Molecular mechanisms involved in the protective effect of pituitary adenylate cyclase-activating polypeptide in an in vitro model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Based on transcriptional profiles of motor cortex samples, in a previous work, we were able to classify two subgroups of sporadic ALS (SALS) patients, named SALS1 and SALS2. A further meta-analysis study has revealed sixteen drug targets commonly deregulated in SALS2 and superoxide dismutase 1 (SOD1) G93A mice. The identified candidate drug targets included pituitary adenylate cyclase-activating polypeptide (PACAP), epidermal growth factor receptor (EGFR) and matrix metallopeptidase-2 (MMP-2). By using a motor neuron-like hybrid cell line (NSC-34) expressing human SOD1 G93A as an in vitro model of ALS, here we investigated the functional correlation among these three genes. Our results have shown that PACAP increases cell viability following serum deprivation. This effect is induced through EGFR transactivation mediated by protein kinase A stimulation. Furthermore, EGFR phosphorylation activates mitogen-activated protein kinases/extracellular signal-regulated kinases 1 and 2 survival signaling pathway and increases MMP-2 expression, significantly reduced by serum starvation. These results suggest that a deeper characterization of mechanisms involved in PACAP/EGFR/MMP-2 axis activation in G93A SOD1 mutated neurons may allow identifying new targets for ALS therapy.

NAP modulates hyperglycemic-inflammatory event of diabetic retina by counteracting outer blood retinal barrier damage.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Prolonged hyperglycemia stimulates inflammatory pathway characterized by the release of some cytokines leading to the impairment of blood retinal barrier (BRB). NAP exerts a protective effect in various eye diseases, including DR. So far, the role of NAP in the modulation of inflammatory event during early phase of this pathology has not been investigated yet. In the current study, we have studied the retinal protective effect of NAP, injected into the eye, in diabetic rats. NAP treatment exerts a dual effect downregulating interleukin (IL)-1ß and its related receptors and upregulating IL-1Ra expression. We have also tested the role of this peptide in human retinal epithelial cells (ARPE19) cultured on a semipermeable support and exposed to hyperglycemic-inflammatory insult, representing a in vitro model of diabetic macular edema, a clinical manifestation of DR. The results have shown that NAP prevents outer BRB impairment by upregulating the tight junctions. In conclusion, deepened characterization of NAP action mechanism on hyperglycemic-inflammatory damage may be useful to develop a new strategy to prevent retinal damage during DR.

NAP counteracts hyperglycemia/hypoxia induced retinal pigment epithelial barrier breakdown through modulation of HIFs and VEGF expression.

Diabetic macular edema (DME) is a common complication leading to a central vision loss in patients with diabetes. In this eye pathology, the hyperglycaemic/hypoxic microenvironment of pigmented epithelium is responsible for outer blood retinal barrier integrity changes. More recently, we have shown that a small peptide derived from the activity-dependent neuroprotective protein (ADNP), known as NAP, counteracts damages occurring during progression of diabetic retinopathy by modulating HIFs/VEGF pathway. Here, we have investigated for the first time the role of this peptide on outer blood retinal barrier (BRB) integrity exposed to hyperglycaemic/hypoxic insult mimicking a model in vitro of DME. To characterize NAP role on disease's pathogenesis, we have analyzed its effect on HIFs/VEGF system in human retinal pigmented epithelial cells, ARPE-19, grown in high glucose and low oxygen tension. The results have shown that NAP prevents outer BRB breakdown by reducing HIF1α/HIF2α, VEGF/VEGFRs, and increasing HIF3α expression, moreover it is able to reduce the percentage of apoptotic cells by modulating the expression of two death related genes, BAX and Bcl2. Further investigations are needed to determine the possible use of NAP in DME treatment.

Caffeine Prevents Blood Retinal Barrier Damage in a Model, In Vitro, of Diabetic Macular Edema.

Diabetic macular edema (DME) is the major cause of vision loss in patients affected by diabetic retinopathy. Hyperglycemia and hypoxia represent the key elements in the progression of these pathologies, leading to breakdown of the blood-retinal barrier (BRB). Caffeine, a psychoactive substance largely consumed in the world, is a nonselective antagonist of adenosine receptors (AR) and it possesses a protective effect in various diseases, including eye pathologies. Here, we have investigated the effect of this substance on BRB integrity following exposure to hyperglycemic/hypoxic insult. Retinal pigmented epithelial cells, ARPE-19, have been grown on semi-permeable supports mimicking an experimental model, in vitro, of outer BRB. Caffeine treatment has reduced cell monolayer permeability after exposure to high glucose and desferoxamine as shown by TEER and FITC-dextran permeability assays. This effect is also mediated through the restoration of membrane's tight junction expression, ZO-1. Moreover, we have demonstrated that caffeine is able to prevent outer BRB damage by inhibiting apoptotic cell death induced by hyperglycemic/hypoxic insult since it downregulates the proapoptotic Bax and upregulates the anti-apoptotic Bcl-2 genes. Although further studies are needed to better comprise the beneficial effect of caffeine, we can speculate that it might be used as an innovative drug for DME treatment. J. Cell. Biochem. 118: 2371-2379, 2017. © 2017 Wiley Periodicals, Inc.

Modulatory role of PACAP and VIP on HIFs expression in lung adenocarcinoma.

Lung adenocarcinoma is the most frequent form of non-small cell lung cancer. Inside the tumor mass, uncontrolled cell proliferation generates hypoxic areas leading to activation of hypoxia-inducible factors (HIFs) responsible for neovascularization and tumor metastasis. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two neuropeptides widely distributed in respiratory organs. Previous studies have demonstrated that these peptides interfere with hypoxic pathways in various diseases, including tumors. However, their modulatory role in HIFs expression in lung adenocarcinomas has not yet been evaluated. In the present paper, we detected the expression profile of PACAP, VIP and related receptors in healthy and adenocarcinoma human lung tissue. To characterize peptides' modulatory effects on HIFs expression, we also exposed A549 lung adenocarcinoma cells and human normal bronchial epithelial BEAS-2B cells to microenvironmental hypoxia by treating them with deferoxamine (DFX). The results showed that PACAP and VIP significantly reduced HIF-1α and HIF-2α levels in both cell lines following hypoxic stress. The HIF-3α expression profile was related to cellular phenotype as it was lower in BEAS-2B and higher in A549 cells under low oxygen tension. In lung adenocarcinoma cells, peptide treatment restored HIF-3 α expression to control levels. These results suggest that endogenous PACAP and VIP exert controversial roles in cellular hypoxic microenvironments depending on the pathophysiological conditions of the lung tissue.

Effect of PACAP on Hypoxia-Induced Angiogenesis and Epithelial-Mesenchymal Transition in Glioblastoma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts different effects in various human cancer. In glioblastoma (GBM), PACAP has been shown to interfere with the hypoxic micro-environment through the modulation of hypoxia-inducible factors via PI3K/AKT and MAPK/ERK pathways inhibition. Considering that hypoxic tumor micro-environment is strictly linked to angiogenesis and Epithelial-Mesenchymal transition (EMT), in the present study, we have investigated the ability of PACAP to regulate these events. Results have demonstrated that PACAP and its related receptor, PAC1R, are expressed in hypoxic area of human GBM colocalizing either in epithelial or mesenchymal cells. By using an in vitro model of GBM cells, we have observed that PACAP interferes with hypoxic/angiogenic pathway by reducing vascular-endothelial growth factor (VEGF) release and inhibiting formation of vessel-like structures in H5V endothelial cells cultured with GBM-conditioned medium. Moreover, PACAP treatment decreased the expression of mesenchymal markers such as vimentin, matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) as well as CD44 in GBM cells by affecting their invasiveness. In conclusion, our study provides new insights regarding the multimodal role of PACAP in GBM malignancy.

Drug Screening and Drug Repositioning as Promising Therapeutic Approaches for Spinal Muscular Atrophy Treatment.

Spinal muscular atrophy (SMA) is the most common genetic disease affecting infants and young adults. Due to mutation/deletion of the survival motor neuron (SMN) gene, SMA is characterized by the SMN protein lack, resulting in motor neuron impairment, skeletal muscle atrophy and premature death. Even if the genetic causes of SMA are well known, many aspects of its pathogenesis remain unclear and only three drugs have been recently approved by the Food and Drug Administration (Nusinersen-Spinraza; Onasemnogene abeparvovec or AVXS-101-Zolgensma; Risdiplam-Evrysdi): although assuring remarkable results, the therapies show some important limits including high costs, still unknown long-term effects, side effects and disregarding of SMN-independent targets. Therefore, the research of new therapeutic strategies is still a hot topic in the SMA field and many efforts are spent in drug discovery. In this review, we describe two promising strategies to select effective molecules: drug screening (DS) and drug repositioning (DR). By using compounds libraries of chemical/natural compounds and/or Food and Drug Administration-approved substances, DS aims at identifying new potentially effective compounds, whereas DR at testing drugs originally designed for the treatment of other pathologies. The drastic reduction in risks, costs and time expenditure assured by these strategies make them particularly interesting, especially for those diseases for which the canonical drug discovery process would be long and expensive. Interestingly, among the identified molecules by DS/DR in the context of SMA, besides the modulators of SMN2 transcription, we highlighted a convergence of some targeted molecular cascades contributing to SMA pathology, including cell death related-pathways, mitochondria and cytoskeleton dynamics, neurotransmitter and hormone modulation.

PACAP and VIP regulate hypoxia-inducible factors in neuroblastoma cells exposed to hypoxia.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two related peptides acting as neurotransmitters/neuromodulators in central and peripheral nervous system. They are also involved in cancer showing a controversial role. Particulary, they are implicated in neuroblastoma differentiation (NB). This pediatric tumor can evolve to a malignant metastatic disease or spontaneously regress towards a benign form, known as ganglioneuroblastoma/ganglioneuroma. A negative hallmark of neoplasia progression is represented by hypoxic microenvironment. Low oxygen tension induces activation of hypoxia-inducible factors (HIFs) promoting cells proliferation and metastasis formation. Moreover, HIFs trigger vascular endothelial growth factor (VEGF) release favouring high-risk NB phenotype development. In the present work, we have investigated for the first time, if PACAP and VIP interfere with NB differentiation through modulation of hypoxic/angiogenic process. To this end, we analyzed their effect in malignant undifferentiated and all-trans retinoic acid (RA) differentiated SH-SY5Y cells, representing the benign form of this tumor. Our results have suggested tha both peptides, but predominantly VIP, induce NB differentiation into benign form by regulating HIFs, VEGF and VEGFRs expression and distribution. All these data give new insight regarding PACAP/VIP regulatory role in NB progression.

Caffeine Effect on HIFs/VEGF Pathway in Human Glioblastoma Cells Exposed to Hypoxia.

BACKGROUND: Caffeine represents the most used psychoactive drug in the world acting through different mechanisms of action and on several molecular targets. It exerts an anti-cancer role in glioblastoma multiforme (GBM). This neoplasia is characterized by extensive hypoxic foci triggering hypoxia-inducible factors (HIFs) expression. Among these factors, HIF-1α performs a crucial role in the induction of vascular endothelium growth factor (VEGF), a key player in angiogenesis and cell migration. METHODS: In this work, we have investigated whether caffeine counteracts GBM progression by modulating hypoxic event. Moreover, we analyzed the activation of phosphoinositide three kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling cascades. RESULTS: Our results have indicated that this psychostimulant drug significantly reduced HIF-1α and VEGF expression in GBM cells exposed to hypoxia. This effect is mediated through inhibition of PI3K/Akt and MAPK/ERK signaling pathways both implied in HIFs regulation. CONCLUSION: The present data give new insight into antitumor activity of caffeine during GBM progression.

Trophic effect of PACAP on human corneal endothelium.

Cornea's posterior surface includes endothelium maintaining stromal hydration and clarity. Due to their limited proliferative capability, the loss of endothelial cells can outcome in permanent opacity. In the last years, different studies have demonstrated the protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in different ocular diseases. However, its role on human corneal endothelial cells (HCECs) has not been investigated, yet. Here, we have developed a culture protocol to differentiate HCECs from donor's cornea. PACAP treatment prevented damage induced by growth factors deprivation of cells grown on transwell supports as revealed by TERR measurements. Moreover, this peptide significantly increased tight junction proteins expression by conferring resistance to endothelial barrier. This effect is also related to promotion of cell viability as demonstrated by MTT assay. Furthermore, PACAP stimulated repairing of corneal endothelium lesion as shown by wound healing analysis. In conclusion, our data suggest that this peptide could represent an important trophic factor in maintaining functionality of human corneal endothelium.

WT1 Alternative Splicing: Role of Its Isoforms in Neuroblastoma.

Wilms tumor 1 (WT1), a tumor suppressor gene, was originally identified in the homonymous renal neoplasm but is also involved in other cancers. Its function is still unclear, since it acts both as a pro- and an anti-oncogene. At least 14 WT1 transcriptional variants have been described; yet most investigations have focused on a small number of isoforms. We describe their structural features and review the evidence of their involvement in cancer with emphasis on neuroblastoma. In future, full characterization of all WT1 isoforms is expected to identify new molecular tumor markers and/or therapeutic targets.

Modulation of IL-1ß and VEGF expression in rat diabetic retinopathy after PACAP administration.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Hyperglycemic/hypoxic microenvironment concurs to aberrant angiogenesis characterizing the pathology and activates many downstream target genes including inflammatory cytokines and vasoactive peptides, such as interleukin-1ß (IL-1ß) and vascular endothelial growth factor (VEGF). It has been largely demonstrated that pituitary adenylate cyclase-activating peptide (PACAP) plays a protective effect in DR. In the present study, we investigated the role of PACAP to protect retinal tissue through IL-1ß and VEGF expression. Diabetes was induced in rats by streptozotocin (STZ) injection, and one week later a single intravitreal injection of 100µM PACAP was administrated. Analyses of IL-1ß and VEGF levels were performed three weeks after diabetes induction. The results demonstrated that a single intraocular administration of PACAP significantly reduced the expression of IL-1ß in diabetic animals. Moreover, it affects VEGF and its receptors (VEGFRs) levels and interferes with their retinal layers distribution as showed by confocal microscopy analysis. In particular, PACAP treatment downregulates VEGF and VEGFRs that are increasingly expressed in STZ-treated animals as compared to controls. These results indicate that PACAP plays an important role to attenuate the early phase of DR.

Nicotine promotes blood retinal barrier damage in a model of human diabetic macular edema.

More than 1 billion world's population actively smokes tobacco containing the bioactive component nicotine (NT). The biological role of this molecule is mediated through the activation of nicotinic cholinergic receptors, widely distributed in various human tissues including retinal pigmented epithelium. The long-term assumption of NT contributes to several diseases development such as diabetic retinopathy. The major complication of this pathology is the diabetic macular edema (DME), characterized by macular area thinning and blood-retinal barrier (BRB) breakdown. Retinal hyperglycemic/hypoxic microenvironment represents one of the main factors favoring DME progression by eliciting the hypoxia-inducible factors (HIFs) expression. The latter induce new vessels formation by stimulating cellular secretion of vascular endothelial growth factor (VEGF). The etiology of DME is multifactorial, but little is known about the risk factors linked to cigarette smoking, in particular to nicotine's contribution. In the present study, we have investigated the NT role in a model, in vitro, of DME, by evaluating its effect on outer BRB permeability and HIFs/VEGF expression following exposure to hyperglycemic/hypoxic insult. Our results have demonstrated that this compound alters outer BRB integrity exposed to high glucose and low oxygen pressure microenvironment by upregulating HIF-1α/HIF-2α, VEGF expression and ERK1/2 phosphorylation. These data have suggested that NT may play a negative role in active smokers affected by DME.

Expression profile of Wilms Tumor 1 (WT1) isoforms in undifferentiated and all-trans retinoic acid differentiated neuroblastoma cells.

Wilms tumor 1 gene (WT1) is a tumor suppressor gene originally identified in nephroblastoma. It is also expressed in neuroblastoma which represents the most aggressive extracranial pediatric tumor. Many evidences have shown that neuroblastoma may undergo maturation, by transforming itself in a more differentiated tumors such as ganglioneuroblastoma and ganglioneuroma, or progressing into a highly aggressive metastatic malignancy. To date, 13 WT1 mRNA alternative splice variants have been identified. However, most of the studies have focused their attention only on isoform of ∼49 kDa. In the present study, it has been investigated the expression pattern of WT1 isoforms in an in vitro model of neuroblastoma consisting in undifferentiated or all-trans retinoic acid (RA) differentiated cells. These latter representing the less malignant phenotype of this tumor. Results have demonstrated that WT1.1-WT1.5, WT1.6-WT1.9, WT1.10 WT1.11-WT1.12 and WT1.13 isoforms are expressed in both groups of cells, but their levels are significantly increased after RA treatment. These data have also been confirmed by immunofluorescence analysis. Moreover, the inhibition of PI3K/Akt and MAPK/ERK, that represent two signalling pathway specifically involved in NB differentiation, induces an overexpression of WT1 isoforms. These data suggest that WT1 isoforms might be involved in differentiation of neuroblastic into mature ganglion cells.