Role of acyl-coenzyme A: cholesterol transferase 1 (ACAT1) in retinal neovascularization

Figures

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  Fig. 1. Expression of low-density lipoprotein receptor (LDLR) is increased during OIR-induced retinal neovascularization (RNV). a, b Western blotting and quantification show increased expression of LDLR in P17 OIR retinas, mean ± SEM, n = 3–6. c LDLR is colocalized with IB4-positive areas of RNV in OIR retina sections (yellow arrows) but is absent in room air (RA) control. n = 4, scale bar = 20 µm

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  Fig. 2. OIR-induced retinal neovascularization (RNV) is accompanied by lipid accumulation and blocked by LDLR deletion. a Oil red O staining of frozen sections shows intense reaction (yellow arrows) in areas of RNV in OIR retinas (indicated in high magnification (HM) image and HM converted to black and white). Intense Oil red O reaction is also evident in the outer plexiform layer (OPL) and photoreceptor (PR) outer segments of the OIR retina. Reaction is also evident in the outer segments of the RA control retina. n = 4, scale bar = 25 μm. b Detection of cholesterol ester (CE) by filipin staining of frozen retina sections shows prominent increases in CE formation in the ganglion cell, outer plexiform and photoreceptor layers of the OIR retina Filipin images are converted to black and white (BW) to show staining better. RA control sample shows a slight reaction to filipin in retinal vessel-like structures (arrows). n = 4, scale bar = 25 μm. c–e OIR-induced RNV (white highlighted areas) is almost completely blocked in LDLR−/− mice and the avascular area (AVA, yellow outline) is significantly reduced. n = 3, scale bar = 300 µm

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  Fig. 3. OIR induces increased expression of ACAT1. Mice were maintained in hyperoxia from P7 to P12 and returned to room air. Retina samples were collected at P17 and prepared for imaging and western blotting. a Representative immunoblot images and quantification showing upregulation of b ACAT1, and no change in c ACAT2 in the OIR retinas compared to RA controls. mean ± SEM, n = 4–6. d Representative retinal images show ACAT1 co-localized with IB4-positive retinal vessels in areas of RNV (yellow arrows). n = 4, scale bar = 60 μm. e High magnification images (×63) of an area of RNV tuft formation show ACAT1 co-localization with LDLR and F4/80 positive macrophage/microglial cells. n = 4, scale bar = 40 μm

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  Fig. 4. ACAT1 is colocalized with ameboid activated macrophage/microglia-like cells in areas of RNV. RNV area in the OIR retina shows colocalization of IB4 with the macrophage/microglia marker Iba1 and ACAT1. Insets show strong immunoreactivity for ACAT1 in ameboid shaped macrophage/microglia in the area of RNV of the OIR retina. ACAT1 expression is weak in Iba1-positive cells outside of the area of RNV of the OIR retina and ramified microglia of the RA control retina. n = 4, scale bar = 60 μm

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  Fig. 5. OIR induces increased expression of pro-inflammatory markers. Mice were maintained in hyperoxia from P7 to P12 and returned to room air. Retina samples were collected at P17 and prepared for imaging and western blotting. a Representative immunoblot images and b–e quantification show upregulation of MCSF (a, b), VEGF (a, c), and TREM1 (a, d) in the OIR retinas compared to RA controls. mean ± SEM, n = 4–6

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  Fig. 6. Inhibiting ACAT1 decreases retinal neovascularization (RNV) and avascular area (AVA) during OIR. a–e OIR mice were treated with the ACAT1 inhibitor K604 or vehicle from P7 to P16. Eyes were enucleated at P17 and retinal vessels were visualized by IB4 labeling of retinal flat mounts. a–c Areas of RNV (white highlighted areas) and AVA (yellow outline) were significantly reduced by treatment with K604. n = 10–11, scale bar = 300 μm. d, e K604 treatment increased vascular sprouting as shown by increased numbers of tip cells with filopodia, as compared to vehicle treated OIR retinas. n = 7–8, scale bar = 40 µm. f–h OIR mice were treated with the ACAT1 inhibitor K604 or vehicle from P12 to P16. Eyes were enucleated at P17 and retinal vessels were visualized by IB4 labeling. Treatment with K604 significantly reduced the formation of RNV (white highlighted areas), but not the AVA (yellow outline). n = 10–11, scale bar = 300 μm. i, j OIR mice were treated with the ACAT1 inhibitor K604 or vehicle from P7 to P11. Eyes were enucleated at P12 and retinal vessels were visualized by IB4 labeling. Treatment with K604 significantly reduced the AVA (yellow outline). n = 6–7, scale bar = 300 μm

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  Fig. 7. ACAT1 inhibition suppresses lipid uptake and CE formation in OIR retinas and plasma. OIR mice were treated with K604 or vehicle from P7 to P16. a, b Frozen sections from vehicle treated OIR retinas show increased reactivity (yellow arrows) for neutral lipids and cholesterol ester (CE) as detected by Oil Red O (a) and filipin staining (b), compared with the RA control retinas. Filipin images are converted to black and white (BW) to show the staining better. Treatment with K604 prevented the lipid accumulation and CE formation in the OIR retina. n = 3, scale bar = 20–25 μm. Cholesterol levels were measured in retinas and plasma from RA, OIR and K604-treated OIR pups at P17. c, d Biochemical analysis of cholesterol levels in retina and plasma samples showed upregulation of cholesterol ester in the vehicle treated OIR animals. K604 treatment completely blocked this effect. mean ± SEM, n = 4–7

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  Fig. 8. ACAT1 inhibition decreases expression of ACAT1 and inflammatory mediators. OIR mice were treated with K604 or vehicle from P7 to P16. Retinal lysates were prepared at P17 for Western blotting. a Representative western blots and b–f their quantification shows that ACAT1 inhibition significantly suppresses expression of MCSF (a, b), ACAT1 (a, c), TREM1 (a, e), and LDLR (a, f), as compared with the vehicle control. However, K604 treatment did not alter levels of VEGF (a, d). mean ± SEM, n = 6

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  Fig. 9. Inhibiting ACAT1 reduces mRNA for ACAT1 and associated inflammatory mediators in OIR retinas. OIR mice were treated with the ACAT1 inhibitor K604 or vehicle from P7 to P16. Retinas were enucleated at P17 and gene expression was analyzed by RT-PCR. a–g Analysis of mRNA levels shows upregulation of a ACAT1, b TREM1, c MCSF, and d DAP12, along with the inflammatory markers e IL1β, f TNFα, and g IL6 in OIR retinas, compared to RA control. K604 treatment significantly reduced their expression as compared to vehicle treated OIR-retinas. mean ± SEM, n = 4

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  Fig. 10. Inhibiting ACAT1 attenuates OGD-induced increases in expression of ACAT1 and inflammatory mediators in HMC3 microglia. Human microglial cells were subjected to OGD (1% O2, glucose-free medium) or normal media (21% O2, 25 mM glucose) with K604 or vehicle for 24 h. a Western blotting, and b–e quantification shows upregulation of MCSF (a, b), ACAT1 (a, c), and TREM1 (a, e) after hypoxia treatment as compared with normoxia. K604 treatment markedly suppressed all except VEGF (a, d). mean ± SEM, n = 6