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.

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on Cell Death.

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated as a hypothalamic peptide based on its efficacy to increase adenylate cyclase (AC) activity. It has a widespread distribution throughout the body including the nervous system and peripheral organs, where PACAP exerts protective effects both in vivo and in vitro through its anti-apoptotic, anti-inflammatory, and antioxidant functions. The aim of the present paper was to review the currently available literature regarding the effects of PACAP on cell death in vitro in neural and non-neural cells. Among others, its effect on apoptosis can be detected in cerebellar granule cells against different toxic stimuli. Different neural cell types from the cerebral cortex are also prevented from cell death. PACAP also shows effects on cell death in cells belonging to the peripheral nervous system and protects both neural and non-neural cells of sensory organs. In addition, cell survival-promoting effect can be observed in different peripheral organ systems including cardiovascular, immune, respiratory, gastrointestinal, urinary, and reproductive systems. The studies summarized here indicate its noteworthy effect on cell death in different in vitro models, suggesting PACAP's potential therapeutic usage in several pathological conditions.

Protective Effects of PACAP in Peripheral Organs.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide widely distributed in the nervous system, where it exerts strong neuroprotective effects. PACAP is also expressed in peripheral organs but its peripheral protective effects have not been summarized so far. Therefore, the aim of the present paper is to review the existing literature regarding the cytoprotective effects of PACAP in non-neuronal cell types, peripheral tissues, and organs. Among others, PACAP has widespread expression in the digestive system, where it shows protective effects in various intestinal pathologies, such as duodenal ulcer, small bowel ischemia, and intestinal inflammation. PACAP is present in both the exocrine and endocrine pancreas as well as liver where it reduces inflammation and steatosis by interfering with hepatic pathology related to obesity. It is found in several exocrine glands and also in urinary organs, where, with its protective effects being mainly published regarding renal pathologies, PACAP is protective in numerous conditions. PACAP displays anti-inflammatory effects in upper and lower airways of the respiratory system. In the skin, it is involved in the development of inflammatory pathology such as psoriasis and also has anti-allergic effects in a model of contact dermatitis. In the non-neuronal part of the visual system, PACAP showed protective effects in pathological conditions of the cornea and retinal pigment epithelial cells. The positive role of PACAP has been demonstrated on the formation and healing processes of cartilage and bone where it also prevents osteoarthritis and rheumatoid arthritis development. The protective role of PACAP was also demonstrated in the cardiovascular system in different pathological processes including hyperglycaemia-induced endothelial dysfunction and age-related vascular changes. In the heart, PACAP protects against ischemia, oxidative stress, and cardiomyopathies. PACAP is also involved in the protection against the development of pre-senile systemic amyloidosis, which is presented in various peripheral organs in PACAP-deficient mice. The studies summarized here provide strong evidence for the cytoprotective effects of the peptide. The survival-promoting effects of PACAP depend on a number of factors which are also shortly discussed in the present review.

Pituitary adenylate cyclase activating polypeptide acts against neovascularization in retinal pigment epithelial cells.

The purpose of this study was to determine whether pituitary adenylate cyclase activating polypeptide (PACAP) could influence the neovascularization processes in hyperosmotic and oxidative stress in retinal pigment epithelial cells. Hyperosmotic conditions and oxidative stress were induced by 200 mM sucrose and 250 µM hydrogen peroxide (H2 O2 ), respectively. Morphology and elasticity of adult retinal pigment epithelial (ARPE-19) cells were measured by atomic force microscopy, while the investigation of junctional molecules, such as occludin and ZO-1, was carried out using immunofluorescence. For cell viability measurement, the MTT test was used. The effect of PACAP on the key angiogenic factors, such as vascular endothelial growth factor, angiogenin, and endothelin-1, was measured by an angiogenesis array and flow cytometry. Hyperosmotic stress-induced reorganization of the cytoskeleton and impairment of the junctions decreased cell viability and upregulated several angiogenic factors. In oxidative stress, we found that opening of the junctions decreased viability and upregulated the expression of angiogenic factors. PACAP was shown to be protective in both conditions. Retinal pigment epithelium cells play an important role in several diseases, such as diabetic retinopathy and macular edema. Therefore, protecting retinal pigment epithelial (RPE) cells with PACAP could be a novel and potential treatment in these diseases.

PACAP Is Protective in a Rat Model of Retinopathy of Prematurity.

The oxygen-induced retinopathy (OIR) is a well-established rodent model of retinopathy of prematurity (ROP), which is one of the most common causes of childhood visual impairment affecting preterm babies. Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to have neuroprotective effects. Several studies have revealed the presence of PACAP and its receptors in the retina and reported its protective effects in ischemic and diabetic retinopathy. In this study, we investigated whether PACAP administration can influence the vascular changes in the rat OIR model. OIR was generated by placing the animals in daily alternating 10/50 oxygen concentrations from postnatal day (PD) 0 to PD14 then returned them to room air. Meanwhile, animals received PACAP or saline intraperitoneally or intravitreally from PD1 to PD8 or on PD11, PD14, and PD17, respectively. On PD19 ± 1, the retinas were isolated and the vessels were visualized by isolectin staining. The percentage of avascular to whole retinal areas and the number of branching points were measured. Change in cytokine expression was also determined. Intravitreal treatment with PACAP remarkably reduced the extent of avascular area compared to the non- and saline-treated OIR groups. Intraperitoneal PACAP injection did not influence the vascular extent. Retinal images of room-air controls did not show vascular alterations. No changes in the number of vessel branching were observed after treatments. Alterations in cytokine profile after local PACAP injection further supported the protective role of the peptide. This is the first study to examine the effects of PACAP in ROP. Although the exact mechanism is still not revealed, the present results show that PACAP treatment can ameliorate the vascular changes in the animal model of ROP.

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion.

Pituitary adenylate cyclase activating polypeptide (PACAP) is known for its potent neuroprotective effects, including the retinoprotective actions in several types of retinal injuries. We have shown earlier that PACAP treatment causes activation of protective pathways and inhibition of pro-apoptotic signaling in excitotoxic retinal lesions. The aim of the present study was to gain insight into the in vivo protective mechanism of PACAP in retinal hypoperfusion injury induced by bilateral common carotid artery occlusion (BCCAO). Rats underwent BCCAO and received intravitreal PACAP (PACAP38) treatment. We investigated the activation level of the protective Akt pathway as well as the different mitogen activated protein kinases (MAPKs) by Western blot analysis and the expression of cytokines using a cytokine array kit. We found that PACAP treatment alone did not influence the phosphorylation of Akt or the MAPKs, but decreased the hypoperfusion-induced activation of both p38MAPK and JNK and increased the activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile was dramatically changed after BCCAO, with most cytokines and chemokines showing an increase, which was attenuated by PACAP (such as CINC, CNTF, fractalkine, sICAM, IL-1, LIX, Selectin, MIP-1, RANTES and TIMP-1). In addition, PACAP increased the expression of VEGF and thymus chemokine. The present results provide further insight into the neuroprotective mechanism induced by PACAP in ischemic retinal injuries, showing that PACAP ameliorates hypoperfusion injury involving Akt, MAPK pathways and anti-inflammatory actions.

PACAP improves functional outcome in excitotoxic retinal lesion: an electroretinographic study.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors occur throughout the nervous system, including the retina. PACAP exerts diverse actions in the eye: it influences ocular blood flow, contraction of the ciliary muscle, and has retinoprotective effects. This has been proven in different models of retinal degeneration. The in vivo protective effects of PACAP have been shown in retinal degeneration induced by kainic acid, optic nerve transection and ischemia. We have previously shown by morphological, morphometrical and immunohistochemical analyses that intravitreal PACAP administration protects against monosodium glutamate (MSG)-induced damage in neonatal rats. The question was raised whether these apparent morphological improvements by PACAP administration also lead to functional amelioration in MSG-induced retinal damage. The aim of the present study was to investigate the functional consequences of MSG treatment and the subsequent PACAP administration using electroretinographic measurements. The histological and morphometrical analyses supported the earlier findings that PACAP protected the retina in MSG-induced excitotoxicity. ERG recordings revealed a marked decrease in both the b- and a-wave values, reflecting the function of the inner retinal layers and the photoreceptors, respectively. In retinas receiving intravitreal PACAP treatment, these values were significantly increased. Thus, the functional outcome, although not parallel with the morphology, was significantly improved after PACAP treatment. The present observations are important from the clinical point of view showing, for the first time, that PACAP treatment is able to improve the functional properties of the retina in excitotoxic damage.