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. 2011 Feb 1;52(1):570-8.
doi: 10.1167/iovs.10-5595. Print 2011 Jan.

The role of RPE cell-associated VEGF₁₈₉ in choroidal endothelial cell transmigration across the RPE

Haibo Wang  1 , Pete Geisen, Erika S Wittchen, Bradley King, Keith Burridge, Patricia A D'Amore, M Elizabeth Hartnett
Affiliations

The role of RPE cell-associated VEGF₁₈₉ in choroidal endothelial cell transmigration across the RPE

Haibo Wang et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: To determine the role of vascular endothelial growth factor 189 (VEGF₁₈₉) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved.

Methods: Using real-time PCR, the expression of VEGF splice variants VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H₂O₂) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF₁₈₉ expression. Migration of CECs across the RPE was determined using fluorescence microscopy.

Results: VEGF₁₈₉ expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF₁₈₉ was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF₁₈₉; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF₁₈₉, VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity.

Conclusions: RPE-derived cell-associated VEGF₁₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.

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Figures

Figure 1.
Figure 1.
Expression of VEGF isoforms in human and mouse RPE. The expression of VEGF isoforms was measured by RT- PCR in (A) human RPE from young (>20 years old) and old (>70 years old) donors. ***P < 0.0001 vs. young (n = 3); the number on the top of the bar means mRNA expression level of VEGF splice variants for real-time PCR. (B) hfRPE treated with H2O2 (600 μM) for 16 hours. ***P < 0.0001 vs. control (n = 6). (C) hfRPE incubated under hypoxic conditions 1% O2. ***P < 0.0001 vs. 21% O2 (n = 3). (D) hfRPE solo, noncontact, and contact with CECs. *P < 0.05, ***P < 0.0001 vs. solo (n = 6). (E) CECs solo, noncontact, and contact with hfRPE (n = 3). (F) Mouse RPE 7 days after laser injury CNV. *P < 0.05 and ***P < 0.0001 vs. control (n = 6). All data are shown as mean ± SEM.
Figure 2.
Figure 2.
RPE-derived VEGF189/188 facilitates CEC migration across RPE. (A) Expression of VEGF splice variants in solo CECs treated with or without H2O2 (n = 3); CEC transmigration was measured in cocultures of RPE and CEC. (B) CEC transmigration was measured when the cocultured hfRPE was incubated in the presence or absence of 600 μM H2O2 for 16 hours H2O2 treatment of hfRPE increased CEC transmigration. **P < 0.001 vs. control (n = 6). (C) CEC migration across the filter when cultured alone (solo) or with mRPE-WT or mRPE188/188. CEC transmigration was highest when cocultured with mRPE188/188. *P < 0.05 and ***P < 0.0001 vs. solo and ###P < 0.0001 vs. mRPE (n = 6). All data are described as mean ± SEM.
Figure 3.
Figure 3.
Knockdown of VEGF189 in RPE decreases CEC transmigration. (A) mRNA levels of VEGF isoforms in ARPE-19 cells transfected with VEGF189 siRNA were measured by RT-PCR. Nontargeting siRNA was used as a negative control. Both VEGF189 siRNA sequences A and B specifically reduced VEGF189 while not affecting expression of the other isoforms. ***P < 0.0001 vs. control (n = 6). (B) CEC transmigration assay was performed using ARPE-19 cell monolayer that had been depleted of VEGF189 by siRNA. CEC transmigration was decreased when cocultured with ARPE-19 with reduced VEGF189. *P < 0.05 vs. control siRNA (n = 6). All data are described as mean ± SEM.
Figure 4.
Figure 4.
CEC contact with hfRPE leads to VEGFR2 increased phosphorylation in CECs. (A) Expression of VEGFR1 and VEGFR2 in CECs (n = 3). Immunoprecipitation of phospho-VEGFR2 and VEGFR1 in CECs. CECs were grown in contact with hfRPE. During the last 16 hours of contact, hfRPE was incubated with 600 μM H2O2. H2O2 treatment of RPE increased the phosphorylation of VEGFR2 in CECs (B) but did not affect VEGFR1 phosphorylation (C; representative blot shown). The bar graph on the right shows phospho-VEGFR2 band density normalized to total VEGFR2. **P < 0.001 vs. control (PBS) (n = 3). (D) Representative blots showing that the phosphorylation of VEGFR1 and VEGFR2 was unchanged in solo CECs treated with H2O2. CECs grown in contact with ARPE-19 cells were transfected with VEGF189 siRNA. After 24 hours of contact, CECs were collected for the detection of phospho-VEGFR2/total VEGFR2 and phospho-VEGFR1/total VEGFR1 by immunoprecipitation and Western blot analysis. VEGFR2 phosphorylation was decreased (E), but VEGFR1 phosphorylation was unchanged in CECs cocultured in contact with RPE with reduced VEGF189 (F). The bar graph on the right shows phospho-VEGFR2 band density normalized to total VEGFR2. *P < 0.05 vs. control siRNA (n = 3) and a representative blot of phospho-VEGFR1.
Figure 5.
Figure 5.
Coculture of CECs with RPE expressing elevated VEGF189/188 leads to increased Rac1 activity. Rac1 activity assay was measured in CECs grown in contact with (A) mRPE-WT and mRPE188. The bar graph on the right shows active-Rac1 band density normalized to total Rac1. *P < 0.05 vs. mRPEWT (n = 3). (B) hfRPE treated with 600 μM H2O2 for 16 hours. After 24 hours of contact, CECs were collected for the detection of active Rac1 and total Rac1 by GST-PBD pull-down and Western blot analysis, as described. Rac1 activity was increased with both treatments. The bar graph on the right shows active-Rac1 band density normalized to total Rac1. **P < 0.001 vs. control (PBS) (n = 3). (C) PI-3K activity using phosphorylation of Akt-1 as a readout was measured in CECs grown in contact with hfRPE treated with H2O2 as in B. CECs were then collected for the detection of phospho-Akt and total Akt by immunoprecipitation and Western blot analysis as described in Experimental Procedures. Upregulation of VEGF189 by H2O2 treatment of RPE does not affect phosphorylation of Akt-1 in CECs. The bar graph on the right shows phospho-Akt (Ser473) band density normalized to total Akt1. P > 0.05 vs. control (PBS) (n = 3). (D) Representative blot showing that Rac1 activity was unchanged in solo CECs treated with H2O2.
Figure 6.
Figure 6.
RPE-derived VEGF189 stimulates CEC transmigration mediated by Rac1 activation. Activities of Rac1 (A) and PI-3K (B) were measured in CECs grown in contact with ARPE-19 cells with reduced VEGF189. Twenty-four hours after contact, CECs were collected for the detection of active Rac1 and phospho-Akt by GST pull-down and immunoprecipitation. Whole cell lysates were used to detect total Rac1 and Akt by Western blot analysis. Knockdown of VEGF189 decreased Rac1 activity compared with control siRNA, whereas p-Akt remained unchanged. The bar graph on the right shows active-Rac1 or phospho-Akt band density normalized to total Rac1 or total Akt1. *P < 0.05 vs. control siRNA (n = 3). (C) CEC transmigration assay was performed using hfRPE and CECs in which Rac1 activity had been inhibited by transfection with GFP-POSH-RBD. Twenty-four hours after transfection, CECs were added, and transmigration was measured after 48 hours. During the last 16 hours of contacting coculture, hfRPE was incubated with 600 μM H2O2. CECs in which Rac1 activity was inhibited by expressing GFP-POSH did not exhibit increased transmigration when cocultured with H2O2-treated hfRPE compared with cells expressing GFP alone. ***P < 0.0001 vs. GFP alone (n = 6).
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