Cyclic stretch-induced VEGF expression is mediated by TGF-β in retinal pigment epithelial cells

Background: Vitreomacular adhesion (VMA) has been theorised to be a prospective risk factor for anti-vascular endothelial growth factor (VEGF) therapy. The mechanisms underlying VMA are not fully understood. Therefore, we investigated whether exposure to cyclic stretch was associated with increasing VEGF and related signalling pathways in vitro. Methods: A cyclic stretch model of adult retinal pigment epithelial cell line-19 (ARPE-19) was conducted using a Flexcell-5000 strain system at a regimen of 10% prolongation stretch (1/2 sine, 1Hz). We observed the effect of cyclic stretch on VEGF and transforming growth factor-β (TGF-β) by using western blot and real-time polymerase chain reaction (PCR) for 3 h, 6 h, 9 h and 12 h. Enzyme-linked immunosorbent assay was used to measure the secretion of VEGF in the supernatant. To quantitatively detect the angiogenesis capacity of VEGF in vitro, tube formation assay was performed. The effects of TGF-β receptor I inhibitor SB335242 on stretch-induced VEGF protein expression and secretion were evaluated by western blot and ELISA. Components of the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) in the stretch group were analysed using western blot. Results: We discovered that cultured ARPE-19 undergoing cyclic stretch significantly increased VEGF and TGF-β in a time-dependent manner and tube formation. Stretch-induced VEGF expression was inhibited by TGF-β receptor I inhibitor SB335242. In vitro, we also verified P38 MAPK and JNK expression. In western blot analysis, mechanical stretch evaluated P38 and JNK protein expression. Conclusion: In this study, we first demonstrated that cyclic stretch induces the increasing VEGF through TGF-β in RPE cells. Mechanical stretch stimulates VEGF in a time-dependent manner and maintains angiogenesis ability. It is thus possible that RPE cells under mechanical stretch, such as VMA, may generate high levels of TGF-β. Sequentially, the overexpressed TGF-β stimulates VEGF production, triggering CNV events and aggravating wAMD.

Similarly, Seko et al. [15] found that in rat RPE cells, VEGF could rapidly increase loaded with 15% elongation.
In this study, we examined the mechanism of stretch-induced VEGF expression in RPE cells. This is the first study to demonstrate that stretch-induced VEGF expression is mediated by a TGF-β dependent pathway. Moreover, RPE cell exposure to cyclic stretch promoted P38 mitogen-activated protein kinase (MAPK) and JNK activation.

Culture of ARPE-19 cells and human umbilical vein endothelial cells (HUVECs)
The ARPE-19 cells were cultured as describe previously [14]. 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% CO 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% CO 2 (Fig.1B). Cells and supernatant were collected at different time points.

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. Realtime 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. 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.

Enzyme-linked immunosorbent assay [31]
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.

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.

Characterisation of stretch-induced VEGFA and TGF-β expression in ARPE-19
cells ARPE-19 cells were subjected to an instance of 10% cyclic stretch for the durations indicated in Fig. 2A. Cyclic stretch maximally increased VEGFA mRNA expression 1.7-fold after 3 h and 6 h (p=0.001). Although VEGF mRNA levels gradually declined after that 6 h, stretch-induced VEGF mRNA significantly upregulated 1.3-fold after 9 h (p=0.023) and 1.2fold after 12 h (p=0.043) compared with the control group. As shown in Fig. 2B, an increase in TGF-β mRNA expression was initially evident at 3 h, which peaked at 6 h when expression was 2.3±0.5-fold greater than in the control group (p=0.005).
To evaluate whether stretch-induced VEGF165 and TGF-β mRNA expression resulted in increased VEGF and TGF-β protein levels, western blot was used for analysis. As displayed in Fig. 3B, cyclic stretch increased VEGF165 expression in a time-dependent manner.
The secretion of VEGFA at different time points was detected by ELISA. The line graph showed that the level of VEGFA increased with the extension of a cyclic stretch (Fig. 3D).

Role of TGF-β in Stretch-induced VEGF Expression
The results in Figures 5A and 5B show that under the action of SB525334, the increase of VEGF165 expression induced by cyclic stretch was inhibited, which was significantly different from that of the non-inhibitor group (P<0.01); at the same time, there were no tension loading and blank control groups. In comparing (weakness without inhibitors), the relative protein expression of VEGF165 was also significantly reduced (P<0.05). With the prolongation of the action time, SB525334 exerted a continuously weakening effect on the traction force-induced VEGF secretion, and there was a statistically significant difference between the time points and the no inhibitor control group (P<0.01). To gain further insight into the potential expression, we detected the media by ELISA (Fig.5C). Treatment with SB525334 for a series time that could significantly inhibited VEGF expression (P<0.01). The average value was 3 h (0.16±0.028 fold), 6 h (0.17±0.013 fold), 9 h (0.2±0.016 fold) and 12 h (0.15±0.024 fold), respectively.

Stretch-induced VEGF stimulated HUVEC tube formation
Since the formation of tube-like structures is essential for angiogenesis [34], the ability of stretch-induced VEGF to promote tube formation was further investigated in Matrigel.
Cultured HUVECs in vitro were incubated with the conditioned medium of the stretch group and control group for 3 h at 37°C in 5% CO 2 . Typical tube-like structures were observed after fluorescent staining (Fig. 6A). The analysis by Image J software showed that the tube of length in the stretch group was significantly longer than that of the control group at 6 h (116±8%, P<0.05), 9 h (115±8%, P<0.05) and 12 h (127±4%, P<0.01), respectively (Fig. 6B).

Discussion
Our study describes that cyclic stretch strongly increases VEGF expression in a timedependent manner and promotes angiogenesis in human RPE cells. Meanwhile, stretchinduced VEGF expression is predominantly dependent on TGF-β and upregulates P38 MAPK and JNK cytokines.
Currently, the gold standard treatment for CNV is an intravitreal injection of anti-VEGF drugs, albeit with limited visual recovery in wAMD combined with VMA[4, 35]. Gao et al. [35] suggested that VMA traction had a significant influence on the treatment of anti-VEGF. Thus, a deeper understanding of VMA traction aetio-pathogeny is warranted.
Stemming from the application of Flexcell-5000 tension system to stimulate ARPE-19 cells, the expression and secretion of VEGF protein were significantly increased over time, while VEGF mRNA peaked at 6 h and then decreased slightly, indicating that mechanical stretch could regulate VEGF expression. The time course of VEGF expression in response to cyclic stretch in ARPE-19 was similar to that reported by Wu et al. [14], who also detected a sharp decrease after withdrawal of cyclic stretch. Seko et al. [15] found that VEGF expression was remarkably upregulated at 24 h in the 0.2 Hz 10% variability group, whereas it was increased at 1 h or 3 h in the 1 Hz 15% variability group in rat RPE cells.
Compared with our results, these discrepancies may relate to the use of the older model device (Flexcell-3000 tension system) and the inconsistent mechanical parameters, or the use of rat cells rather than human cells.
In addition, we noticed that the higher concentration of VEGF measured by ELISA could significantly induce the differentiation of endothelial cells to tube-like cells, and length of the tube was dramatically longer than that of the control group. Since the formation of tube-like structures is essential for angiogenesis, these results indicate that cyclic stretch could induce high expression of VEGF and has the potential to promote angiogenesis. CNV

Availability of data and materials
The datasets generated and analyzed during the current study available from the corresponding author on reasonable request.  Figure 1 Schematic diagram of one well of a culture plate (A) shown from above, the rubbe membrane coated with Collagen type I is drawn in non-stretch. Fig.1B showed that vacuum drawn through the Loading post pulls the rubber membrane downward in a cyclic stretch regimen of 10% prolongation stretch (1/2 sine, 1Hz).
Thereafter, cells and supernatant were collected at different time points.

Figure 2
Cyclic stretch increases VEGFA mRNA and TGF-mRNA expression VEGF mRNA (A) and TGF-β mRNA (B) expression showed a statistically significant time-dependent increase after normalization to the control group. The experiment was repeated three times with similar results. *P < 0.05, **P < 0.01.

Figure 3
Effect of cyclic stretch on VEGF and TGF-protein A Western blotting analysis of stretch induced VEGF165 protein and TGF-β protein undergo stretch and nonstretch group. The location of a 24kd and 44kd are respectively shown in figure.
Results are expressed as relative VEGF165 protein (B) and TGF-β (C) protein compared with control levels. D The collected supernatant was analyzed by ELISA to examine the levels of secreted VEGFA. Data was from three independent experiments. *P < 0.05, **P < 0.01.

Figure 4
Cyclic stretch increases P38 MAPK and JNK expression A Western blotting analysis of stretch induced P38 MAPK, JNK, Smad2/3 and ERK1/2 in stretch and non-stretch group. As is shown, P38 MAPK and JNK are respectively significant increasing compared with control group. Smad2/3 and ERK1/2 were not increased in any samples of stretch group. B-C Quantitative analysis of P38 MAPK and JNK abundance. Data was from three independent experiments. *P < 0.05, **P < 0.01.

Figure 5
Cyclic stretch-induced VEGF expression is TGF-β dependent ARPE-19 was exposed Fluorescence microscopy imaging and quantification of tube formation Typical tube formation was seen in any of the samples with conditioned medium (A). The length of tube-like formation was evaluated by image J software. Quantitative data are presented as mean ± SEM (n = 3) in (B). *P < 0.05, **P < 0.01.