Retinoprotective Effects of TAT-Bound Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase Activating Polypeptide

Abstract

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.

Keywords: Bio-barriers; Eye drops; Retinal protection; TAT.

Fig. 1

The efficiency of FITC labeled PACAP/PACAP-TAT (a, b) and VIP/VIP-TAT (c, d) traversing to retina given in eye drops. The retina was separated 2 h after the eye drops and submitted to the fluorescence microscopic observation of FITC fluorescence signal (A, C) and the calculation of Efficiency of Traversing Eye to Retina (EtE) (b, d). The data are means ± SEM of four experiments

Fig. 2

cAMP assay results showing the effects of PACAP/PACAP-TAT (a) and VIP/VIP-TAT (b) on the activation of PAC1-R. The intracellular cAMP accumulation in PAC1-CHO cells induced by PACAP38(), PACAP-TAT(), VIP() and VIP-TAT () in their respective effective working concentration was plotted as the percentage (%) of the maximum cAMP level induced by PACAP27. The data are means ± SEM of four experiments

Fig. 3

Light microphotographs of retinal sections. Retinal tissue from BCCAO+SOCB (d) showed severe degeneration compared to SHAM+SOCB (a), SHAM + PACAP-TAT (b) or SHAM + VIP-TAT (c). The retinal layers of BCCAO+SOCB rats following treatment with eye drops containing PACAP-TAT (e) or VIP-TAT (f) showed only mild degeneration. Abbreviations: PL, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 μm

Fig. 4

Quantification of retinal layers in SHAM+SOCB, in SHAM+PACAP-TAT, in BCCAO+SOCB, and in BCCAO+PACAP-TAT animals; the right eye was treated with PACAP-TAT eye drops, the left eye served as controls receiving only SOCB. Comparison of all retinal layers (a–e). Morphometric analysis showed that treatment with PACAP-TAT eye drops improved the structure of all the retinal layers. Statistical significance (*p < 0.05 vs. SHAM+SOCB retinas, #p < 0.05 vs. BCCAO+SOCB retinas) was calculated by two-way ANOVA followed by Fischer’s post hoc test

Fig. 5

Quantification of the number of cells/100 μm GCL length (a), the number of cells/500 μm² ONL (b) and INL (c) areas in SHAM+SOCB, in SHAM+PACAP-TAT, in BCCAO+SOCB, and in BCCAO+PACAP-TAT animals. Statistical significance (*p < 0.05 vs. SHAM+SOCB retinas, #p < 0.05 vs. BCCAO+SOCB retinas) was calculated by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 6

Quantification of retinal layers in SHAM+SOCB, in SHAM+VIP-TAT, in BCCAO+SOCB, and in BCCAO+VIP-TAT animals; the right eye was treated with VIP-TAT eye drops, the left eye served as control receiving only SOCB. Comparison of all retinal layers (a–e). Morphometric analysis showed that treatment with VIP-TAT eye drops improved the structure of all the retinal layers. Statistical significance (*p < 0.05 vs. SHAM+SOCB retinas, #p < 0.05 vs. BCCAO+SOCB retinas) was calculated by two-way ANOVA followed by Fischer’s post hoc test

Fig. 7

Quantification of the number of cells/100 μm GCL length (a), the number of cells/500 μm² ONL (b) and INL (c) areas in SHAM+SOCB, in SHAM+VIP-TAT, in BCCAO+SOCB, and in BCCAO+VIP-TAT animals. Statistical significance (*p < 0.05 vs. SHAM+SOCB retinas, #p < 0.05 vs. BCCAO+SOCB retinas) was calculated by two-way ANOVA followed by Bonferroni’s post hoc test