Investigating the Role of PPARβ/δ in Retinal Vascular Remodeling Using Pparβ/ δ-Deficient Mice

Abstract

Peroxisome proliferator-activated receptor (PPAR)β/δ is a member of the nuclear receptor superfamily of transcription factors, which plays fundamental roles in cell proliferation and differentiation, inflammation, adipogenesis, and energy homeostasis. Previous studies demonstrated a reduced choroidal neovascularization (CNV) in Pparβ/δ-deficient mice. However, PPARβ/δ's role in physiological blood vessel formation and vessel remodeling in the retina has yet to be established. Our study showed that PPARβ/δ is specifically required for disordered blood vessel formation in the retina. We further demonstrated an increased arteriovenous crossover and wider venous caliber in Pparβ/δ-haplodeficient mice. In summary, these results indicated a critical role of PPARβ/δ in pathological angiogenesis and blood vessel remodeling in the retina.

Keywords: PPARβ/δ; angiogenesis; arteriovenous crossover; blood vessel remodeling; pericytes; vessel caliber.

Figure 1

Peroxisome proliferator-activated receptor (PPAR)β/δ is highly expressed in mouse retina. (a) Representative Western blot of PPARβ/δ, retinal pigment epithelium-specific protein 65 kDa (RPE65) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) in the retina (n = 4) and choroid/RPE (n = 4) compartments of adult C57BL/6 mice. Relative gene expression of Pparβ/δ in (b) the retina (n = 10) and choroid/RPE (n = 3) compartments of C57BL/6 mice; (c) the retina of fasted (n = 4) and re-fed (n = 4) adult C57BL/6 mice; (d) the retina of P2 (n = 4) and P21 (n = 4) C57BL/6 mice, as determined by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Relative expressions of (e) Pparβ/δ and (f) Carnitine palmitoyltransferase 1A (Cpt1a) in P12 hyperoxic retina of C57BL/6 mice subjected to oxygen-induced retinopathy (OIR) (n = 4) as compared to those in age-matched normoxic retina (n = 4), as determined by RT-qPCR analysis. Relative expressions of (g) Pparβ/δ and (h) Cpt1a in P13 hypoxic retina of C57BL/6 mice subjected to OIR (n = 4) as compared to those in age-matched normoxic retina (n = 4), as determined by RT-qPCR analysis. Gene expressions are quantified relative to the housekeeping gene, Gadph, as detailed in the Materials and Methods. Data are expressed as mean ± standard error of the mean (SEM). Unpaired, two-tailed t-test was used for statistical analysis; *** p < 0.001, * p < 0.05.

Figure 2

Pparβ/δ deletion does not affect normal blood vessel formation. (a) Representative images and quantification of retinal vessel density of P10 Pparβ/δ^+/− (n = 4) and wild type (n = 3) retina. Scale bar: 50 µm. (b) Representative images and quantitative analysis of vessel outgrowth from P10 aortic rings isolated from Pparβ/δ^+/− (n = 10 explants) and wild-type littermate controls (n = 5 explants). Scale bar: 200 µm. (c) Representative images and quantitative analysis of vessel outgrowth from P3 choroid explants isolated from Pparβ/δ^+/− (n = 49 explants) and wild-type littermate controls (n = 34 explants). Scale bar: 200 µm. Data are expressed as mean ± SEM. Unpaired, two-tailed t-test was used for statistical analysis.

Figure 3

PPARβ/δ inhibition by 10h does not affect angiogenesis. (a) Relative gene expression of Angiopoietin-like 4 (ANGPTL4), CPT1A, and Pyruvate Dehydrogenase Kinase 4 (PDK4) in human retinal microvascular endothelial cells (ECs) treated with 100 nM of 10h for 24 h compared to that in dimethyl sulfoxide (DMSO) vehicle control, as determined by RT-qPCR analysis (n = 3). (b) Representative images and quantitative analysis of vessel outgrowth from P3 C57BL/6 aortic ring explants treated with 100 nM of 10 h (n = 9 explants) and DMSO control (n = 11 explants). Scale bar: 200 µm. (c) Representative images and quantitative analysis of vessel outgrowth from P3 C57BL/6 choroidal explants treated with 100nM of 10h (n = 15 explants) and DMSO control (n = 34 explants). Scale bar: 200 µm. Data are expressed as mean ± SEM. Unpaired, two-tailed t-test was used for statistical analysis; *** p < 0.001.

Figure 4

PPARβ/δ mediates retina blood vessel remodeling. (a) Representative retina images of flatmounted P10 Pparβ/δ^+/− and wild type retinas, where arteriovenous crossings are highlighted by white arrowheads. Corresponding quantification of arteriovenous crossings in Pparβ/δ^+/− (n = 4) and wild type (n = 5) retinas. Scale bar: 500 µm. (b) Representative images of the vein (red box) and artery (yellow box) in P10 Pparβ/δ^+/− and wild type retinas. Corresponding quantification of the vessel diameter of the radial veins and arteries of Pparβ/δ^+/− (n = 4) and wild type retinas (n = 5). Two points of at least 4 veins/arteries were measured per retina. (c) Representative confocal images for NG2 (red) and CD31 (green) staining of the retinal vasculature in adult Pparβ/δ^−/− and wild type retinas. Scale bar: 50 µm. Corresponding quantification of the vessel density and pericyte coverage in wild type (n = 3) and Pparβ/δ^−/− (n = 5) retinas. Relative gene expression of (d) Vegf, (e) Tgfβ1, (f) Tie2, and (g) Pdgfrβ mRNA of adult Pparβ/δ^−/− and wild type retinas (n ≥ 3), as determined by RT-qPCR analysis. Data are expressed as mean ± SEM. Unpaired, two-tailed t-test was used for statistical analysis; * p < 0.05.

Figure 5

PPARβ/δ is specifically required for pathological retinal angiogenesis. (a) Representative immunofluorescence images of CD1 stained (green) retinal flatmount of P17 Pparβ/δ^+/− and wild type retinas subjected to OIR. Scale bar: 500 μm. Corresponding quantification of retinal vaso-obliteration in P17 OIR retinas (n = 6). (b) Pathological neovascular tufts (delineated in higher magnification by red boundary line) and the corresponding quantification of tufts (normalized to retinal area) in Pparβ/δ^+/− and wild type P17 OIR retinas (n ≥ 3). Scale bar: 100 μm. Data are expressed as mean ± SEM. Unpaired, two-tailed t-test was used for statistical analysis; * p < 0.05.