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.

The Protective Effects of Endogenous PACAP in Oxygen-Induced Retinopathy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP.

PARP Inhibitor Protects Against Chronic Hypoxia/Reoxygenation-Induced Retinal Injury by Regulation of MAPKs, HIF1α, Nrf2, and NFκB.

Purpose: In the eye, chronic hypoxia/reoxygenation (H/R) contributes to the development of a number of ocular disorders. H/R induces the production of reactive oxygen species (ROS), leading to poly(ADP-ribose) polymerase-1 (PARP1) activation that promotes inflammation, cell death, and disease progression. Here, we analyzed the protective effects of the PARP1 inhibitor olaparib in H/R-induced retina injury and investigated the signaling mechanisms involved. Methods: A rat retinal H/R model was used to detect histologic and biochemical changes in the retina. Results: H/R induced reductions in the thickness of most retinal layers, which were prevented by olaparib. Furthermore, H/R caused increased levels of Akt and glycogen synthase kinase-3ß phosphorylation, which were further increased by olaparib, contributing to retina protection. By contrast, H/R-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinases (MAPK) phosphorylation and activation were reduced by olaparib, via mitogen-activated protein kinase phosphatase 1 (MKP-1) expression. In addition, H/R-induced hypoxia-inducible factor 1α (HIF1α) levels were decreased by olaparib, which possibly contributed to reduced VEGF expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was slightly increased by H/R and was further activated by olaparib. Nuclear factor-κB (NFκB) was also activated by H/R through phosphorylation (Ser536) and acetylation (Lys310) of the p65 subunit, although this was significantly reduced by olaparib. Conclusions: Olaparib reduced H/R-induced degenerative changes in retinal morphology. The protective mechanisms of olaparib most probably involved Nrf2 activation and ROS reduction, as well as normalization of HIF1α and related VEGF expression. In addition, olaparib reduced inflammation by NFκB dephosphorylation/inactivation, possibly via the PARP1 inhibition-MKP-1 activation-p38 MAPK inhibition pathway. PARP inhibitors represent potential therapeutics in H/R-induced retinal disease.

Protective Role of Endogenous PACAP in Inflammation-induced Retinal Degeneration.

PURPOSE: Pituitary adenylate Cyclase-Activating Polypeptide (PACAP) is a neuroprotective peptide that has been shown to exert protective effects in different models of neurodegenerative diseases, including retinal degenerations. Data obtained from PACAP-deficient (PACAP KO) mice provide evidence that endogenous PACAP has a neuroprotective role in different pathologies. PACAP KO mice show enhanced sensitivity to different insults, such as oxidative stress, hypoxia and inflammation. The aim of the present study was to investigate the protective effects of endogenous PACAP in retinal inflammation. METHODS: Endotoxin-induced eye inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in PACAP KO and wild-type (Wt) mice. After LPS treatment, retinas were processed for histological examination. To detect the alterations of different proteins and cytokines, immunohistochemical, western blot and cytokine array were used. We also performed dark-adapted electroretinography (ERG) to detect the functional differences. RESULTS: The thickness of nearly all layers was significantly less in LPS-injected PACAP KO mice compared to Wt animals. Increased expression of Glial Fibrillary Acidic Protein (GFAP) was induced in Müller glial cells after LPS treatment, which was more intense in PACAP KO mice. The levels of pAkt and pGSK were decreased in PACAP KO group during inflammation. LPS treatment significantly increased cytokines (sICAM-1, JE, TIMP-1) in both treated groups, but it was more expressed in PACAP KO animals. Furthermore, ERG responses were disturbed after LPS injection in PACAP KO mice. CONCLUSION: Our results showed that endogenous PACAP has a protective role in LPS-caused retinal inflammation.

Protective effects of PACAP in ischemia.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an ubiquitous peptide involved, among others, in neurodevelopment, neuromodulation, neuroprotection, neurogenic inflammation and nociception. Presence of PACAP and its specific receptor, PAC1, in the trigeminocervical complex, changes of PACAP levels in migraine patients and the migraine-inducing effect of PACAP injection strongly support the involvement of PACAP/PAC1 receptor in migraine pathogenesis. While antagonizing PAC1 receptor is a promising therapeutic target in migraine, the diverse array of PACAP's functions, including protection in ischemic events, requires that the cost-benefit of such an intervention is well investigated by taking all the beneficial effects of PACAP into account. In the present review we summarize the protective effects of PACAP in ischemia, especially in neuronal ischemic injuries, and discuss possible points to consider when developing strategies in migraine therapy interfering with the PACAP/PAC1 receptor system.

Passage through the Ocular Barriers and Beneficial Effects in Retinal Ischemia of Topical Application of PACAP1-38 in Rodents.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP's neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP's retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases.

Ocular Delivery of PACAP1-27 Protects the Retina From Ischemic Damage in Rodents.

Purpose: Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in neuronal injuries. Bilateral common carotid artery occlusion (BCCAO) causes chronic hypoperfusion-induced degeneration in the rat retina, where we proved the retinoprotective effect of intravitreal PACAP. Although this route of administration is a common clinical practice in several diseases, easier routes are clinically important. Our aim was to investigate the potential retinoprotective effects of PACAP eye drops in BCCAO-induced ischemic retinopathy. Methods: After performing BCCAO in rats, the right eyes were treated with PACAP1-27 eye drops (1 µg/drop, 2 × 1 drops/day for 5 days), containing different vehicles: saline, water for injections, thiomersal or benzalkonium solution for ophthalmic use (SOCB). Histology and immunohistochemistry were performed 2 weeks after surgery, while molecular analysis was performed 24 hours after BCCAO. Passage of PACAP1-27 through the ocular layers was tested with radioactive PACAP-SOCB in mice. Results: Bilateral common carotid artery occlusion led to a severe degeneration of all retinal layers. Solution for ophthalmic use was the most effective vehicle for delivering PACAP (PACAP-SOCB), significantly ameliorating BCCAO-induced damage. The massive upregulation of GFAP was not observed in retinas treated with PACAP-SOCB eye drops. PACAP-SOCB treatment also increased activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile showing upregulation in different cytokines was attenuated by PACAP-SOCB. Radioactive PACAP reached the retina when delivered in SOCB-containing eye drops. Conclusions: PACAP1-27, delivered in the SOCB vehicle as eye drops, was retinoprotective in ischemic retinopathy, providing the basis for future therapeutic administration.

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.

Analysis of nonderivatized steroids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using C70 fullerene as matrix.

Neutral steroid hormones are currently analyzed by gas or liquid chromatography/mass spectrometry based methods. Most of the steroid compounds, however, lack volatility and do not contain polar groups, which results in inadequate chromatographic behavior and low ionization efficiency. Derivatization of the steroids to form more volatile, thermostable, and charged products solves this difficulty, but the derivatization of compounds with unknown chemical moieties is not an easy task. In this study, a rapid, high-throughput, sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method is described using C(70) fullerene as a matrix compound. The application of the method is demonstrated for five general sex steroids and for synthetic steroid compounds in both negative and positive ionization modes.

Presence of pituitary adenylate cyclase activating polypeptide-38 in human plasma and milk.

OBJECTIVE: Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide widely distributed throughout the body. It is involved in the regulation of various physiological and pathophysiological processes, such as reproduction, thermoregulation, motor activity, brain development, neuronal survival, inflammation and pain. Since little is known about its distribution in humans, our aim was to examine PACAP-38 in human plasma. Furthermore, based on the presence of vasoactive intestinal peptide, structurally the closest to PACAP, in milk and PACAP and its receptors in the mammary gland, our aim was to study PACAP-38 in human milk. DESIGN AND METHODS: The presence of PACAP-38 was determined by mass spectrometry in plasma samples from healthy male and female volunteers (age: 20-40), as well as in plasma and milk samples from lactating women (age: 20-35). PACAP concentration was measured with a specific and sensitive RIA. RESULTS: Our results revealed that PACAP-38 is present in human plasma, its concentration is relatively stable in healthy volunteers and it is not significantly altered by gender, age, food intake or hormonal cycle in females. However, PACAP-38 plasma levels significantly increased in lactating women having 1-6 month-old babies. Moreover, this study is the first which provides evidence for the presence of PACAP-38 in the human milk with levels 5-20-fold greater in the milk whey than in the respective plasma samples. CONCLUSIONS: We found PACAP-38 in human plasma and its increase during the first 6 months of the lactation period. A prominent, nearly 10-fold higher concentration of this peptide was detected in human milk. Based on the literature, several important actions of milk-derived PACAP-38 can be suggested such as mammary gland proliferation, nutrient transfer as well as regulation of growth/differentiation of certain tissues of the neonates. The novelty of the present descriptive data provides a basis for further investigations on the mechanism of PACAP-38 secretion in human milk and its functional significance.