PPARβ/δ selectively regulates phenotypic features of age-related macular degeneration

Results

The PPARβ/δ pathway is biologically active in AMD vulnerable cells

The activity of PPARβ/δ in AMD vulnerable cells was examined in human primary RPE cells, the human derived ARPE19, and the macaque derived RF/6A choroidal endothelial cell lines. This was performed by examination of the (i) expression of PPARβ/δ and its binding partners the retinoid X receptor alpha and beta (RXRα and β), (ii) receptor transcriptional activity, by measuring the binding of the receptor-ligand complex to the gene-response element, and (iii) expression of known PPARβ/δ target genes. Freshly isolated human RPE cells and choroid from aged donor eyes, along with primary human RPE cultured cells, ARPE19 and RF/6A cell lines, expressed PPARβ/δ and its binding partners the RXRs (Figure 1A). Additionally, ligand activation of PPARβ/δ with GW0742 (10μM) caused increased transcriptional activity in human primary RPE (Figure 1B), RF/6A cells (Figure 1C) and ARPE19 cells (Figure S1) [16]. These changes were mitigated by the PPARβ/δ antagonist, GSK0660 (10μM), and siRNA-mediated knockdown of PPARβ/δ (Figure 1, B and C). Similarly, ligand activation of PPARβ/δ increased expression of the PPARβ/δ target genes, angiopoietin-like 4 (ANGPTL4) and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), in primary RPE cells (Figure 1, D and E), RF/6A cells and ARPE19 cells (Figure 1, F, G, S1 and S2), an effect that was diminished by treatment with a PPARβ/δ antagonist or PPARβ/δ knockdown.

Figure 1. PPARβ/δ signaling pathway is functional in AMD vulnerable cells. (A) Agarose gel image of PCR amplification products of PPARβ/δ and its obligate binding partners RXRα and RXRβ in primary human RPE cells [R], freshly isolated human RPE cells [hR], ARPE19 cells [A], human choroid [hC], and RF/6A cells [C], 36B4 was used as loading control. PPARβ/δ activity in primary RPE (1°RPE) cells (B) and RF/6A cells (C) transfected with the DR1 luciferase reporter and siC or siPPARβ/δ; cells were treated with PPARβ/δ agonist, GW0742 (10μM) or antagonist, GSK0660 (10μM) or DMSO as vehicle control (n = 3): a: p < 0.05 relative to DMSO treated cells; b: p < 0.05 relative to drug+siC treated cells (p <  0.05; two way ANOVA, Sidak’s multiple comparisons test). Expression of ANGPTL4 and PDK4 mRNA in primary RPE (1°RPE) cells (D and E) and RF/6A (F and G) in siC and siPPARβ/δ (100 pmoles/250,000 cells) treated cells in response to GW0742, GSK0660, or DMSO as a control (n = 3); a: p < 0.05 relative to DMSO treated cells; b: p < 0.05 relative to drug+siC treated cells (Two way ANOVA, Sidak’s multiple comparisons test).

Loss of PPARβ/δ results in selective regulation of dry and wet AMD related pathogenic pathways

The discovery of multiple genetic, systemic, and environmental risk factors associated with AMD ontology has resulted in the identification of several AMD-pathogenic pathways. These pathways include, but are not limited to, impairment of extracellular matrix turnover [3,17], increased angiogenesis [18], inflammation [19,20], and dysregulation of lipid processing pathways [21]. Since modulation of the PPARβ/δ pathway has been shown to regulate collagen synthesis in vitro and in vivo [22–24], the effect of PPARβ/δ knockdown (siPPARβ/δ) on the expression of extracellular matrix-related genes was assessed. Deletion of PPARβ/δ expression caused upregulation of collagen type 1A1 (COL1A1) and vitronectin (VTN) in human primary RPE cells, and no significant effect on expression levels of collagen type 4A4 (COL4A4) and fibronectin (FN1) (Figure 2A). In RF/6A cells, deletion of PPARβ/δ resulted in downregulation of the extracellular matrix genes COL1A1, COL4A4 and FN1 (Figure 2B). Increased deposition of collagen type 1A1, collagen 4A4 and vitronectin is characteristic of Bruch’s membrane and human sub-RPE deposits typically observed in dry AMD [25], while endothelial cells require extracellular matrix molecules such as COL4A4, for pericyte recruitment and vessel stabilization during angiogenesis [26,27]. These results demonstrate selective roles for PPARβ/δ in AMD vulnerable cells suggesting it regulates extracellular matrix turnover in RPE cells similar to that reported for dry AMD, yet inhibits an angiogenic phenotype in endothelial cells. Evaluation of the expression of growth factors that regulate vessel stabilization following PPARβ/δ knockdown confirmed this variability in AMD vulnerable cells. A significant decrease in the expression of platelet-derived growth factor receptor beta (PDGFRB), vascular endothelial growth factor A (VEGFA) and transforming growth factor beta 1 (TGFB1) in both primary RPE and RF/6A cells transfected with siPPARβ/δ, as compared to control siRNA (Figure 2, C and D) suggests that disruption of PPARβ/δ expression in both of these AMD-vulnerable cells leads to an anti-angiogenic environment in the RPE and choroid. Interestingly, receptor knockdown resulted in a downregulation of the expression of the neurotrophic agent, pigment epithelial-derived factor (PEDF or SERPINF1) in RPE cells but not in RF/6A cells (Figure 2, C and D). Since modulation of the PPARβ/δ pathway has been shown to regulate inflammation in vitro and in vivo, the effect of PPARβ/δ knockdown on the expression of molecular markers of inflammation was also examined [23,28,29]. Genetic knockdown of PPARβ/δ resulted in the formation of a pro-inflammatory environment in the outer retinal cells, which was evident by the upregulation of inflammatory genes such as, prostaglandin-endoperoxide synthase 2 (PTGS2), interleukin-1 beta (IL1B), chemokine ligand 2 (CCL2) and tumor necrosis factor alpha (TNFA) in RPE cells (Figure 2E); and secreted phosphoprotein 1 (SPP1) and TNFA in RF/6A cells (Figure 2F). Given the role of PPARβ/δ in regulating lipid processing pathways [30], the expression of genes involved in lipid metabolism and previously shown to be altered in AMD was examined. Increased expression of apolipoprotein E (APOE), A (APOA) and low density lipoprotein receptor (LDLR) in human RPE cells (Figure 2G), along with decreased expression of several lipid transfer genes in choroidal endothelial cells (Figure 2H) following PPARβ/δ knockdown was observed. Extracellular and intracellular accumulation of lipids and lipofuscin are characteristics of dry AMD. Good animal models demonstrating significant lipid accumulation in Bruch’s membrane and/or deposits, and not requiring aging mice for long periods of time are currently not available. Therefore, in lieu of that, we examined the effect of activating or antagonizing PPARβ/δ in an in vitro culture model of lipid-loaded RPE cells. Ligand activation of PPARβ/δ resulted in a significant decrease in RPE lipid accumulation (Figure 2I), suggesting a potential therapeutic avenue to pursue in the treatment of early dry AMD, in which removal of extra- and intra-cellular lipids is a goal. Collectively, these data suggest that though PPARβ/δ drives several of the pathogenic pathways associated with AMD development, it may have selective detrimental and beneficial effects in AMD vulnerable cells. To determine the role of PPARβ/δ on the posterior eye, the ocular phenotype of wild-type (Pparβ/δ^+/+) and Pparβ/δ-null (Pparβ/δ^–/–) mice was examined.

Figure 2. PPARβ/δ regulates both dry- and wet-AMD related pathogenic pathways. Effect of siRNA mediated knockdown of PPARβ/δ on mRNA expression of extracellular matrix genes COL1A1, COL4A4, FN1 and VTN; angiogenesis and fibrosis genes, VEGFA, PDGFB, TGFB1 and SERPINF1; inflammation-related genes, PTGS2, IL1B, CXCL8, CCL2, SPP1, and TNFA; and lipid processing genes APOE, ABCA1, APOA1, LCAT and LDLR in 1°RPE cells (A, C, E, and G) and RF/6A (B, D, F and H) cells. (mean and S.E.M.; n = 3; *p < 0.05, ns: not significant, two way ANOVA, Sidak’s multiple comparisons test); siC, control siRNA; siPPARβ/δ, PPARβ/δ siRNA. Quantification of intracellular lipid accumulation after lipid loading followed by incubation with PPARβ/δ agonist, GW0742 or antagonist, GSK0660 in (I) 1°RPE. (*, p<0.05, compared to DMSO Control; ** p<0.05, compared to DMSO; ns: not significant, n=3, two way ANOVA, Sidak’s multiple comparisons test); Control: DMSO vehicle, αLA: α-linolenic acid, DHA: docosahexaenoic acid, AA: arachidonic acid, and PA: palmitic acid.

Aged Pparβ/δ^–/– mice exhibit several phenotypic features of dry AMD

Gene specific differences in the weights of 18-month old Pparβ/δ^+/+ and Pparβ/δ^–/– mice were not observed (Figure S3). The overall architecture of the retina and RPE/choroid was evaluated and no differences in the morphology of the inner retina and/or thickness of the inner and outer nuclear layers were found (Figure S4). In the outer retina, thin patchy sub-RPE deposits were observed in both 18-month old Pparβ/δ^+/+ and Pparβ/δ^–/– mice with varying degrees of thickness and length (Figure 3, A, B, C and D). Detailed quantification of the length of deposit relative to length of Bruch’s membrane revealed deposits were present at a higher frequency in Pparβ/δ^–/– mice compared to Pparβ/δ^+/+ mice (ratio of deposits/BrM length: 0.42$\pm 0.02$versus 0.31$\pm 0. 03$ for Pparβ/δ^–/– and Pparβ/δ^+/+ mice, respectively; Figure 3E; 100% of Pparβ/δ^-/- mice developed deposits, while only 25% of Pparβ/δ^+/+ mice developed deposits). Transmission electron microscopy was used for further detailed analysis of the ultrastructure of the RPE-choroid complex. Eighteen-month old Pparβ/δ^+/+ mice exhibited normal RPE morphology with organized basal infoldings (Figure 3F), along with some thin continuous “age-related” sub-RPE deposits (Figure 3G). By contrast, eyes from age-matched Pparβ/δ^–/– mice exhibited regions with both normal and abnormal RPE morphology. Specifically, the RPE degenerative changes observed included hypo- and hyper-pigmentation (Figure 3, H, I, J, K and L), loss of basal infoldings (Figure 3, J, K and L), thickened Bruch’s membrane (0.59$\pm 0.03 \mathrm{\mu }\mathrm{m}$ versus 0.45$\pm 0.01 \mu m$for Pparβ/δ^–/– and Pparβ/δ^+/+ mice, respectively; Figure 3M), and a higher frequency of continuous thin and thick sub-RPE deposits (Figure 3, J, K, L and S5) compared to age-matched Pparβ/δ^+/+ mice. Gene specific differences in the morphology and overall structure of the choriocapillaries, including fenestration of the vessels, were not noted (Figure S5). Given the observed increase in APOE expression following PPARβ/δ knockdown in vitro, apoE protein deposition was determined by immunolocalization in retinal sections. ApoE immunoreactivity was found to be increased within Bruch’s membrane and sub-RPE deposits in Pparβ/δ^–/– mice compared to Pparβ/δ^+/+ controls (Figure 3, N, O and P). RPE autofluorescence, which reflects lipofuscin accumulation, was also examined and found to be significantly higher throughout retinal sections of Pparβ/δ^–/– mice as compared to age-matched Pparβ/δ^+/+ mice (Figure S6). Interestingly, evaluation of visual function revealed that the average electroretinographic (ERG) responses from 14-16 month old dark-adapted Pparβ/δ^–/– and Pparβ/δ^+/+ controls displayed identical dark-adapted a-wave responses, but a slight increase in sensitivity of b-wave amplitudes compared to Pparβ/δ^+/+ mice (Figure S7 A and B). Examination of the localization of markers for inner retinal cells including photoreceptors and bipolar cells by immunohistochemistry failed to demonstrate any gene specific differences (Figure S7 C and D). Overall, these data highlight that PPARβ/δ is essential for the outer retina at the level of the RPE cells, and may regulate sub-deposit formation and RPE degenerative changes as seen in the early stages of dry AMD.

Figure 3. 18-month old Pparβ/δ^−/− mice exhibit dry-AMD pathology. Toluidine blue stained images of plastic sections from 18-month old (A)Pparβ/δ^+/+ and (B)Pparβ/δ^−/− mice displaying retinal layers (IPL: Inner plexiform layer, INL: Inner nuclear layer, OPL: Outer plexiform layer, ONL: Outer nuclear layer, OS: photoreceptor outer segments, RPE: Retinal pigment epithelium). Toluidine blue stained images of the outer retina of (C)Pparβ/δ^+/+ and (D)Pparβ/δ^−/− mice, which have sub-RPE deposits (Scale bar = 10 μm). (E) Quantification of deposits per Bruch’s membrane (BrM) length in plastic sections (mean and S.E.M., n=10 images/animal, n=4/genotype, * p<0.05). Electron micrographs of RPE/Bruch’s membrane/choroidal junction in 18-mo-old Pparβ/δ^+/+ mice display (F) normal RPE morphology (G) with some age related deposits (dotted line), whereas 18-mo-old Pparβ/δ^−/− mice show (H) RPE hypo-pigmentation, (I) hyper-pigmentation and (J) abnormal basal infoldings with thin sub-deposits, (K) loss of basal infoldings with thin and (L) thick sub-RPE deposits (dotted line). (M) Quantification of Bruch’s membrane thickness in electron micrographs of Pparβ/δ^+/+ and Pparβ/δ^−/− mice (n = 10 images per mouse, n = 4 mice per genotype, two tailed t-test). Scale bars in panels F-L: 1 μm. Images of apolipoprotein E (apoE; red) stained sections from (N)Pparβ/δ^+/+ and (O)Pparβ/δ^−/− mice (Scale bar: 20 μm). Nuclei are stained with Hoescht (blue). (P) Quantification of ratio of apoE stained regions/Bruch’s membrane length (mean and S.E.M., n=4 per group, * p < 0.05, two tailed t-test).

PPARβ/δ promotes laser-induced CNV

Since PPARβ/δ knockdown in both RPE and choroidal endothelial cells in vitro resulted in an anti-angiogenic phenotype, the role of PPARβ/δ in development of CNV in vivo was examined. No evidence of spontaneous CNV or overt vascular changes were observed in aged Pparβ/δ^–/– mice. However, laser-induced CNV lesions in 18-20 month old Pparβ/δ^–/– were smaller as compared to those observed in Pparβ/δ^+/+ mouse eyes (Figure 4, A, B and C). Additionally, examination of the distribution of the lesions in flatmounts of the posterior eye cups revealed a genotype-dependent difference in the number of mouse eyes with single versus merged lesions. Specifically, while Pparβ/δ^+/+ mice presented with two or three merged lesions, no eyes from the Pparβ/δ^–/– cohort presented with three merged lesions, and only one eye displayed two merged lesions (Figure 4B). Quantitatively, measurement of CNV lesion volume determined from z-stacks and three-dimensional reconstructions confirmed a significantly smaller volume in Pparβ/δ^–/– mice compared to Pparβ/δ^+/+ mice (Figure 4D). These data demonstrate a functional role for the PPARβ/δ gene in angiogenesis and the development of CNV lesions.

Figure 4. Genetic disruption of PPARβ/δ attenuates laser-induced CNV. (A) Choroidal flat-mounts were prepared from 18–20 month-old laser-induced CNV mice (Pparβ/δ^+/+, n = 10 eyes, 40 lesions; Pparβ/δ^−/−, n=5 eyes, 20 lesions) and stained with isolectin GS-IB₄ (ON, optic nerve). Representative images from three eyes/genotype are shown to demonstrate individual and merged CNV lesions. Dotted and solid line circles demarcate lesions and optic nerves, respectively. (B) Distribution of number of eyes with individual versus merged lesions in Pparβ/δ^+/+ and Pparβ/δ^−/−. (C) Relative lesion area/animal was measured using ImageJ (mean and S.E.M.; *p < 0.01, two tailed t-test). (D) Relative lesion volume/animal (mean and S.E.M. for each group; *p < 0.05, two tailed t-test).

PPARβ/δ regulates extracellular matrix deposition and immune cell infiltration in CNV lesions

PPARβ/δ has been shown to regulate extracellular matrix and inflammation [31]. With this in mind, extracellular matrix regulation, extracellular matrix deposition and immune cell localization within CNV lesions from aged Pparβ/δ^+/+ and Pparβ/δ^–/– mice were characterized by examining cross sections of the retina/RPE/choroid. Collagen type 4 (COL4) and fibronectin (FN1) are known components of Bruch’s membrane, sub-RPE deposits [32–34], and associated with CNV lesions [33]. Furthermore, fibronectin is known to be produced in response to vessel injury [35]. Quantitatively, significantly lower staining intensity for both COL4 and FN1 in CNV lesions was measured in aged Pparβ/δ^–/– mice compared to age-matched Pparβ/δ^+/+ controls (Figure 5, A and B). As mentioned earlier, PPARβ/δ is known to influence inflammation [31], and this is one of the major pathways that regulates the pathogenesis of AMD [29,36]. Thus, cross sections of the retina/RPE/choroid containing CNV lesions were probed with antibodies to ionized calcium binding adaptor molecule 1 (Iba1), which labels microglial cells and macrophages, and adhesion G protein-coupled receptor E1, also known as F4/80, which labels mature macrophages (Figure 5 C and S8). Interestingly, a significant increase in the localization of both F4/80⁺ and Iba1⁺ cells within the neovascular lesions of Pparβ/δ^–/– mice as compared to age-matched Pparβ/δ^+/+ controls was quantified (Figure 5 D and S8). This is a noteworthy observation given the distinct phenotypes that macrophages can differentiate into: 1) the broadly characterized M1 macrophage, which is reported to contribute to pro-fibrotic and pro-inflammatory phenotypes, and 2) M2 macrophages, which also have 4 different subtypes 37]; characterized as having immunosuppressive and tissue remodeling properties [19,38]. This finding warrants future detailed analysis of the polarity of macrophages in the Pparβ/δ^–/– CNV lesions. Combined, these results show that the loss of PPARβ/δ expression causes decreased extracellular matrix deposition associated with increased acute immune cell infiltration within small CNV lesions, and may reflect decreased fibrosis modulated by PPARβ/δ.

Figure 5. PPARβ/δ regulates extracellular matrix deposition and immune-cell infiltration in CNV lesions. (A) FN1 (green) and COL4 (red) immunolocalization in CNV lesions of Pparβ/δ^+/+ and Pparβ/δ^−/− mice (dotted oval demarcates the lesion area; nuclei are stained blue with Hoechst; representative images are shown; scale bar = 50 µm). (B) FN1 and COL4 staining intensity was quantified in the CNV lesions of Pparβ/δ^+/+ and Pparβ/δ^−/− mice using ImageJ (mean and S.E.M.; n = 3/group; **p < 0.01, two tailed t-test). (C) Laser CNV lesions from Pparβ/δ^−/− mice display a higher number of F4/80 (green) and Iba1 (red) immunopositive cells (dotted oval demarcates the lesion area; nuclei are stained blue with Hoechst; representative images are shown; scale bar = 50 µm). (D) The numbers of F4/80⁺ and Iba1⁺ cells in the CNV lesions of Pparβ/δ^+/+ and Pparβ/δ^−/− mice were counted using ImageJ (mean and S.E.M.; n = 3/group; *p < 0.01, two tailed t-test).

Pharmacological antagonism of PPARβ/δ inhibits endothelial cell migration and tube formation in vitro

Cellular organization is a central process in neovascularization, which involves endothelial cell migration and tube formation [39,40]. Since disruption of the PPARβ/δ gene caused smaller CNV lesions in vivo, whether pharmacological antagonism of PPARβ/δ could inhibit angiogenesis, using functional assays measuring endothelial migration and tube formation, was determined. Basic fibroblast growth factor (bFGF) was used to induce migration in a scrape wound assay in cultures of choroidal endothelial cells in the presence of the PPARβ/δ agonist, GW0742 or the PPARβ/δ antagonist GSK0660 (Figure 6A). Choroidal endothelial cell migration into the wound was inhibited by antagonism of PPARβ/δ with GSK0660 (Figure 6, A and B), while ligand activation of PPARβ/δ with GW0742 did not influence cell migration. The effect of ligand activation and pharmacological antagonism of PPARβ/δ on the ability of endothelial cells to form a three-dimensional network, indicative of vascular morphogenesis was also assessed (Figure 6C). Similarly, pharmacological antagonism of PPARβ/δ with GSK0660 inhibited tube formation (tube length) in choroidal endothelial cells compared to control and growth factor induced tube formation, whereas ligand activation of PPARβ/δ did not affect tube formation (Figure 6, C and D). In cell viability assays no significant differences were seen between control and PPARβ/δ agonist or antagonist-treated endothelial cells, indicating that the changes in cell migration and tube formation are not due to PPARβ/δ agonist or antagonist-induced cell-death (Figure S9).

Figure 6. Antagonism of PPARβ/δ blocks endothelial cell migration and tube formation. (A) The effect of ligand activation or antagonism of PPARβ/δ on migration of RF/6A cells was analyzed in a bFGF induced wound-healing assay (n = 3, representative images at t = 36 hours are shown); dotted lines demarcate the boarders of the scrape wound. Ctrl: media only, bFGF: basic fibroblast growth factor (10 μg/ml), bFGF+GW: bFGF plus GW0742 (10μM), bFGF+GSK0660: bFGF plus GSK0660 (10 μM). (B) The cells migrating into the wound were counted using ImageJ (mean and S.E.M.; n = 3; a, p < 0.01 relative to Ctrl; b, p < 0.01 relative to bFGF, one way ANOVA, Tukey’s multiple comparisons test). (C) The effect of ligand activation or antagonism of PPARβ/δ on bFGF-induced tube formation in RF/6A cells was analyzed by an angiogenesis assay in Geltrex™ (n = 3; representative images at t = 3 hours are shown). Ctrl: media only, bFGF: basic fibroblast growth factor (10 μg/ml), bFGF+GW: bFGF plus GW0742 (10 μM), bFGF+GSK0660: bFGF plus GSK0660 (10 μM). Suramin, an inhibitor of tube formation, was used as a negative control (data not shown). (D) Quantification of tube length in Geltrex™ using ImageJ (mean and S.E.M.; n = 3; a, p < 0.01 relative to Ctrl; b, p < 0.01 relative to bFGF, one way ANOVA, Tukey’s multiple comparisons test).

Pharmacological antagonism of PPARβ/δ attenuates CNV lesions in vivo

The therapeutic potential of inhibiting PPARβ/δ activity on CNV formation in 12-13 month old aged Pparβ/δ^+/+ mice following experimentally induced laser injury to the back of the eye was examined. Mice were treated with vehicle control, GW0742 (0.5 mg/kg/day) or GSK0660 (1 mg/kg/day) (Figure 7A). The distribution, area, and volume of the lesions in flatmounts of posterior eye-cups stained with isolectin GS-IB₄ were assessed. A treatment dependent difference in the distribution of merged lesions was observed. Antagonism of PPARβ/δ with GSK0660 caused a higher proportion of individual neovascular lesions as compared to vehicle or agonist treated mice (Figure 7B). Additionally, the area and volume of the lesions from animals treated with the PPARβ/δ antagonist were significantly lower relative to vehicle control and PPARβ/δ agonist treated mice (Figure 7, C and D). Finally, examination of cross-sections of the retina/RPE/choroid containing CNV lesions revealed decreased deposition of COL4 within the lesions of mice treated with the PPARβ/δ antagonist, while no difference was observed in the distribution of FN1 (Figure 8, A, B and C). Interestingly, this acute treatment with agonist and antagonist did not effect the distribution of Iba1⁺ and F4/80⁺ cells within the neovascular lesion of aged Pparβ/δ^+/+ mice (data not shown). Noteworthy, this short-term treatment with PPARβ/δ agonist or antagonist, also, did not affect the integrity of the RPE tight junctions (Figure S10). Overall, these results are consistent with the in vitro studies and demonstrate that inhibition of PPARβ/δ activity results in a less severe CNV phenotype and may therefore be beneficial in attenuating lesion formation in vivo.

Figure 7. Antagonism of PPARβ/δ pathway attenuates CNV. (A) Choroidal flat-mounts were prepared from 12-13 month-old C57BL/6J (Pparβ/δ^+/+) mice subjected to laser CNV and treated with vehicle control (1% DMSO in saline), GW0742 (0.5 mg/kg/day, i.p.), and GSK0660 (1 m/kg/day, i.p.) and stained with isolectin-GS-IB₄ (n=10 eyes/group, 40 lesions; ON, optic nerve). Representative images from three eyes/treatment are shown to demonstrate individual and merged CNV lesions (dotted and solid line circles demarcate lesions and ONs, respectively). (B) Distribution of number of eyes with individual versus merged lesions in vehicle control, GW0742 and GSK0660. (C) Relative lesion area/animal was measured using ImageJ (n=10 eyes per group, mean and S.E.M.; *p < 0.01, one way ANOVA, Tukey’s multiple comparisons test). (D) Relative lesion volume/animal (n=10 eyes per group, mean and S.E.M. for each group; *p < 0.05. ns: not significant, one way ANOVA, Tukey’s multiple comparisons test).

Figure 8. Antagonism of PPARβ/δ decreases accumulation of collagen type 4 in CNV lesions. (A) FN1 (green) and COL4 (red) immunolocalization in CNV lesions of mice treated with vehicle control (1% DMSO in saline), GW0742 (0.5mg/kg/day, i.p.), or GSK0660 (1m/kg/day, i.p.) (dotted oval demarcates the lesion area; nuclei are stained blue with Hoechst; representative images are shown; scale bar = 50 µm). (B) COL4 and (C) FN1 staining intensity was quantified in the CNV lesions using ImageJ (Mean and S.E.M.; n = 3/group; *p < 0.01; ns: not significant, one way ANOVA, Tukey’s multiple comparisons test).

Discussion

These studies demonstrate for the first time a role of PPARβ/δ in regulating different aspects of extracellular matrix turnover, angiogenesis, inflammation, and lipid processing in the eye. PPARβ/δ affects the RPE and choroidal endothelium differentially, selectively impacting the development of several fundamental AMD phenotypes. These data show that PPARβ/δ activity is functionally important in RPE and choroidal endothelial cell models systems; cells that are compromised during the initiation and progression of AMD. Knockdown of PPARβ/δ expression led to upregulation of extracellular matrix gene expression in primary RPE cells but a downregulation in choroidal endothelial cells. Additionally, the observed PPARβ/δ-dependent downregulation of the expression of factors critical for angiogenesis, including VEGFA, PDGFRB and TGFB, in both cell populations, supports the hypothesis that PPARβ/δ regulates signaling pathways important in development of neovascular lesions. Consistent with the in vitro studies, a significant decrease in area and volume of laser induced neovascular lesions was observed in both aged Pparβ/δ^-/- mice and in Pparβ/δ^+/+ mice following pharmacological antagonism of the receptor. In contrast to these findings, morphological characterization of the ocular phenotype of aged Pparβ/δ^–/– mice revealed the exacerbation and development of several features of the early dry AMD phenotype including continuous sub-RPE deposits, increased RPE autofluorescence, Bruch’s membrane thickening, RPE pigmentary changes and disorganized basal infoldings. These in vivo results are supported by in vitro data demonstrating increased dysregulation of extracellular matrix molecules following PPARβ/δ knockdown in human RPE cells. Collectively, these studies illustrate, for the first time, cell-specific effects of PPARβ/δ in two populations of AMD vulnerable cells (Figure 9). Our findings correlate with the concept of selective modulation of PPARs and other nuclear receptors such as the estrogen receptor, in which ligand binding may lead to differential gene expression and biological responses in different cells and tissues [41,42]. Additionally, part of the variability observed in the eye, may be due to differential expression of PPARβ/δ in the RPE versus choroidal endothelial cells (Figure 1A); differential expression of the receptor co-regulatory proteins including co-activators and co-repressors; differential binding affinities of the co-regulatory proteins; and/or varying receptor conformational changes induced by endogenous ligand binding in epithelial versus endothelial cells [43]. This is an area of ongoing investigation.

Figure 9. Summary model of selective regulation of dry and wet AMD related pathogenic pathways by PPARβ/δ in AMD vulnerable cells. Ligand activation of PPARβ/δ inhibits lipid accumulation by RPE cells and may have therapeutic effect in dry-AMD. In contrast, antagonism of PPARβ/δ inhibits neovascularization possibly by regulating inflammation and fibrosis in outer-retina.

The increased influx of immune cells within the neovascular lesion in the absence of PPARβ/δ expression is an interesting finding that requires further investigation and may be clarified with full characterization of the polarization status of these immune cells. Recently, PPARβ/δ has been shown to be involved in alternative activation of macrophages in mice [44]. Reportedly, both IL-13 and IL-4 induce expression of PPARβ/δ and modulate the ability of adipose tissue and liver macrophages to transition to the M2 phenotype. It has also been shown that PPARβ/δ coordinates the immune phenotype of alternatively activated macrophages, both in vitro and in vivo [45]. These reports suggest that the PPARβ/δ regulates macrophage polarization in animal models of liver-injury and atherosclerosis. However, it is important to note that to date, no one has critically examined the specific subtype of M2 macrophages actually regulated by PPARβ/δ, and since different subtypes have different functions [37], the precise effect of this particular change requires further investigation. Based on these studies it is plausible that the population of Iba1⁺ cells observed in the CNV lesions of Pparβ/δ^–/– mice are transient and may express markers of the alternatively activated macrophage phenotype, which are involved in immunosuppression and tissue remodeling [19,38]. CNV lesions in Pparβ/δ^–/– mice also displayed a reduced deposition of COL4 and FN1 in the lesion supporting a decrease in fibrosis. This is a major factor in responsiveness to anti-VEGF treatment for neovascular AMD [46,47]. To our knowledge this is the first study to report the involvement of the PPARβ/δ pathway in CNV formation.

To date, no differences in PPARβ/δ expression between normal and AMD cohorts have been reported. This may be due to the fact that evaluating the activity of PPARβ/δ along with expression of co-factors and specific target genes is more informative than measuring expression of the receptor in silos. Nevertheless, the purported role of PPARβ/δ in other tissues [13,48–50] in combination with our data from PPARβ/δ knockdown in vitro, and genetic ablation in vivo, gave rise to the hypotheses that pharmacologic targeting of PPARβ/δ may therapeutically improve choroidal neovascularization and lipid accumulation. To test our first hypothesis, the postulate that pharmacologic antagonism of PPARβ/δ could therapeutically improve choroidal neovascularization, the effect of ligand activation and pharmacological antagonism of PPARβ/δ on endothelial migration and tube formation in the choroidal endothelial cell line, RF/6A was determined. Pharmacological antagonism of PPARβ/δ activity inhibited choroidal endothelial cell migration as well as tube formation in a three-dimensional matrix. By contrast, ligand activation of PPARβ/δ had no effect in these angiogenesis assays. These results are consistent with previous studies by others using human retinal microvascular endothelial cells and a rat model of oxygen-induced retinopathy [51]. Modeling retinal neovascularization, it was shown that pharmacological antagonism of PPARβ/δ significantly decreased serum-induced retinal endothelial cell proliferation and tube formation in a dose-dependent manner, and in vivo decreased retinal neovascularization in young rats. Another study from the same group showed that pharmacological antagonism of PPARβ/δ stabilized tight junctions in retinal microvascular endothelial cells treated with VEGF, and reduced VEGFR1/2 expression, suggesting a role in retinal vascular permeability [52]. The present study examined the effect of ligand activation and pharmacological antagonism of PPARβ/δ on choroidal neovascularization, in which the highly fenestrated choriocapillaris; vasculature supporting the outer retina; grow through Bruch’s membrane under the RPE, in vivo, using a laser-induced CNV model. Antagonism of PPARβ/δ provided a therapeutic effect on laser-induced lesion formation, whereas ligand activation of PPARβ/δ had no effect, similar to results obtained from our in vitro analyses. Interestingly, antagonizing PPARβ/δ activity did not negatively impact the integrity of the RPE cell tight junctions. This may be due to the fact that antagonism of PPARβ/δ was acute rather than long term. Equally likely is that genetic ablation of the receptor (as seen in vivo in our mice, but not reported in humans) is detrimental to the RPE cells rather than receptor antagonism itself.

Our second hypothesis supports the notion that it is critical to investigate the therapeutic potential of targeting PPARβ/δ activity in the pathogenesis of dry AMD. Specifically, whether PPARβ/δ activity can be targeted for removal of lipid-rich deposits within Bruch’s membrane and sub-RPE deposits should be examined in more detail. Several studies have found that ligand activation of PPARβ/δ inhibits lipid accumulation as well as expression of pro-inflammatory cytokines in THP-1 macrophages and mouse monocytes [53,54]. Though, the sub-RPE deposits in the aged Pparβ/δ^–/– mice contain apoE, they did not stain positively for oil red o or filipin and do not appear to contain neutral lipids or cholesterol (data not shown). As proof of concept, using an in vitro model of lipid-loaded RPE cells, it was found that ligand activation of PPARβ/δ decreased lipid accumulation. Taken together, these results support the need to test the effect of PPARβ/δ agonists in conditions where there are systemic and/or dietary factors contributing to development of early AMD. Though there is selective modulation of the PPARβ/δ in RPE and choroidal endothelial cells, based on the in vitro and in vivo data, ligand activation of PPARβ/δ is beneficial to RPE cells and does not appear to have a negative impact on healthy choroidal endothelial cells. Currently there are no FDA-approved PPARβ/δ drugs, but the recent development of highly specific and selective agonists and antagonists [selective PPARβ/δ modulators (SPPARMs)] such as GW501516, GW0742, GSK0660 and GSK3787 have led to improved approaches to study the PPARβ/δ pathway in disease models including obesity and atherosclerosis [55]. In future studies, it will be imperative to construct PPARβ/δ-selective drug-dose response curves to describe cell-selective responses in a quantitative manner and test the suitability of pharmacological agents in long-term studies using aged animal models of AMD that develop lipid accumulation within Bruch’s membrane.

This is the first study to report on the PPARβ/δ pathway in RPE and choroidal endothelial cells and its potential role in the pathogenesis of AMD. The results indicate, for the first time, selective modulation of a nuclear receptor in the eye. Specifically, that inhibition of PPARβ/δ activity may successfully attenuate neovascular lesion formation concomitant with decreased expression of extracellular matrix molecules and decreased angiogenic factors, in vivo, while activation of PPARβ/δ activity may target lipid accumulation. The results of this treatment may be attributable to PPARβ/δ regulation of several distinct AMD pathogenic pathways: fibrosis, inflammation and lipid metabolism. The Pparβ/δ^–/– mice also displayed early dry AMD like pathology, establishing these mice as a model to further study the initiation and progression of the early sub-type of the disease. Additional studies should be conducted to study the contribution of other PPARβ/δ regulatory pathways that may influence disease pathology and the therapeutic potential of selectively targeting PPARβ/δ as a means to inhibit inflammation, fibrosis and lipid accumulation (Figure 9).

Acknowledgements

We thank Mr. Edwin Meade for help with immunohistochemistry, and Mr. Peter Saloupis for assistance with animal experiments, Dr. Donald P. McDonnell and Dr. Ching-Yi Chang for plasmids used in transcriptional activity assays and valuable discussion. Many thanks to Mrs. Ying Hao for assistance with electron microscopy. We thank Dr. Scott Cousins for allowing us to utilize his slit lamp and laser.

Funding

This work was supported by the National Eye Institute grant EY02868 (GM), NEI P30 EY005722 (Duke Eye Center) and Research to Prevent Blindness Core Grant (Duke Eye Center).

Conflicts of Interest

The authors declare that no conflict of interest exists.

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