Agata Grazia D’Amico

Ameliorative effect of PACAP and VIP against increased permeability in a model of outer blood retinal barrier dysfunction.

Breakdown of outer blood retinal barrier (BRB) due to the disruption of tight junctions (TJs) is one of the main factors accounting for diabetic macular edema (DME), a major complication of diabetic retinopathy. Previously it has been shown that PACAP and VIP are protective against several types of retinal injuries. However, their involvement in the maintenance of outer BRB function during DME remains uncovered. Here, using an in vitro model of DME, we explored the effects of both PACAP and VIP. Human retinal pigment epithelial cells (ARPE19) were cultured for 26 days either in normal glucose (5.5 mM, NG) or in high glucose (25 mM, HG). In addition, to mimic the inflammatory aspect of the diabetic milieu, cells were also treated with IL-1β (NG+IL-1β and HG+IL-1β). Effects of PACAP or VIP on cells permeability were evaluated by measuring both apical-to-basolateral movements of fluorescein isothyocyanate (FITC) dextran and transepithelial electrical resistance (TEER). Expression of TJ-related proteins was evaluated by immunoblot. Results demonstrated that NG+IL-1β and, to a greater extent, HG+IL-1β significantly increased FITC-dextran diffusion, paralleled by decreased TEER. PACAP or VIP reversed both of these effects. Furthermore, HG+IL-1β-induced reduction of claudin-1 and ZO-1 expression was reversed by PACAP and VIP. Occludin expression was not affected in any of the conditions tested. Altogether, these finding show that both peptides counteract HG+IL-1β-induced damage in ARPE19 cells, suggesting that they might be relevant to the maintenance of outer BRB function in DME.

Antiproliferative Effects of PACAP and VIP in Serum-Starved Glioma Cells

Emerging evidence have suggested that calorie restriction (CR) is a reliable method to decrease cancer development since it produces changes in tumor microenvironment that interfere with cell proliferation, tissue invasion, and formation of metastases. Studies on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in cancer cells indicate that their influence on cell growth is either cell type specific or dependent on culture conditions. Evidence showing the effect of PACAP and VIP in glioma cells grown under conditions mimicking CR are currently unavailable. Therefore, we explored the effects of both PACAP and VIP in C6 glioma cells either grown in a normal growth medium or exposed to serum starvation, to resemble an acute condition of CR. Cell viability, expression of proteins related to cell proliferation (cyclin D1), apoptosis (Bcl2, p53, and cleaved caspase-3), and cell malignancy (GFAP and nestin) were assessed by MTT assay, immunoblot, and immunolocalization, respectively. Results demonstrated that CR significantly decreased cell proliferation, reduced levels of cyclin D1 and Bcl2, and increased the expression of p53 and cleaved caspase-3. Surprisingly, all of these CR-driven effects were further exacerbated by PACAP or VIP treatment. We also found that PACAP or VIP prevented GFAP decrease caused by CR and further reduced the expression of nestin, a prognostic marker of malignancy. In conclusion, these data demonstrate that PACAP and VIP possess antiproliferative properties against glioma cells that depend on the specific culture settings, further supporting the idea that CR might offer new avenues to improve peptide-oriented glioma cancer treatment.

Different Retinal Expression Patterns of IL-1α, IL-1β, and Their Receptors in a Rat Model of Type 1 STZ-Induced Diabetes.

Diabetic retinopathy (DR), a common complication of diabetes, remains a major cause of blindness among population. Considerable amounts of evidences suggest the involvement of inflammatory process in this pathology. Increased levels of proinflammatory cytokines, including interleukin-1β (IL-1β), were found in the vitreous of diabetic patients and in the retina of diabetic rats. However, in this context, no attention has been given to the other main IL-1 family members: IL-1α, two transmembrane receptors IL-1RI and IL-1RII and the natural antagonist receptor IL-1Ra. Despite that they actively participate in the IL-1-mediated inflammation process, their implication in DR has not been described. Thus, we investigated by Western blot and confocal laser scanning microscopy analysis the effect of hyperglycemia on expression of IL-1 family members in retinal layers, using an in vivo model of type 1 diabetes. It was induced in adult rats by intraperitoneal injection of streptozotocin (STZ). Exposure to hyperglycemia induces a significant increase in the protein expression of IL-1β, IL-1RI, IL-RII and IL-1Ra but not of IL-1α. Moreover, high glucose alters their distribution pattern in the rat’s retinal layers. Among these latter, the most compromised are the photoreceptor, the inner plexiform and ganglion cell layers. These findings support previous data demonstrating the involvement of inflammation in DR and suggest new pharmacological approaches for the treatment of this pathology.

Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro

Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson’s disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression.

Dopamine D3 receptor deletion increases tissue plasminogen activator (tPA) activity in prefrontal cortex and hippocampus

Considerable evidence indicates that dopamine (DA) influences tissue plasminogen activator (tPA)-mediated proteolytic processing of the precursor of brain-derived neurotrophic factor (proBDNF) into mature BDNF (mBDNF). However, specific roles in this process for the dopamine D3 receptor (D3R) and the underlying molecular mechanisms are yet to be fully characterized. In the present study, we hypothesized that D3R deletion could influence tPA activity in the prefrontal cortex and hippocampus. Using D3R knockout (D3(-/-)) mice, we show that receptor inactivation is associated with increased tPA expression/activity both in the prefrontal cortex and, to a greater extent, in the hippocampus. Augmented tPA expression in D3(-/-) mice correlated with increased BDNF mRNA levels, plasmin/plasminogen protein ratio and the conversion of proBDNF into mBDNF, as well as enhanced tPA and mBDNF immunoreactivity, as determined by quantitative real time polymerase chain reaction (qRT-PCR), immunoblot and immunohistochemistry. In addition, when compared to wild-type controls, D3(-/-) mice exhibited increased basal activation of the canonical cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-driven Akt/cAMP-response element-binding protein (CREB) signaling cascade, as determined by the increased Akt phosphorylation both at Thr304 and Ser473 residues, of DA and cAMP-regulated protein of 32kDa (DARPP-32) at Thr34 and a phosphorylation state-dependent inhibition of glycogen synthetase kinase-3β (GSK-3β) at Ser9, a substrate of Akt whose constitutive function impairs normal CREB transcriptional activity through phosphorylation at its Ser129 residue. Accordingly, CREB phosphorylation at Ser133 was significantly increased in D3(-/-) mice, whereas the GSK-3β-dependent phosphorylation at Ser129 was diminished. Altogether, our finding reveals that mice lacking D3Rs show enhanced tPA proteolytic activity on BDNF which may involve, at least in part, a potentiated Akt/CREB signaling, possibly due to hindered GSK-3β activity.

PACAP and VIP Inhibit the Invasiveness of Glioblastoma Cells Exposed to Hypoxia through the Regulation of HIFs and EGFR Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) through the binding of vasoactive intestinal peptide receptors (VIPRs), perform a wide variety of effects in human cancers, including glioblastoma multiforme (GBM). This tumor is characterized by extensive areas of hypoxia, which triggers the expression of hypoxia-inducible factors (HIFs). HIFs not only mediate angiogenesis but also tumor cell migration and invasion. Furthermore, HIFs activation is linked to epidermal growth factor receptor (EGFR) overexpression. Previous studies have shown that VIP interferes with the invasive nature of gliomas by regulating cell migration. However, the role of VIP family members in GBM infiltration under low oxygen tension has not been clarified yet. Therefore, in the present study we have investigated, for the first time, the molecular mechanisms involved in the anti-invasive effect of PACAP or VIP in U87MG glioblastoma cells exposed to hypoxia induced by treatment with desferrioxamine (DFX). The results suggest that either PACAP or VIP exert an anti-infiltrative effect under low oxygen tension by modulating HIFs and EGFR expression, key elements involved in cell migration and angiogenesis. These peptides act through the inhibition of PI3K/Akt and MAPK/ERK signaling pathways, which are known to have a crucial role in HIFs regulation.

Hippocampal Neurofibromin and Amyloid Precursor Protein Expression in Dopamine D3 Receptor Knock-out Mice Following Passive Avoidance Conditioning

Passive avoidance (PA) conditioning is a fear motivated task able to initiate a cascade of altered gene expression within the hippocampus, a structure critical to learning and memory. We have previously shown that neurofibromin (NF1) and amyloid precursor protein (APP), two genes implicated in cognitive function, are differentially expressed in brain of dopamine D3 receptor knock-out mice (D3R−/−), suggesting that the receptor might have a role in their trascriptional regulation. Here in this study, we hypothesized that during acquisition of PA conditioning the expression of NF1 and APP genes could be influenced by D3Rs. To address this issue, we analyzed the expression of NF1 and APP in the hippocampus of both wild-type (WT) and D3R−/− mice subjected to the single trial step-through PA paradigm. Our finding demonstrated that (1) D3R−/− mice exhibit increased cognitive performance as compared to WT mice in the step-through PA trial; (2) acquisition of PA increased D3R and NF1, but not APP expression in WT mice hippocampus; (3) PA-driven NF1 induction in WT was abrogated in D3R−/− mice and finally that (4) the heightened basal APP expression observed in naive D3R−/− mice was totally reversed by acquisition of PA. In conclusion, the present finding show for the first time that both D3R and NF1 genes are upregulated following PA conditioning and suggest that hippocampal D3Rs might be relevant to NF1 transcriptional regulation in the hippocampus.

Expression pattern of parkin isoforms in lung adenocarcinomas

The parkin gene has been shown to be genetically altered in a wide variety of human tumors including lung cancer. Although many parkin splice variants have been identified, to date, most of the studies have only been focused on originally cloned isoforms. In this work, for the first time, the expression profile of parkin isoforms in human lung adenocarcinomas has been analyzed to identify their involvement in lung cancer. Their contribution in some biological conditions, such as proteasomal degradation or mitophagy or cell death, has been analyzed in human lung cells. The expression profile of parkin isoforms has been investigated in paraffin-embedded samples of human lung adenocarcinomas by using Western blot analysis. Their expression has also been evaluated in human lung adenocarcinoma and in human normal bronchial epithelial cell lines following treatment with a proteasome inhibitor or mitochondrial depolarizing agent, or in serum starvation. Parkin proteins were detected on blot by using two antibodies, AbI and AbII, which recognize different domains of originally cloned parkin. Furthermore, parkin immunolocalization has been visualized in both cell lines by using immunofluorescence analysis. Results have shown that H1 and/or H5, H14, H4 and/or H8 and/or H17 and H3 and/or H12 isoforms are expressed in human lung adenocarcinomas. Some of them are also present in A549 cell line, whereas they are absent or faintly expressed in BEAS-2B cells. Furthermore, their expression changed after treatment. Human lung adenocarcinomas express different parkin isoforms, which might represent markers of malignancy and could be linked to specific biological functions.

Nap Interferes with Hypoxia-Inducible Factors and VEGF Expression in Retina of Diabetic Rats

The retinal microvascular damage is a complication of diabetic retinopathy (DR). Hyperglycemia and hypoxia are responsible of aberrant vessel’s proliferation. The cellular response to hypoxia is mediated through activation of hypoxia-inducible factors (HIFs). Among these, HIF-1α modulates expression of its target gene, VEGF, whose upregulation controls the angiogenic event during DR development. In a previous study, we have demonstrated that a small peptide, NAP, is able to protect retina from hyperglycemic insult. Here, we have demonstrated that its intraocular administration in a rat model of diabetic retinopathy has reduced expression of HIF-1α, HIF-2α, and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization study by confocal microscopy. Although these evidences need to be further deepened to understand the molecular mechanism involved in the protective NAP action, the present data suggest that this small peptide may be effective to prevent the development of this ocular pathology.

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

HIGHLIGHTSPACAP modulates the inflammatory cytokines IL‐1&bgr; release in STZ‐injected rats.PACAP exogenous administration is able to downregulate VEGF, VEGFR1 and VEGFR2 expression in retina of diabetic rats.Hyperglycaemia and hypoxia concur to trigger a signalling cascade responsible of microvasculature dysfunction. ABSTRACT 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‐1&bgr; (IL‐1&bgr;) 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‐1&bgr; and VEGF expression. Diabetes was induced in rats by streptozotocin (STZ) injection, and one week later a single intravitreal injection of 100 &mgr;M PACAP was administrated. Analyses of IL‐1&bgr; 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‐1&bgr; 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.