2.1 Culture of ARPE-19 cells and human umbilical vein endothelial cells (HUVECs)
The ARPE-19 cells were cultured as describe previously. The normal ARPE-19 cells purchased from the American Type Culture Collection (ATCC) were cultured in Dulbecco’s Modified Eagle Medium/Ham’s Nutrient Mixture F12 (1:1) (DMEM/F12, Gibco, Grand Island, NY) containing 10% foetal bovine serum (FBS, Gibco, Australia) and 1% penicillin-streptomycin (Gibco, Grand Island, NY) in a humidified incubator at 37°C at 5% CO2. The medium was replaced every two or three days.
HUVECs were purchased from ATCC and were grown under the manufacturer’s recommendation using Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY) supplemented with 10% foetal bovine serum (FBS, Gibco, Australia) and 1% penicillin-streptomycin (Gibco, Grand Island, NY). Cells of passages 2 through 7 were used for experiments. Cells were grown at 37°C in a humidified incubator with 5% CO2.
2.2 Application of cyclic stretch
After removal of culture medium, ARPE-19 cells were harvested for seedlings on 6-well Flexcell culture plates coated with collagen type I (Flexcell International Corporation, Burlington, CA, USA). ARPE-19 cells were seeded in rubber membrane (Fig.1A). And then using DMEM with 1% FBS serum starved until reaching subconfluency. Subsequently, ARPE-19 cells in fresh 1% FBS media were subjected to a cyclic stretch regimen of 10% prolongation stretch (1/2 sine, 1Hz) using a Flexcell® FX-5000 Tension system (Flexcell International Corporation, Burlington, CA, USA). Vacuum drawn through the Loading post pulls the rubber membrane downward at 37°C in a 5% CO2 (Fig.1B). Cells and supernatant were collected at different time points.
2.3 Quantitative real-time PCR
According to the manufacturer’s instructions, total RNAs were extracted using Trizol (Invitrogen, USA). The purity and concentration of RNA were measured by the ultraviolet spectrometer at an optical density ratio of 260/280 (OD260/OD280). Then, total RNA was treated with RNase-Free DNase (Thermo, Pittsburgh, PA). First-strand cDNA was synthesised by reverse transcriptase from 5 μg total RNA with oligo-d (T) primers. Real-time PCR was performed by using an iCycler IQ real-time PCR detection system (Thermo, Pittsburgh, PA) to measure the fluorescence produced by SYBR Green I (Thermo, Pittsburgh, PA). The negative control was obtained by performing PCR without cDNA. The thermal cycling conditions were: 3 min at 95°C, followed by 40 cycles at 95°C for 30 sec, 57°C for 30 sec and 72°C for 30 sec. All PCR reaction products were verified by melting curve analysis. TGF-β mRNA is consistent with TGF-β1 mRNA and TGF-β2 mRNA. TGF-β and VEGFA mRNA expression levels were quantified by calculating the average value of triplicate reactions, normalised by the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences are shown in Table 1.
2.4 Western blot
Antibodies used in this study are listed as follows: VEGF165 (1:1000, ab1316, Abcam) , TGF-β 1+2 ( 1:1000, ab124894, Abcam) , P38 MAPK (1:2000, 8690S, Cell Signalling Technology), JNK (1:2000, 9252S, Cell Signalling Technology), Smad2/3 (1:1000, ab207447, Abcam), ERK1/2 (1:1000, ab36991, Abcam) and GAPDH (1:5000, ab8245, Abcam)
The protein concentration of the supernatant was measured by a BCA Protein Assay Kit (CWBIO, CHINA). Equivalent amounts of total proteins (30 ug) were loaded on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel after transferring to polyvinylidene difluoride (PVDF) membrane. Then, the membranes were incubated with primary antibody. The secondary antibody was goat anti-rabbit immunoglobulin G/horseradish peroxidase (IgG HRP)-conjugated (1:5,000) or goat anti-mouse IgG (HRP)-conjugated (1:5,000) All western blots were developed by HRP with the detection reagent Western Lightning Plus ECL (Merck, Waltham, MA). Band densities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). These data were normalised by GAPDH expression.
2.5 Enzyme-linked immunosorbent assay 
Samples were performed using the Quantikine Human VEGF ELISA Kit (R&D Systems, Minneapolis, MN). An aliquot of 200 μl of conditioned medium was used per well. The levels of antigenic VEGF in the serial dilutions of ARPE-19 supernatants were quantitated by modification of a double ligand ELISA.
2.6 Tube formation assay
Tube formation assay was performed by the published protocol by Bai. Aliquots of 100 μl of Matrigel (Cat#356231, BD Sciences) were added to each well of the 48-well plates, followed by incubation at 37°C for 30 min. The HUVECs (5 × 104) were seeded to the top of solidified Matrigel with 150 ul of conditioned medium for 3 h at 37°C. Before start of the assay, the HUVECs can be treated with calcein AM (ab141420，Abcam) to visualise the cells using fluorescence. On five photographs in each group, fields of the Matrigel were randomly chosen under the microscope, and the length of the tubes was easier quantitated using Image J Pro (National Institutes of Health, Bethesda, MD). The experiments were repeated three times.
2.7 Statistical analysis
Data are expressed as mean ± SEM. Significant differences between groups were ascertained by one-way analysis of variance (ANOVA) in combination with all pair wise multiple comparison procedures using Bonferroni test. In all cases, values of p < 0.05 were considered as statistically significant.