Heterotypic RPE-choroidal endothelial cell contact increases choroidal endothelial cell transmigration via PI 3-kinase and Rac1

Abstract

Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that "contact" with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD.

Figure 1. Contact between CECs and underlying RPE increases CEC transmigration

Transmigration of CECs was quantified after 24, 48, or 72 hr in coculture or solo culture. There is a significant increase in CEC transmigration when CECs are cultured in contact with RPE versus solo CEC culture. Data represent the mean of 3 experiments +/−SEM (n=3) *p=0.012, **p=0.004, ***p=0.016, Student’s t-Test.

Figure 2. Contact between CECs and underlying RPE increases Rac1 activity; an effect which is dependent on PI 3-K activity

(A) Schematic representation of coculture assay used for biochemical analysis in CECs. Solo culture indicates CECs are plated on the upper surface of the Transwell insert, with no RPE present. For contacting coculture assays, CECs are grown in a Transwell insert with small (1 µm) pores that permit CEC processes to contact the basal aspect of an RPE monolayer grown on the underside of the Transwell insert. In non-contacting coculture, RPE are grown in the lower well with CECs in the insert. (B) CECs grown in solo, contacting, or non-contacting coculture for 24 hr were pulsed for 30 min with LY294002 (50 µM) or DMSO vehicle control, rinsed, then assayed for Rac1 activity 20 min later. The levels of active Rac1 increase with contacting coculture, and this increase is blocked by LY294002 pretreatment. A representative blot of active Rac1 sedimented by GST-PBD versus total Rac1 from whole cell lysates is shown. (C) Quantification of blots: average relative Rac1 activity (+/− SEM) is obtained by averaging the ratio of active to total Rac1 from 4 independent experiments. Overall ANOVA, p=0.0217, post-hoc Student-Newman-Keuls Multiple Comparison showed significant differences between contacting CECs (*) and solo p<0.05) or non-contacting CECs (p<0.05) in the absence of LY294002.

Figure 3. Contact between CECs and underlying RPE activates Akt1 via PI 3-K

(A) CECs in solo, contacting, or non-contacting coculture (as in Figure 2A) were grown for 24 hr and assayed for activation of PI3K/Akt1. Akt1 immunoprecipitates were analyzed for either total Akt1 levels or active Akt1, using a phospho-specific antibody. Contact between CECs and RPE increased Akt1 phosphorylation, and this increase was blocked when CECs were pulse-treated with the PI 3-K inhibitor LY294002 as described (30 min, 50 µM). A typical blot of total versus active (phosphorylated) Akt1 is shown. (B) Graph represents the average relative phosphorylation of Akt1 (+/− SEM) from 3 independent experiments. Overall ANOVA, p=0.0039, post-hoc Student-Newman-Keuls Multiple Comparison tests showed significant differences between contacting CECs and solo (p<0.01) or non-contacting CECs (p<0.001) in the absence of LY294002.

Figure 4. Inhibition of PI 3-K reduces CEC transmigration

(A) Schematic representation of transmigration assays. CECs and RPE are grown in coculture on either side of a Transwell insert with large enough pores (8 µm) to permit transmigration. RPE are plated in the lower well to provide chemoattractants. (B) CECs grown 24 hr in transmigration assays were pulsed for 30 min with 50 µM LY294002, 2 µM PP2, or DMSO-only control. After 24 hr, transmigrated CECs were quantified. Transmigration was inhibited specifically when PI 3-K/Akt1 activity was blocked. Graph represents mean +/− SEM of 3 independent experiments. *p < 0.01

Figure 5. Inhibition of Rac1 activation reduces CEC transmigration

CECs were nucleofected to express either GFP alone, or GFP-POSH-RBD and GFP-17N Rac1 to inhibit Rac1 activity, and then grown in contacting co-cullture with RPE. After 72 hr, transmigration of CECs expressing these constructs was quantified by counting only green cells from the underside of the filter. Inhibition of Rac1 by expressing both GFP-POSH-RBD and GFP-17N Rac1 significantly reduced transmigration compared to GFP-expressing cells. Graph represents mean +/− SEM of 4 independent experiments with GFP-POSH-RBD and 3 experiments with GFP-17N Rac1. p<0.001 GFP compared to GFP-POS-RBD or GFP-17N Rac1.

Figure 6. A Rac1 and PI 3-K-dependent signaling pathway is central to the contact-induced migration of CECs

Putative signals which stimulate CECs to migrate include: chemotactic factors such as soluble VEGF secreted by RPE, contact between CECs and RPE, or contact with RPE extracellular matrix. Rac1 GTPase is activated via a PI 3-K-dependent mechanism in response to any or all of these stimuli, with the end result being transmigration of CECs across the RPE.