Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration - PubMed (original) (raw)
Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration
Haibo Wang et al. Invest Ophthalmol Vis Sci. 2016.
Abstract
Purpose: This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV).
Methods: Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA.
Results: Thy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin-labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC-induced Rac1 and Src activation.
Conclusions: Thy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted.
Figures
Figure 1
Thy-1 is expressed in CECs and upregulated by AMD-related stresses. (A) Western blot of Thy-1 protein in cultured HRPE (top panel) and in CECs before passaging (p0) and after in vitro culturing (passages 2 [p2] and p5) (bottom panel); Western blot of Thy-1 protein (top panel) and qPCR of Thy-1 mRNA (bottom panel) (B) in CECs treated with CCL11 (10 ng/mL) or VEGF (20 ng/mL) overnight (*P < 0.05 vs. PBS, _n_ = 6) and (**C**) in RPE/choroids isolated from young (20- to 40-year-old) and old (>60-year-old) donor eyes (***P < 0.001 vs. Young, n = 3).
Figure 2
Thy-1 is expressed at human choroids and CNV lesions. Immunostaining of Thy-1 (A) in retinal cryosections from a 25-year-old donor (red, Thy-1; green, VE-cadherin; autofluorescence of RPE shown in Alexa Fluor 568 and no primary control); (B) in paraffin-embedded sections from a 79-year-old donor with neovascular AMD (blue, 4′,6-diamidino-2-phenylindole [DAPI]; green, VE-cadherin; red, Thy-1); and (C, D) in paraffin-embedded sections from 75-year-old donor with neovascular AMD and an age-matched control donor without AMD (C) (blue, DAPI; green, Thy-1; magnification 20×) and at VE-cadherin–labeled CNV lesions (D) (green, Thy-1; red, VE-cadherin; blue, TO-PRO3, magnification, 40×; arrows point to CNV lesion).
Figure 3
Thy-1 is upregulated in laser-treated RPE/choroids and expressed at CNV lesions. (A) Quantitative real time PCR of Thy-1 mRNA in RPE/choroids from nonlasered control eyes or laser-treated eyes at day 5 after laser (*P < 0.05 vs. nonlasered, n = 5); immunostaining of Thy-1 in retinal cryosections of nonlasered eyes or lasered eyes (B) 5 days after laser (blue, DAPI; green, Thy-1) or (C) 7 days after laser (green, Thy-1; red, lectin; gray, TO-PRO3) (magnification, 20×; n = 3).
Figure 4
Knockdown of Thy-1 inhibits VEGF-mediated Rac1 activation and angiogenic effects in CECs. (A) Rac1 activity assay, (B) CEC migration, (C) CEC proliferation, and (D, E) cytoskeleton stress fibers stained by rhodamine phalloidin (D, representative images; E, quantification of fluorescent density of stress fiber staining [normalized to control siRNA/PBS]) in CECs transfected with Thy-1 siRNA and treated with VEGF (20 ng/mL) for 30 minutes. Note intracellular fibers induced by VEGF and reduction in Thy-1 siRNA-transfected CECs compared to control siRNA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS of control siRNA; ††P < 0.01, †††P < 0.001 vs. VEGF of control siRNA; ‡P < 0.05 vs. PBS of Thy-1 siRNA; n = 6–9).
Figure 5
Inhibition of Thy-1 inhibits VEGF-mediated Rac1 activation and angiogenic effects in CECs. (A) Rac1 activity assay (left panel, representative images; right panel, quantification of densitometry of GTP-Rac1 to total Rac1 [normalized to PBS of control]), (B) CEC migration, (C) CEC proliferation, and (D, E) cytoskeleton stress fibers stained by rhodamine phalloidin (D, representative images; E, quantification of fluorescent density of stress fiber staining [normalized to control/PBS]) were measured in CECs pretreated with Thy-1 blocking peptide (Thy-1Inh) for 30 minutes prior to incubation with VEGF (20 ng/mL) for additional 30 minutes. Note intracellular fibers induced by VEGF and reduction in CECs pretreated with Thy-1 blocking peptide compared to control (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS of control; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. VEGF of control; n = 6–9).
Figure 6
Knockdown or inhibition of Thy-1 inhibits VEGF-induced activation of VEGFR2, integrin β3, and Src. Western blots of (A, B) phosphorylated VEGFR2 (top panel, representative images; bottom panel, quantification of densitometry), (C, D) phosphorylated integrin β3 (top panel, representative images; bottom panel, quantification of densitometry), and (E, F) p-Src (top panel, representative images; bottom panel, quantification of densitometry) in CECs transfected with Thy-1 siRNA (A, C, E: *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS of control siRNA; †P < 0.05, ††P < 0.01 vs. VEGF of control siRNA) or pretreated with Thy-1 blocking peptide (Thy-1 Inh) (B, D, F: ‡P < 0.05 vs. PBS of control; #P < 0.05, ##P < 0.01 vs. VEGF of control) with 30 minutes incubation with VEGF (20 ng/mL) (n = 3–6).
Figure 7
Thy-1 regulates extracellular matrix protein 7-KC–induced Rac1 activation. (A) Rac1 activity assay in CECs treated with 7-KC at different doses for 30 minutes (top panel, representative images; bottom panel, quantification of densitometry; *P < 0.05 vs. 7-KC at 0 μM) and (B) Rac1 activity assay and phosphorylation of Src in CECs transfected with Thy-1 siRNA or control siRNA and treated with 7-KC (10 μM) or vehicle for 30 minutes (n = 3–6).
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