The analysis of miRNA expression profiling datasets reveals inverse microRNA patterns in glioblastoma and Alzheimer's disease.

There is recent evidence to indicate the existence of an inverse association between the incidence of neurological disorders and cancer development. Concurrently, the transcriptional pathways responsible for the onset of glioblastoma multiforme (GBM) and Alzheimer's disease (AD) have been found to be mutually exclusive between the two pathologies. Despite advancements being made concerning the knowledge of the molecular mechanisms responsible for the development of GBM and AD, little is known about the identity of the microRNA (miRNAs or miRs) involved in the development and progression of these two pathologies and their possible inverse expression patterns. On these bases, the aim of the present study was to identify a set of miRNAs significantly de­regulated in both GBM and AD, and hence to determine whether the identified miRNAs exhibit an inverse association within the two pathologies. For this purpose, miRNA expression profiling datasets derived from the Gene Expression Omnibus (GEO) DataSets and relative to GBM and AD were used. Once the miRNAs significantly de­regulated in both pathologies were identified, DIANA­mirPath pathway prediction and STRING Gene Ontology enrichment analyses were performed to establish their functional roles in each of the pathologies. The results allowed the identification of a set of miRNAs found de­regulated in both GBM and AD, whose expression levels were inversely associated in the two pathologies. In particular, a strong negative association was observed between the expression levels of miRNAs in GBM compared to AD, suggesting that although the molecular pathways behind the development of these two pathologies are the same, they appear to be inversely regulated by miRNAs. Despite the identification of this set of miRNAs which may be used for diagnostic, prognostic and therapeutic purposes, further functional in vitro and in vivo evaluations are warranted in order to validate the diagnostic and therapeutic potential of the identified miRNAs, as well as their involvement in the development of GBM and AD.

Prognostic significance of deregulated microRNAs in uveal melanomas.

Uveal melanoma (UM) represents the most frequent primary tumor of the eye. Despite the development of new drugs and screening programs, the prognosis of patients with UM remains poor and no effective prognostic biomarkers are yet able to identify high­risk patients. Therefore, in the present study, microRNA (miRNA or miR) expression data, contained in the TCGA UM (UVM) database, were analyzed in order to identify a set of miRNAs with prognostic significance to be used as biomarkers in clinical practice. Patients were stratified into 2 groups, including tumor stage (high­grade vs. low­grade) and status (deceased vs. alive); differential analyses of miRNA expression among these groups were performed. A total of 20 deregulated miRNAs for each group were identified. In total 7 miRNAs were common between the groups. The majority of common miRNAs belonged to the miR­506­514 cluster, known to be involved in UM development. The prognostic value of the 20 selected miRNAs related to tumor stage was assessed. The deregulation of 12 miRNAs (6 upregulated and 6 downregulated) was associated with a worse prognosis of patients with UM. Subsequently, miRCancerdb and microRNA Data Integration Portal bioinformatics tools were used to identify a set of genes associated with the 20 miRNAs and to establish their interaction levels. By this approach, 53 different negatively and positively associated genes were identified. Finally, DIANA­mirPath prediction pathway and Gene Ontology enrichment analyses were performed on the lists of genes previously generated to establish their functional involvement in biological processes and molecular pathways. All the miRNAs and genes were involved in molecular pathways usually altered in cancer, including the mitogen­activated protein kinase (MAPK) pathway. Overall, the findings of the presents study demonstrated that the miRNAs of the miR­506­514 cluster, hsa­miR­592 and hsa­miR­199a­5p were the most deregulated miRNAs in patients with high­grade disease compared to those with low­grade disease and were strictly related to the overall survival (OS) of the patients. However, further in vitro and translational approaches are required to validate these preliminary findings.

N-hydroxymethylglycinate with EDTA is an efficient eye drop preservative with very low toxicity: an in vitro comparative study.

PURPOSE: Preservatives are used in multi-dose ophthalmic topical medications in order to prevent contamination by bacteria and fungi. However, prolonged use of preserved eye drops, as it may happen in dry eye or glaucoma, may damage cells of the ocular surface. Therefore, an important goal is to find preservatives with low toxicity which are mild to host cells, still able to prevent drug contamination so to maintain their sterility and efficacy. Hence, aim of this study has been to compare the relative toxicity on a rabbit corneal cell line of a new preservative, made by the association of N-hydroxy-methyl-glycinate (NIG) with disodium-ethylene diamine tetra-acetate (EDTA), with other known and widely used eye-drops preservatives. MATERIALS AND METHODS: Rabbit corneal cells (SIRC) were tested either in 96-well plates or in suspension culture. Treatments with preservatives (used at known bacteriostatic concentrations) included: benzalkonium chloride (BAK), polyquaternium-1 (PQ-1), sodium perborate (SP: NaBO3 * H2O), and NIG ± EDTA at different concentrations (0.001% and 0.002%), and different treatment times (from 30 minutes to 120 hours). At the end of treatment, cell survival was evaluated by a specific spectrophotometric method through the metabolic conversion of MTT [3-(4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] into formazan crystals. RESULTS: Almost no cell toxicity was evident for NIG and SP at either concentration (0.001% or 0.002%), while a low toxicity was observed for PQ-1 (62% at the highest dose at 120 hours). BAK, as expected, showed the highest toxicity (60-80% at 30 minutes, and over 90% from eight hours onward). EDTA 0.1% alone or in combination with NIG 0.002%, showed no toxicity at 24 hours, and even resulted in cell growth promotion (46% and 38%, respectively), after 48 hours of treatment. CONCLUSIONS: These data show that the new preservative NIG/EDTA, at doses known to have effective antimicrobial properties, has a very low toxicity on corneal cells, and so it can be safely used in multi-dose eye drops.

Gabapentin Attenuates Ocular Inflammation: In vitro and In vivo Studies.

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1ß, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 µg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

Immobilization of Neurotrophin Peptides on Gold Nanoparticles by Direct and Lipid-Mediated Interaction: A New Multipotential Therapeutic Nanoplatform for CNS Disorders.

Neurotrophins are essential proteins for the development and maintenance of neural functions as well as promising drugs in neurodegenerative disorders. Current limits in their effective clinical applications can be overwhelmed by the combined use of peptidomimetic and nanomedicine approaches. Indeed, neurotrophin-mimicking peptides may allow minimizing the adverse side effects of the whole protein drug. Moreover, the immobilization of such peptides on nanomaterials may offer additional advantages, including protection against degradation, enhanced permeability of barrier membranes, and intrinsic therapeutic properties of the nanoparticles (e.g., antiangiogenic and plasmonic features of gold nanoparticles (AuNPs)). In the present article, we scrutinize the functionalization of spherical AuNPs of diameter 12 nm by peptides because of the N-terminal domains of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF), NGF1-14 and BDNF1-12, respectively. The hybrid gold-peptide nanobiointerface was investigated, both in the direct physisorption and in the lipid-bilayer-mediated adsorption processes, by a multitechnique study that included UV-vis and X-ray photoelectron spectroscopies, dynamic light scattering, zeta-potential analyses, and atomic force microscopy. Both peptide- and lipid-dependent features were identified, to have a modulation in the peptide coverage of nanoparticles as well as in the cellular uptake of NGF and BDNF peptides, as investigated by confocal microscopy. The promising potentials of the neurotrophins to cross the blood-brain barrier were demonstrated.

Anti-angiogenic Therapy in Cancer: Downsides and New Pivots for Precision Medicine.

Primary solid tumors originate close to pre-existing tissue vasculature, initially growing along such tissue blood vessels, and this phenomenon is important for the metastatic potential which frequently occurs in highly vascularized tissues. Unfortunately, preclinic and clinic anti-angiogenic approaches have not been very successful, and multiple factors have been found to contribute to toxicity and tumor resistance. Moreover, tumors can highlight intrinsic or acquired resistances, or show adaptation to the VEGF-targeted therapies. Furthermore, different mechanisms of vascularization, activation of alternative signaling pathways, and increased tumor aggressiveness make this context even more complex. On the other hand, it has to be considered that the transitional restoration of normal, not fenestrated, microvessels allows the drug to reach the tumor and act with the maximum efficiency. However, these effects are time-limited and different, depending on the various types of cancer, and clearly define a specific "normalization window." So, new horizons in the therapeutic approaches consist on the treatment of the tumor with pro- (instead of anti-) angiogenic therapies, which could strengthen a network of well-structured blood vessels that facilitate the transport of the drug.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier.

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

UV-O3-treated and protein-coated polymer surfaces facilitate endothelial cell adhesion and proliferation mediated by the PKCalpha/ERK/cPLA2 pathway.

We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O(3) treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid-base behaviour for PHMS. UV-O(3) treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O(3) irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O(3) irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Calpha (p-PKCalpha, phospho-ERK1/2, and phospho-cytosolic phospholipase A(2) (p-cPLA(2)), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O(3)-treated PHMS, Western blot analyses showed increased levels of p-PKCalpha, p-ERK1/2 and p-cPLA(2) in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O(3)-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree.

MAPKs mediate the activation of cytosolic phospholipase A2 by amyloid beta(25-35) peptide in bovine retina pericytes.

We have previously shown that, in bovine retina pericytes, amyloid beta(1-42) and its truncated form containing amino acids 25-35, after 24 h treatment, stimulate arachidonic acid (AA) release and phosphatidylcholine hydrolysis, by activation of both cytosolic (cPLA(2)) and Ca(2+)-independent (iPLA(2)) phospholipase A(2). A putative role for MAP kinases in this process emerged. Here we studied the role of the MAP-kinase family as well as both cPLA(2) and iPLA(2) mRNA expression by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the same sublethal model of amyloid-beta (Abeta) damage to pericytes in vitro. Abeta(25-35) peptide evoked AA release as well as stimulated phosphorylation of ERK1/2, p38 MAPKs and cPLA(2), but not c-Jun N-terminal kinase (JNK/SAPK). PD98059, an inhibitor of ERK-activating kinase MEK-1, and SB203580, an inhibitor of p38 protein kinase, abolished the stimulation of AA release and MAPK activities. In cells stimulated by Abeta(25-35) peptide, Western blotting and confocal microscopy analyses confirmed either an increase in the phosphorylated form of ERKs and p38 or their nuclear translocation. A complete inhibition of MAPK activation and AA release was also observed when pericytes were treated with GF109203X, a general PKC inhibitor, indicating the important role of both PKC and the two MAPKs in mediating the Abeta peptide response. Compared with samples untreated or treated with reverse Abeta(35-25) peptide, pretreatment with 50 microM Abeta(25-35) for 24 h significantly increased the level of constitutively expressed iPLA(2) mRNA by 25%, which seems to depend on the activation of kinases. By contrast, the level of cPLA(2) mRNA remained unchanged. Together, these data link either the stimulation of PKC-ERK-p38 cascades or PLA(2) activity by Abeta peptide to prooxidant mechanism induced by amyloid, which may initially stimulate the cell reaction as well as metabolic repair, such as during inflammation.

Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1.

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, Km, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4 +/- 1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80 +/- 1.5 nmol/min/mg protein) compared to that estimated in liver and brain (1-3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the transfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10(-6) and 10(-4) M showed significant reduction in 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.

Cytosolic phospholipase A2 mediates arachidonoyl phospholipid hydrolysis in immortalized rat brain endothelial cells stimulated by oxidized LDL.

We tested the hypothesis that oxidized low-density lipoprotein (oxLDL), administered in sublethal doses to the culture medium of immortalized rat brain endothelial cells (ECs, GP8.39), acts as a prooxidant signal to stimulate peroxidation processes and membrane phospholipid hydrolysis. ECs were grown at confluence in a medium with or without native LDL (nLDL) or oxLDL (1.5 mg/dish; up to 350-450 nmol hydroperoxides/mg protein) for two temporally distinct phases (short incubation period up to 1 h, or long incubation period spanning 24 h). Peroxidation parameters (conjugated dienes, MDA, hydroperoxides and LDH release) and arachidonic acid (AA) release were determined. Cell lysates and subcellular fractions were assayed for cPLA(2) while the cytotoxic effect and apoptosis were monitored by morphological changes, trypan blue dye exclusion, MTT reduction test, caspase-3 activity, COMET and laser confocal fluorescence microscopy (LCFM) analyses. Effects of alpha-tocopherol and 85-kDa PLA(2) inhibitor (AACOCF(3)), alone or in combination, were also tested. Immunoblot analysis of cPLA(2) was carried out on cell fraction proteins. After incubation for 1 or 24 h, oxLDL (100-200 microM hydroperoxides), but not nLDL, markedly increased lipid peroxidation, cPLA(2) activity and AA release in a dose-dependent manner. AACOCF(3) and antioxidant alpha-tocopherol (1 mM) strongly inhibited the prooxidant-stimulated AA release. Long-term exposure (24 h) to oxLDL (100 microM) had no effect on the cPLA(2) protein content as tested by Western immunoblot analysis, while showing a sharp cytotoxic effect on the cells. Caspase-3 activity and LCFM analysis indicated that oxLDL (100/200 microM) were able to trigger an apoptotic process. The results suggest that (i) ECs may be the target of extensive oxidative damage caused by oxLDL; (ii) activation of cPLA(2) mediates liberation of AA; (iii) cPLA(2) expression was not stimulated by long-term exposure to oxLDL; (iv) oxidized specific constituents of oxLDL, acting as regulatory signals, increase the ability of ECs to degrade membrane phospholipids, end products of which are linked to the development of atherosclerotic lesions.