Investigation of PACAP38 and PAC1 Receptor Expression in Human Retinoblastoma and the Effect of PACAP38 Administration on Human Y-79 Retinoblastoma Cells.

Retinoblastoma represents the most prevalent malignant neoplasm affecting the eyes in childhood. The clear-cut origin of retinoblastoma has not yet been determined; however, based on experiments, it has been suggested that RB1 loss in cone photoreceptors causes retinoblastoma. Pituitary adenylate-cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide which has been shown to be affected in certain tumorous transformations, such as breast, lung, kidney, pancreatic, colon, and endocrine cancers. This study aimed to investigate potential changes in both PACAP38 and PAC1 receptor (PAC1R) expression in human retinoblastoma and the effect of PACAP38 administration on the survival of a human retinoblastoma cell line (Y-79). We analyzed human enucleation specimens removed because of retinoblastoma for PACAP38 and PAC1R immunostaining and the effect of PACAP38 on the survival of the Y-79 cell line. We described for the first time that human retinoblastoma cells from patients showed only perinuclear, dot-like immunopositivity for both PACAP38 and PAC1R, irrespective of laterality, genetic background, or histopathological features. Nanomolar (100 nM and 500 nM) PACAP38 concentrations had no effect on the viability of Y-79 cells, while micromolar (2 µM and 6 µM) PACAP38 significantly decreased tumor cell viability. These findings, along with general observations from animal studies showing that PACAP38 has strong anti-apoptotic, anti-inflammatory, and antioxidant effects on ocular tissues, together suggest that PACAP38 and its analogs are promising candidates in retinoblastoma therapy.

Protective Effects of Pituitary Adenylate-Cyclase-Activating Polypeptide on Retinal Vasculature and Molecular Responses in a Rat Model of Moderate Glaucoma.

Despite the high probability of glaucoma-related blindness, its cause is not fully understood and there is no efficient therapeutic strategy for neuroprotection. Vascular factors have been suggested to play an important role in glaucoma development and progression. Previously, we have proven the neuroprotective effects of pituitary adenylate-cyclase-activating polypeptide (PACAP) eye drops in an inducible, microbeads model in rats that is able to reproduce many clinically relevant features of human glaucoma. In the present study, we examined the potential protective effects of PACAP1-38 on the retinal vasculature and the molecular changes in hypoxia. Ocular hypertension was induced by injection of microbeads into the anterior chamber, while control rats received PBS. PACAP dissolved in vehicle (1 µg/drop) or vehicle treatment was started one day after the injections for four weeks three times a day. Retinal degeneration was assessed with optical coherence tomography (OCT), and vascular and molecular changes were assessed by immunofluorescence labeling. HIF1-α and VEGF-A protein levels were measured by Western blot. OCT images proved severe retinal degeneration in the glaucomatous group, while PACAP1-38 eye drops had a retinoprotective effect. Vascular parameters were deteriorated and molecular analysis suggested hypoxic conditions in glaucoma. PACAP treatment exerted a positive effect against these alterations. In summary, PACAP could prevent the severe damage to the retina and its vasculature induced by ocular hypertension in a microbeads model.

Distribution of PACAP and PAC1 Receptor in the Human Eye.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution and diverse biological functions. Several studies show that PACAP has strong cytoprotective effects mediated mostly through its specific PAC1 receptor (PAC1-R) and it plays important roles in several pathological conditions. Its distribution and altered expression are known in various human tissues, but there is no descriptive data about PACAP and its receptors in the human eyebulb. Since PACAP38 is the dominant form of the naturally occurring PACAP, our aim was to investigate the distribution of PACAP38-like immunoreactivity in the human eye and to describe the presence of PAC1-R. Semiquantitative evaluation was performed after routine histology and immunohistochemical labeling on human eye sections. Our results showed high level of immunopositivity in the corneal epithelium and endothelium. Within the vascular layer, the iris and the ciliary body had strong immunopositivity for both PACAP and PAC1-R. Several layers of the retina showed immunoreactivity for PACAP and PAC1-R, but the ganglion cell layer had a special pattern in the immunolabeling. Labeling was observed in the neuropil within the optic nerve in both cases and glial cells displayed immunoreactivity for PAC1-R. In summary, our study indicates the widespread occurrence of PACAP and its specific receptor in the human eye, implying that the results from in vitro and animal studies have translational value and most probably are also present in the human eye.

Retinoprotective Effects of PACAP Eye Drops in Microbead-Induced Glaucoma Model in Rats.

Glaucoma is associated with increased intraocular pressure (IOP), causing the apoptosis of retinal ganglion cells (RGCs) and the loss of their axons leading to blindness. Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in several neural injuries, including retinopathies. The aim of this study was to investigate the effects of PACAP1-38 eye drops in a model of glaucoma. IOP was elevated bilaterally by injections of microbeads to block the aqueous humor outflow. The control groups received the same volume of saline. Animals were treated with PACAP1-38 (1 µg/drop, 3 × 1 drop/day) or vehicle for 4 weeks starting one day after the injections. Retinal morphology by histology and optical coherence tomography, function by electroretinography, and IOP changes were analyzed. Animals were sacrificed 8 weeks after the injections. Microbeads injections induced a significant increase in the IOP, while PACAP1-38 treatment lowered it to normal levels (~10 mmHg). Significant retinal degeneration and functional impairment were observed in the microbead-injected group without PACAP1-38 treatment. In the microbeads + PACAP1-38 group, the retinal morphology and functionality were close to the normal values. In summary, our results show that PACAP1-38, given in form of eye drops, is neuroprotective in glaucoma, providing the basis for potential future therapeutic administration.