Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells

Abstract

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell-to-cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT-PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.

Keywords: VEGF receptors; angiogenesis; exosomes; oxidative stress; retinal pigment epithelium.

Figure 1

Exosome biogenesis. Formation of MVBs was observed in untreated cells (arrow in A) (higher magnification in the inset). Exosomes released from ARPE‐19 cells exhibit the classical morphology and size (50–150 nm). They were detected in extracellular medium from untreated cells (B) and from 80 mM ethanol‐treated cells (C). Flow cytometry exosome detection was performed targeting CD9. Size‐distribution analysis of exosomes was performed by Nanosight (D). Control ARPE‐19 cells released a lower number of exosomes (first plot in E) than those cells treated with 40 mM (second plot in E), and 80 mM (third plot in E). Quantification is shown in the bar graph. Scale bars represent 500 μm in A, 500 nm in the inset, and 100 nm in B and C. Values are expressed as mean ± S.E.M. (N = 3). Significance levels: P < 0.05 (*).

Figure 2

VEGF and VEGFR‐1 from ARPE‐19 cells. ARPE‐19 cells were incubated for 24 hrs in the absence (control) or presence of ethanol, and the result was analysed by Western blot. Non‐treated cells secreted a baseline amount of VEGF into the medium (first band in A), whereas ethanol‐treated cells secreted a significantly higher concentration of VEGF. Those observations were made using 40 mM ethanol (second band in A) and 80 mM ethanol (third band in A). Conversely, VEGFR‐1 did not show a significant change in release when cells were exposed to ethanol damage (B). All experiments were normalized to the loading control (B‐actin or α‐tubulin). Values are expressed as mean ± S.E.M. (N = 3). Significance levels: P < 0.01 (**).

Figure 3

Effects of ethanol in exosomal VEGFRs content. ARPE‐19 cells were incubated for 24 hrs in the absence (control) or presence of different ethanol concentrations. Released exosomes were scrutinized by flow cytometry and Western blot. VEGFR‐1 protein was detected in control and ethanol groups, being significantly different at 80 mM of ethanol (A). VEGFR‐2 protein was also found in exosomes released from untreated cells, and its levels significantly rose in an ethanol concentration‐dependent manner (at 40 and 80 mM) (B). Presence of VEGFR‐1 in exosomes released by ARPE‐19 was also observed by Western blot, and results were in agreement with cytometry experiments (C). Values are expressed as mean ± S.E.M. (N = 3). Significance levels: P < 0.05 (*) and P < 0.01 (**).

Figure 4

Exosomes released from ethanol‐exposed ARPE‐19 cells are highly enriched in VEGFR‐1 and VEGFR‐2 mRNAs. VEGFR‐1 and VEGFR‐2 transcript levels in exosomes released by ARPE‐19 were also analysed using reverse transcription assay followed by qPCR. ARPE‐19 cells were incubated for 24 hrs in the absence (control) or presence of different ethanol concentrations. RNA was extracted from the exosome released from ARPE‐19 cells. Presence of mRNA of VEGFR‐1 was found in control and ethanol groups, being its level significantly higher at 40 and 80 mM of ethanol (A). VEGFR‐2 mRNA was also detected in exosomes derived from untreated cells, and its amount increased in an ethanol concentration‐dependent manner (B). Values are expressed as mean ± S.E.M. (N = 3). *Significance level: P < 0.05.

Figure 5

Vasculogenesis capacity of exosomes‐treated HUVEC cells. Exosome‐treated and ‐untreated HUVEC cells were seeded on Matrigel, showing specific tube formation after 5 hrs. Non‐treated HUVEC cells showed basal tube formation (A). Tube formation appeared less effective when HUVEC cells were treated with exosomes derived from non‐treated ARPE‐19 cells (B). When HUVEC cells were treated with exosomes derived from EtOH‐treated (80 mM) ARPE‐19 cells, tube building was more efficient (C). Node formation (D) and length of the new blood vessels (E) was quantified. After 5 hrs, HUVEC cells were pulled and VEGFR‐1 and VEGFR‐2 protein expression were studied by Western Blot (F and G). Loading control with B‐actin was performed for VEGFR‐1 and ‐2 (F). Amounts of mRNA were measured in HUVEC cells before and after exosome treatment, showing an enhancement when endothelial cells were treated with stressed exosomes (H). Scale bars: 500 μm. Values are expressed as mean ± S.E.M. (N = 3). Significance levels, when compared to control: P < 0.05 (*); when compared to Exo (control): P < 0.05 (#) and P < 0.01 (##).