Targeting the tight junction protein, zonula occludens-1, with the connexin43 mimetic peptide, αCT1, reduces VEGF-dependent RPE pathophysiology

Abstract

A critical target tissue in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from attenuation/disruption of intercellular tight junctions. Zonula occludens-1 (ZO-1) is a major structural protein of intercellular junctions. A connexin43-based peptide mimetic, αCT1, was developed to competitively block interactions at the PDZ2 domain of ZO-1, thereby inhibiting ligands that selectively bind to this domain. We hypothesized that targeting ZO-1 signaling using αCT1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions. RPE-cell barrier dysfunction was generated in mice using laser photocoagulation triggering choroidal neovascularization (CNV) or bright light exposure leading to morphological damage. αCT1 was delivered via eye drops. αCT1 treatment reduced CNV development and fluid leakage as determined by optical coherence tomography, and damage was correlated with disruption in cellular integrity of surrounding RPE cells. Light damage significantly disrupted RPE cell morphology as determined by ZO-1 and occludin staining and tiling pattern analysis, which was prevented by αCT1 pre-treatment. In vitro experiments using RPE and MDCK monolayers indicated that αCT1 stabilizes tight junctions, independent of its effects on Cx43. Taken together, stabilization of intercellular junctions by αCT1 was effective in ameliorating RPE dysfunction in models of AMD-like pathology.

Key message: The connexin43 mimetic αCT1 accumulates in the mouse retinal pigment epithelium following topical delivery via eye drops. αCT1 eye drops prevented RPE-cell barrier dysfunction in two mouse models. αCT1 stabilizes intercellular tight junctions. Stabilization of cellular junctions via αCT1 may serve as a novel therapeutic approach for both wet and dry age-related macular degeneration.

Keywords: Age-related macular degeneration; Choroidal neovascularization; Connexin43; Light damage; Retinal pigment epithelium; Tight junctions; Vascular endothelial growth factor.

Figure 1. αCT1 detection in murine RPE flatmounts

The αCT1 peptide has an amino acid sequence that is a mimetic of the Cx43 C-terminal sequence. Thus, the peptide can be detected via a Cx43 antibody that recognizes the C-terminal domain. The eyes of the mouse that received the peptide were enucleated 4 hours after eye drop administration and stained for Cx43. The αCT1 peptide could be clearly detected in the animal that received the treatment drops (D), when compared to vehicle-treated animals (B). No primary antibody (A) and antibody preabsorption with 10× molar excess of peptide (C) were used as controls. Scale bar: 50 µm.

Figure 2. Choroidal neovascularization and fluid leakage in αCT1- versus vehicle-treated mice

Animals were analyzed post laser-photocoagulation by SD-OCT. The cross-sectional area of the hyporeflective spot seen in the fundus image (A–C) as well as the area of fluid accumulation in the outer retina (D–F) were determined and representative OCT images taken from the vehicle (A, D), αCT1-treated (B, E) and JM2-treated animals (C, F) are depicted. CNV area and fluid accumulation were determined from SD-OCT images. Quantifications of the cross-sectional areas of the lesions (I–M, left-hand column) as well as areas of fluid accumulation (I–M, right-hand column) were measured in pixels for the individual treatment groups. CNV size and area of fluid accumulation in αCT1-treated animals was significantly reduced, compared to the vehicle group for the continuous (J) and early (I) treatment paradigms. No significance was noted between the vehicle and the two groups for the late αCT1 treatment study (K, L). Angiogenesis was confirmed in flat-mount preparations of RPE-choroid stained with isolectin B, with αCT1significantly reducing the size of the lesion (H) when compared to control (G). (K) JM2-treatment, a Cx43 peptide that includes the microtubule-binding sequence and targets hemichannels, resulted in an increase in CNV size and area of fluid accumulation. Data are expressed as mean ± SEM (n = 7–23 animals per treatment group). Scale bar: 100 pixels or 160 µm.

Figure 3. RPE integrity in αCT1 compared to vehicle-treated mice

On day 6 after the induction of CNV, eyes were enucleated and RPE/choroid eyecups were stained for two different cell junction markers, ZO-1 (A, B) and occludin (C, D). Representative images for each cell junction marker are presented, depicting the differences in the diameter of unhealthy cells (peri-lesion area) surrounding the lesion (LES) in the control group compared to the αCT1-treated animals. Morphometric analyses were obtained from images as depicted in Figure 2, comparing two measures of RPE cell shape, cell eccentricity (E) and form factor (F) in 4 steps of 35 µm each (bins 1–4) for both ZO-1 (left-hand column) and occludin (right-hand column). Cell eccentricity measures the deviation of a shape from an ellipse; form factor measures the deviation from a perfect circle. RPE cells from C57BL/6J mice exhibit a form factor of ~0.82 and an eccentricity value of ~0.65. RPE cells from animals treated with the αCT1 peptide exhibited form factor and eccentricity values closer to normal, closer to the edge of the lesions when compared to mice treated with vehicle. Data are expressed as mean ± SEM (n = 7–8 animals per cell junction marker). Scale bar: 50 µm.

Figure 4. RPE morphology following light-damage

Balb/c mice were exposed to bright light (3000 lux) for 3 hours, sacrificed and eyes enucleated after 24 hours. RPE morphology was analyzed by immunohistochemistry for ZO-1 (A–C) and occludin (D–F) on RPE/choroid flatmounts from the respective treatment groups, no light damage controls (A, D), light damage treated with vehicle (B, E) and light damage treated with αCT1 peptide (C, F). Scale bar: 50 µm.

Figure 5. Effects of αCT1 on VEGF-mediated loss in barrier function; analysis of mechanism of action

ARPE-19 and primary mouse RPE cells were grown on Transwell plates for >3 weeks after which they formed a monolayer with stable transepithelial resistance (TER). TER was measured via a volt-ohm meter with an STX2 electrode. The integrity of the monolayer was confirmed in ARPE-19 cells, demonstrating the presence of β-actin filament distribution in the form of circumferential bundles (A), cell-junction markers at the cell-borders, ZO-1 (B) and occludin (C), and co-labeling of ZO-1 and phalloidin (D). (E) In ARPE-19 cells, VEGF (10 ng/mL) significantly (P <0.05) reduced TER by 2 and 4 hours post-application. Pretreatment with 100 µM αCT1 ameliorated the drop in TER at both time points. αCT1 alone had no effect over the four hour time course. (F) The protective effect of αCT1 was confirmed in RPE monolayers derived from mouse. (G) Treatment of the ARPE-19 cell monolayers for 12 days starting at confluency (day 0) resulted in higher TER levels in the αCT1 when compared to the control group (P =0.03), reaching maximal TER levels 4 days earlier (indicated by the slope). (H) The gap-junction blocker, 18-β-GCA (0.1 mM), did not reduce the protective effect of αCT in ARPE-19 cells, neither did two modulators of hemichannels, apyrase and JM2 as shown by TER measurements at the 4 hour time point (I, J). Data are expressed as mean ± SD for ARPE-19 cells, mean ± SEM for primary mouse RPE cells (n = 3 per treatment group). Scale bar: 50 µm.

Figure 6. Effects of αCT1 on calcium-chelation-mediated loss in barrier function of MDCK cells

Barrier function was measured in MDCK cells in response to calcium chelation during the damage phase (transepithelial resistance measurements [TER] using a volt-ohm meter with an STX2 electrode) or during the recovery phase (Electric Cell-Substrate Impedance Sensing [ECIS] system). (A) MDCK cells were grown in Transwell plates for >3 weeks to measure barrier function by TER. Measurements were obtained at baseline and 20 and 40 minutes after the addition of 1.2 mM EGTA in αCT1-pretreated (30 µM) and untreated cells. EGTA treatment decreased barrier function within 20 minutes; but significantly less so in the αCT1 group. Data are expressed as mean ± SEM (n = 6 per treatment group). (B) MDCK cells grown in 8W10E+ ECIS dishes were used to measure barrier function using an ECIS system. Impedence was measured at baseline, after the addition of 0.5 mM EGTA and during the recovery phase upon restoration of physiological levels of calcium, in αCT1- and untreated cells. Calcium chelation significantly decreased barrier function, which recovered upon re-addition of calcium. Revovery of impendance was expressed as the rate of barrier function following EGTA treatment to the mean barrier function at 1 hour after αCT1 or vehicle treatment (C). Recovery was significantly accelerated in αCT1- when compared to un-treated cells Data are expressed as mean ± SEM (n = 6–8 per treatment group).