While the mechanisms leading to increased expression of RPE-derived angiogenic factors and the resulting angiogenesis during CNV in AMD are not entirely clear, physical changes in the RPE contribute to the elevated expression of angiogenic factors [1,18,21]. To better understand the role that mechanical stress plays in AMD pathogenesis, we investigated the effect of mechanical stress on the expression of angiogenic and EMT factors and in vitro angiogenesis using a novel in vitro device (Fig. 1).
Mechanical stress in many cell types, including periodontal ligament fibroblasts (HPLF), mesenchymal stem cells and HUVECs, has been found to affect angiogenesis [22–24]. ARPE-19 cells, a human RPE cell line, express increased levels of angiogenic cytokines, such as VEGF and IL6, involved in various retinal diseases in response to cyclic mechanical stress [1,25]. However, ARPE-19 cells lack important RPE characteristics, such as RPE-specific marker expression and high transepithelial resistance . A more realistic RPE model is needed to characterize the effect of mechanical stress-derived changes to the RPE secretome and angiogenesis. To this end, we produced a realistic in vitro model of the RPE by growing freshly isolated porcine RPE cells on Transwell membranes. Porcine RPE has been used previously as a realistic in vitro model to study many RPE functions, including VEGF expression and in vitro angiogenesis, demonstrating its suitability for the purpose of this study [27–29]. Our in vitro porcine RPE cultures demonstrated several characteristics similar to native RPE, such as junctional localization of b-catenin, F-actin, and ZO-1, expression of the RPE-specific RPE65, and high TEER in 4-week-old RPE cultures (Fig. 2), indicating that the substrate supports proper maturation of RPE cells. Next, using a novel device, mechanical stress was added to the RPE monolayer to determine changes in gene and protein expression of major angiogenic and EMT factors. Lastly, an in vitro angiogenesis assay was performed to determine if pro-angiogenic factor production outweighed anti-angiogenic factor production and induced angiogenesis.
Our results showed elevated mRNA expression of angiogenic factors, VEGF, HIF-1α, IL-6, IL-8, ANG2, and anti-angiogenic PEDF (Fig. 3q), and increased protein expression of VEGF, IL-6, IL-8, and ANG2 in mechanically stressed RPE cells (Fig. 3b-p, s, t). ICC results also revealed a remarkable disruption of the actin cytoskeleton in RPE sites with higher VEGF, IL-6 and IL-8 expression. The stressed areas of the RPE showed diffusely distributed actin fibers, while in the non-stressed areas, actin was localized in cell-cell junctions (insets in Fig. 3c, h, m). According to the FEA (Fig. 3a), in these areas of impact, strain distribution pattern correlated with the disruption of actin structure and increased VEGF, IL-6, and IL-8 expression, supporting the hypothesis that the increase in mechanical stress is responsible for elevated angiogenic factor expression.
The increase in IL-8 and VEGF expression (Fig. 3q, s, t) was consistent with previous reports of IL-8 and VEGF induction in human RPE cells after RPE injury [30,31]. It has previously been shown that actin polymerization activation induces IL-8 expression and different VEGF isoforms [32,33] . It is therefore possible that disrupting the actin cytoskeleton activates actin polymerization, leading to the increased expression of IL-8 and different VEGF isoforms. Unlike VEGF121 and VEGF165, VEGF189 was not sensitive to the mechanical stress levels used in our experiments (Fig. 3q). The two soluble VEGF isoforms, VEGF121 and VEGF165, are regulated by low frequency stress while insoluble VEGF189 is more susceptible to high frequency mechanical stress . The single pulse of mechanical stress used in our experiment may be lower than required for VEGF189 mRNA overexpression. Both soluble VEGF isoforms, VEGF121 and VEGF165, have been implicated in in vitro and in vivo neovascularization [34–37]. Hence, their overexpression induced by mechanical stress may contribute to CNV development.
We also observed an increase in the expression of HIF-1α in response to mechanical stress (Fig. 3q). HIF-1α is an inducer of VEGF, IL-6, and IL-8 under hypoxic conditions [38,39], and its mRNA expression was activated by mechanical stress, suggesting that the mechanisms of mechanical stress-induced VEGF, IL-6, and IL-8 overexpression may be similar to hypoxic conditions. Further investigation is needed to accurately assess the involvement of HIF-1α in inducing angiogenic factors in RPE cells due to mechanical stress.
Our gene expression results also showed an increase in the expression of TNF-α in response to mechanical stress (Fig. 3q). Mechanical stress can induce oxidative stress in RPE cells, which in turn activates TNF-a transcription [40,41]. Our qRT-PCR results confirm these previous findings by implicating mechanical stress in inducing TNF-α gene expression. However, ELISA results showed undetectably low levels of TNF-α in RPE supernatants before and after applying mechanical stress, suggesting that the low level of mechanical stress used in this study may not be enough to activate TNF-α protein expression in RPE cells. Further experiments with different mechanical stress levels will elucidate the mechanism of mechanical stress-induced TNF-α expression in RPE cells and explain the lack of TNF-α protein expression despite increased transcription.
The qRT-PCR results showed that mechanical stress promoted an EMT-like phenotype in RPE cells, as demonstrated by an increase in the expression of mesenchymal markers, CDH2, VIM, and FN1, and a decrease in the expression of the RPE-specific RPE65 (Fig. 3r). Previous studies have shown that VEGF, IL-6 and IL-8 can trigger EMT [15–17]. Our experiments demonstrated that mechanical stress induced all three of these cytokines, confirming the hypothesis that mechanical stress may induce EMT in the RPE through the induction of EMT promoters during stages of CNV development.
The expression of PEDF, a major anti-angiogenic gene, also increased due to mechanical stress (Fig. 3q). Previous work has shown that the balance between VEGF and PEDF must change for choroidal angiogenesis initiation , The in vitro angiogenesis assay determined if pro-angiogenic protein levels outweigh anti-angiogenic factors and promote angiogenesis. The increased angiogenic response of the HUVECs to the mechanically stressed RPE secretome (Fig. 4) suggests that the increase in the PEDF expression was either not sufficient to neutralize the angiogenic activity of the overexpressed angiogenic factors or did not lead to significant protein secretion. However, the higher increase in the angiogenic potential of the apical supernatant compared to that of the basal supernatant (Fig. 4) could be due to the elevated PEDF expression, as the expression of PEDF is mainly basolateral. Further protein expression analyses are needed to better understand the dynamics of the VEGF/PEDF angiogenic switch under mechanical stress. The choroid is adjacent to the basal side of the RPE and our experiments showed that media from both sides of mechanically-stressed RPE cultures increased in vitro angiogenesis (Fig. 4), suggesting that mechanical stress promotes angiogenesis by inducing the overexpression of angiogenic factors. These results deepen our understanding of the role that mechanical stress may play in the initiation and development of CNV and open potential avenues to more effective therapeutic interventions for neovascularization in AMD.