Journal: Theranostics

Article Title: System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy

doi: 10.7150/thno.72947

Figure Lengend Snippet: ADAM10's activator EGCG inhibits the activation of human retinal endothelial cells (HRECs) in vitro and vessel outgrowth in ex vivo models. (A and B) ADAM10 activity impairment in ocular disease models are shown. (A) ADAM10 activity in retinae from control mice and OIR mice. n = 3. (B) ADAM10 activity in choroid/RPE from control mice and CNV mice. n = 4. Data are presented as mean ± s.e.m. Statistical analysis was determined by unpaired, two-tailed Student's t -test; * p < 0.05. (C) AlamarBlue assay demonstrated inhibition of HRECs viability by 50 µM EGCG following 24 h treatment (n = 3). (D) DAPI staining demonstrated inhibition of HRECs migration by 50 µM EGCG following 4 h treatment (n = 3). (E) EGCG inhibits choroidal vessel outgrowth in a dose-dependent manner. Representative images (left) and quantitative analysis (right) of microvessel formation from mouse choroidal explants demonstrating a significant inhibitory effect of EGCG (n = 3 independent experimental groups, n ≥ 6 explants per treatment group). (F) EGCG inhibits aortic vessel sprouts at the dosage of 50 µM. Representative images (left) and quantitative analysis (right) of macrovessel formation from mouse aortic explants demonstrating a significant inhibitory effect of EGCG (n = 3 independent experimental groups, n ≥ 6 explants per treatment group). Scale bar: 100 µm. All images shown are representative, and data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test or unpaired, two-tailed Student's t -test; * p < 0.05 and *** p < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) were purchased from Angioproteomie (Boston, MA, USA) and maintained in endothelial growth media 2 (EGM2), which contained EBM2 basal media and Endothelial Cell Growth Medium BulletKits TM (Lonza, Basel, Switzerland) or endothelial basal media 2 (EBM2), which contained 0.2 % FBS according to the supplier's instruction.

Techniques: Activation Assay, In Vitro, Ex Vivo, Activity Assay, Control, Two Tailed Test, Alamar Blue Assay, Inhibition, Staining, Migration, Comparison

Journal: Theranostics

Article Title: System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy

doi: 10.7150/thno.72947

Figure Lengend Snippet: AXL is ADAM10's downstream substrate and ADAM10's function is dependent on the activity of AXL. (A) Full-length AXL protein levels in HREC with 50 µM ADAM10 activator EGGC or 10 µM ADAM10 inhibitor GI 254023X is shown at both 4- and 24-h post treatment. GAPDH is used as loading control (B) Combination treatment of AXL inhibitor R428 and ADAM10 activator EGCG have no additive effects on HRECs viability. AlamarBlue assay demonstrated inhibition of HRECs viability by 50 µM EGCG, 1 µM R428 or combination treatment of 50 µM EGCG, 1 µM R428 following 24 h treatment (n = 3). (C) Combination treatment of AXL inhibitor R428 and ADAM10 activator EGCG have no additive effects on HRECs migration. DAPI staining demonstrated inhibition of HRECs migration by 50 µM EGCG, 5 µM R428 or combination treatment of 50 µM EGCG, 5 µM R428 following 4 h treatment (n = 3). (D) R428 reverses the promoting effect of EGCG inhibitor GI 254023X on HRECs viability. AlamarBlue assay demonstrated effects of HRECs viability by 10 µM GI 254023X, 1 µM R428 or combination treatment of 10 µM GI 254023X, 1 µM R428 following 24 h treatment (n = 3). (E) R428 reverses the promoting effect of GI 254023X on HRECs migration. DAPI staining demonstrated effects of HRECs migration by 10 µM GI 254023X, 5 µM R428 or combination treatment of 10 µM GI 254023X, 5 µM R428 following 4 h treatment (n = 3). Scale bar: 100 µm. All images shown are representative, and data are presented as means ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test; ** p < 0.01 and *** p < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) were purchased from Angioproteomie (Boston, MA, USA) and maintained in endothelial growth media 2 (EGM2), which contained EBM2 basal media and Endothelial Cell Growth Medium BulletKits TM (Lonza, Basel, Switzerland) or endothelial basal media 2 (EBM2), which contained 0.2 % FBS according to the supplier's instruction.

Techniques: Activity Assay, Control, Alamar Blue Assay, Inhibition, Migration, Staining, Comparison