health Research and Charles A. King Trust Award (to P.S.); William Randolph Hearst Award, March of Dimes Foundation, and a Simeon Burt Wolbach Research Fellowship (to K.M.C.); Juvenile Diabetes Research Foundation International, Knights Templar Eye Foundation, and CHB Manton Center for Orphan Disease (to J.C.); NIH grants EY017017 and EY017017-S1, V. Kann Rasmussen Foundation, RoFAR, CHB Mental Retardation and Developmental Disabilities Research Center, an RPB Senior Investigator Award, Alcon Research Institute Award, and the MacTel Foundation (to L.E.H.S.).
In May 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
Non-standard Abbreviations and Acronyms
- angiopoietin 2
- endothelial cell
- endothelial selectin
- intercellular adhesion molecule
- oxygen-induced retinopathy
- postnatal day
- peroxisome proliferator-activated receptor
- long-chain polyunsaturated fatty acid
- tumor necrosis factor
- vascular cell adhesion molecule
- vascular endothelial growth factor
- Received April 2, 2010.
- Revision received June 11, 2010.
- Accepted July 1, 2010.
- © 2010 American Heart Association, Inc.
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Novelty and Significance
What Is Known
Omega3 long-chain polyunsaturated fatty acids (ω3-PUFAs) are angiogenic modulators that can attenuate the severity of retinal neovascularization (NV).
Whether ω3-PUFAs exert a direct effect on retinal neovessel formation and what mechanisms might be involved, remained unknown.
What New Information Does This Article Contribute
By specifically targeting the neovascular phase of proliferative retinopathy (phase II), this study identifies, for the first time, a direct beneficial effect of ω3-PUFAs on retinal NV formation during the active proliferative phase.
This study identifies peroxisome proliferator-activated receptor (PPAR)γ as a major pathway through which ω3-PUFAs exert their beneficial effects on NV formation.
Activation of PPARγ by ω3-PUFAs affected the inflammatory state of the retina and attenuated angiogenic activation of retinal endothelial cells.
We have shown previously that ω3-PUFAs given before the onset of retinopathy can attenuate disease severity. In many patients, however, retinopathy is diagnosed when the disease has already progressed to active neovascularization. This study investigated whether the known endogenous ω3-PUFA receptor PPARγ is involved in mediating a potential direct effect of ω3-PUFAs on retinal neovessel formation. Our study demonstrates that ω3-PUFAs given selectively during the neovascular phase of retinopathy directly reduce retinal neovessel formation without altering vasoobliteration or regrowth of normal vessels. Mechanistically, we identify PPARγ as a major mediator for the ω3-PUFA effect that acts independent of VEGF through modulation of inflammatory mediators and reduced angiogenic activation of retinal endothelial cells. ω3-PUFAs might thus be considered as an additive treatment to anti-VEGF therapies.