Alessandro Castorina

PACAP and VIP prevent apoptosis in schwannoma cells

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.

Early changes in pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide and related receptors expression in retina of streptozotocin-induced diabetic rats.

The retinal expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) and their receptors was investigated in early streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection (60 mg/kg i.p.). PACAP, VIP and their receptors in nondiabetic control and diabetic retinas were assayed by quantitative real-time PCR and Western blot 1 and 3 weeks after STZ injection. Effects of intravitreal treatment with PACAP38 on the expression of the two apoptotic-related genes Bcl-2 and p53 were also evaluated. PACAP and VIP, as well as VPAC1 and VPAC2 receptors, but not PAC1 mRNA levels, were transiently induced in retinas 1 week following STZ. These findings were confirmed by immunoblot analyses. Three weeks after the induction of diabetes, significant decreases in the expression of peptides and their receptors were observed, Bcl-2 expression decreased and p53 expression increased. Intravitreal injection of PACAP38 restored STZ-induced changes in retinal Bcl-2 and p53 expression to nondiabetic levels. The initial upregulation of PACAP, VIP and related receptors and the subsequent downregulation in retina of diabetic rats along with the protective effects of PACAP38 treatment, suggest a role for both peptides in the pathogenesis of diabetic retinopathy.

Effects of PACAP and VIP on hyperglycemia-induced proliferation in murine microvascular endothelial cells.

Hyperglycemia is implicated both in micro- and macro-vascular complications in diabetes mellitus. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two known nonclassic regulators of angiogenesis, although their biological role on endothelial cell proliferation remains poorly defined. In the present study we hypothesized that either peptides might play an inhibitory role on hyperglycemia-induced cell growth. To this end, we investigated the effect of both PACAP and VIP on cell proliferation in murine microvascular endothelial cells (H5V) cultured both under euglycemic and hyperglycemic conditions (5 and 25 mM glucose, respectively) for 24, 48 h, 7 and 15 days. Results demonstrated that high glucose treatment induced a time-dependent increase in cell viability after 48 h (p<0.05), which was much more evident after 7 and 15 days (p<0.001). Similar effects were observed in cell proliferation, although significant changes were obtained after prolonged exposures to high glucose (7 and 15 days; p<0.001). The proliferative response to the glucose-enriched environment was correlated to changes in the expression of PAC1 and, to a minor extent, to VPAC2, but not VPAC1 receptors, as measured by quantitative real-time PCR. These results were further confirmed by Western blot and immunofluorescence analyses. Interestingly, 10⁻⁷ M PACAP or VIP treatment significantly attenuated hyperglycemia-induced increase in cell viability and proliferation after 7 and 15 days. Taken together, our findings demonstrate that both PACAP and VIP peptides exert an inhibitory activity on hyperglycemia-induced endothelial cell proliferation, thus suggesting that the effect might be mediated by PAC1 and VPAC2 receptors.

Degenerative disc disease of herniated intervertebral discs is associated with extracellular matrix remodeling, vimentin-positive cells and cell death

We studied patients with degenerative disc disease (DDD) to demonstrate that i) remodeling of the extracellular matrix (ECM) in the intervertebral disc (IVD), particularly the elastic fiber system, of subjects with herniated discs is dysregulated and that ii) it is accompanied by accelerated elastin degradation due to increased expression of matrix metalloprotease-9 (MMP-9). Moreover we wanted to obtain a deeper insight into the pathogenesis of DDD through the study of ECM calcification, DNA fragmentation using TUNEL analysis, BAX, bcl-2 and vimentin immunopositive cells. We studied herniated discs from patients of three age groups (group 1=30-40 years; group 2=40-50 years; and group 3=50-65 years) to evaluate the oxytalan fiber systemMMP-9, apoptosis and vimentin immunopositive cells. The results demonstrated the presence of oxytalan fibers in the annulus fibrosus (AF) and the nucleus pulposus (NP) of herniated discs. In the AF oxytalan fibers replaced disrupted mature elastic fibers in calcified areas, while in the NP they were mostly found in nests at the periphery of chondrocytes. MMP-9 was prevalently observed in NP nests above all in group 1 and group 3 discs while group 2 exhibited a lower MMP-9 immunostaining. Activation of the apoptotic process was demonstrated by upregulated BAX expression in group 3. BAX immunopositivity was inversely mirrored by a significant decrease in bcl-2 expression. Intermediate filament protein vimentin was strongly expressed only in group 1 samples. A large number of apoptotic TUNEL+ cells was observed in group 3 specimens. The presence of oxytalan fibers may be the result of a process of incomplete elastogenesis, or a response to mechanical stress trying to functionally replace the lack of elastic fibers. MMP-9 expression seems to relate to disc damage, while chondrocyte BAX upregulation and TUNEL+ cell staining revealed apoptosis activation regardless of patient age. Vimentin immunopositivity was clearly detected in group 1 annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs.

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.

Dopamine D3 Receptor Is Necessary for Ethanol Consumption: An Approach with Buspirone

Mesolimbic dopamine (DA) controls drug- and alcohol-seeking behavior, but the role of specific DA receptor subtypes is unclear. We tested the hypothesis that D3R gene deletion or the D3R pharmacological blockade inhibits ethanol preference in mice. D3R-deficient mice (D3R−/−) and their wild-type (WT) littermates, treated or not with the D3R antagonists SB277011A and U99194A, were tested in a long-term free choice ethanol-drinking (two-bottle choice) and in a binge-like ethanol-drinking paradigm (drinking in the dark, DID). The selectivity of the D3R antagonists was further assessed by molecular modeling. Ethanol intake was negligible in D3R−/− and robust in WT both in the two-bottle choice and DID paradigms. Treatment with D3R antagonists inhibited ethanol intake in WT but was ineffective in D3R−/− mice. Ethanol intake increased the expression of RACK1 and brain-derived neurotrophic factor (BDNF) in both WT and D3R−/−; in WT there was also a robust overexpression of D3R. Thus, increased expression of D3R associated with activation of RACK1/BDNF seems to operate as a reinforcing mechanism in voluntary ethanol intake. Indeed, blockade of the BDNF pathway by the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and strongly decreased the striatal expression of D3R. Finally, we evaluated buspirone, an approved drug for anxiety disorders endowed with D3R antagonist activity (confirmed by molecular modeling analysis), that resulted effective in inhibiting ethanol intake. Thus, DA signaling via D3R is essential for ethanol-related reward and consumption and may represent a novel therapeutic target for weaning.

Ameliorative Effects of PACAP against Cartilage Degeneration. Morphological, Immunohistochemical and Biochemical Evidence from in Vivo and in Vitro Models of Rat Osteoarthritis

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1β in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1β-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways.

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.

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.

Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride

In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl3) enhanced the neurotoxicity of the amyloid beta(25-35) fragment (Abeta(25-35)) in neuroblastoma cells and affected the expression of Alzheimers disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A2A adenosine receptors (A2AR), although it also inhibits A1Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta(25-35)+AlCl3) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta(25-35)+AlCl3 were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A1R and A2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl3 treatment exacerbated Abeta(25-35) toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta(25-35) only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta(25-35)+AlCl3-induced damage, suggesting that A1Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A1R inhibition in the setting of combined Abeta(25-35)+AlCl3 neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum.