7-ketocholesterol induces endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment

Abstract

Oxidized cholesterols and lipids accumulate in Bruch's membrane in age-related macular degeneration (AMD). It remains unknown what causal relationship exists between these substances and AMD pathophysiology. We addressed the hypothesis that a prevalent form, 7-ketocholesterol (7KC), promotes choroidal endothelial cell (CEC) migration and macular neovascularization in AMD. Compared to control, 7KC injection caused 40% larger lectin-stained lesions, but 70% larger lesions measured by optical coherence tomography one week after laser-injury. At two weeks, 7KC-injected eyes had 86% larger alpha smooth muscle actin (αSMA)-labeled lesions and more collagen-labeling than control. There was no difference in cell death. 7KC-treated RPE/choroids had increased αSMA but decreased VE-cadherin. Compared to control-treated CECs, 7KC unexpectedly reduced endothelial VE-cadherin, CD31 and VEGFR2 and increased αSMA, fibroblast activation protein (FAP) and transforming growth factor beta (TGFβ). Inhibition of TGFβ receptor-mediated signaling by SB431542 abrogated 7KC-induced loss of endothelial and increase in mesenchymal proteins in association with decreased transcription factor, SMAD3. Knockdown of SMAD3 partially inhibited 7KC-mediated loss of endothelial proteins and increase in αSMA and FAP. Compared to control, 7KC-treatment of CECs increased Rac1GTP and migration, and both were inhibited by the Rac1 inhibitor; however, CECs treated with 7KC had reduced tube formation. These findings suggest that 7KC, which increases in AMD and with age, induces mesenchymal transition in CECs making them invasive and migratory, and causing fibrosis in macular neovascularization. Further studies to interfere with this process may reduce fibrosis and improve responsiveness to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

Keywords: 7-ketocholesterol; Age-related macular degeneration; Choroidal endothelial cells; Endothelial-mesenchymal transition; Macular fibrosis; Non-responsive (or unresponsive) neovascular AMD.

Figure 1.. Intravitreal injections of 7KC increase CNV lesion in a laser-induced CNV model.

(a) Representative images of RPE/choroidal flat mounts stained with lectin (scale bar: 100 μm), (b) fold increase in lectin-stained CNV volume; (c) representative OCT images lesions and (d) fold increase in lesion volume measured by OCT in wild type mice one week post laser treatment (*p<0.05, **p<0.01 vs. HPBCD; Results were Mean ± SEM).

Figure 2.. Intravitreal injections of 7KC promote fibrosis of lesions induced by laser treatment.

(a) Representative images of RPE/choroidal flat mounts stained by lectin and αSMA (Green: lectin; Red: αSMA; scale bar: 100 μm)) and (b) quantification of lesion volume stained by αSMA; (c) immunostaining of collagen I in retinal cryosections (scale bar: 100 μm) and (d) quantification of collagen I density at lesions of wild type mice 2 weeks post laser treatment (Green: lectin; Red: Collagen I; Gray: Topro3 to stain the nuclei; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; the white arrows point to lesions; the areas labeled with dashed lines indicate the places where integrated density of collagen I was quantified) (p=0.08, *p<0.05 vs. HPBCD; Results were Mean ± SEM).

Figure 3.. Intravitreal injections of 7KC does not increase cell death determined by TUNEL staining in the retina of wild type mice two weeks post laser treatment.

(a) Representative images of TUNEL staining in retinal cryosections (Red: TUNEL positive cells; Blue: DAPI to stain the nuclei; scale bar: 100 μm) and (b) quantification of TUNEL positive cells (Results were Mean ± SEM).

Figure 4.. Intravitreal injections of 7KC increase mesenchymal proteins and reduce endothelial proteins in the RPE/choroids from wild type mice one week after laser treatment.

Western blots of (a) αSMA, (b) FAP, (c) VE-cadherin and (d) VEGFR2 top panels: representative gel images; bottom panels: quantification of densitometry) (p=0.175, *p<0.05, **p<0.01 vs. HPBCD; Results were Mean ± SEM ).

Figure 5.. 7KC increases mesenchymal cell markers in choroidal endothelial cells (CECs).

Western blots of (a and b) αSMA, (c and d) FAP and (e and f) TGFβ in CECs treated with 7KC or HPBCD for 24, 48 or 72 hours (a, c and d: representative gel images and b, d and f: quantification of densitometry) (***p<0.001 vs. HPBCD at 48 hr; ^‡p<0.05 and ^‡‡‡p<0.001 vs. HPBCD at 72 hr; Results were Mean ± SD).

Figure 6.. 7KC reduces endothelial cell markers in choroidal endothelial cells (CECs).

Western blots of (a and b) VEGFR2, (c and d) VE-cadherin and (e and f) CD31 in CECs treated with 7KC or HPBCD for 24, 48 or 72 hours (a, c and d: representative gel images and b, d and f: quantification of densitometry) (***p<0.001 vs. HPBCD at 24 hr; ^†p<0.05 and ^††p<0.01 vs. HPBCD at 48 hr and ^‡p<0.05 and ^‡‡‡p<0.001 vs. HPBCD at 72 hr; Results were Mean ± SD ).

Figure 7.. 7KC promotes EndMT of CECs via TGFβ-dependent signaling.

Western blots of (a) VEGFR2, VE-cadherin and CD31 and (b) αSMA, FAP and TGFβ in CECs treated with 7KC or TGFβ or respective control for 72 hr; Western blots of (c) VEGFR2, VE-cadherin and CD31, and (d) αSMA, FAP and TGFβ in CECs in CECs pretreated with SB431541 and incubated with 7KC or TGFβ for 72 hr.

Figure 8.. Upregulation of SMAD3 is involved in 7KC-mediated EndMT of CECs.

Western blots of (a) SMAD3 in CECs pretreated with control or SB431542 and treated with HPBCD or 7KC for 72 hours, and (b) SMAD3 (c) VEGFR2, VE-cadherin and CD31, and (d) αSMA, FAP and TGFβ in CECs transfected with SMAD3 siRNA or ControlsiRNA and treated with 7KC for 72 hours.

Figure 9.. 7KC promotes cell migration via Rac1GTP dependent signaling, but does not induce tube formation.

(a) Rac1 activity assay and (b) cell migration assay were performed in CECs after treatment with 7KC or HPBCD for 72 hr in the presence of the Rac1 inhibitor or vehicle control (***p<0.001 vs. HPBCD of Vehicle control; ^††p<0.01 vs. 7KC of Vehicle control; Results were Mean ± SEM); (c and d) tube formation assay were performed in CECs treated with 7KC or HPBCD for 72 hours (c, representative images of tube formation; ‡p<0.05 vs. HPBCD; Results were Mean ± SD)