Luisa Pieroni

In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors.

Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected. The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice. In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain. Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice. Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.

Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations.

Archival formalin‐fixed paraffin‐embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI‐TOF imaging MS (MALDI‐IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo‐preserved tissues. We have developed an in vitro approach using “tissue surrogate” samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI‐TOF MS and nLC‐MSE analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI‐IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.

Lenalidomide restrains motility and overangiogenic potential of bone marrow endothelial cells in patients with active multiple myeloma

Purpose: To determine the in vivo and in vitro antiangiogenic power of lenalidomide, a “lead compound” of IMiD immunomodulatory drugs in bone marrow (BM) endothelial cells (EC) of patients with multiple myeloma (MM) in active phase (MMEC). Experimental Design: The antiangiogenic effect in vivo was studied using the chorioallantoic membrane (CAM) assay. Functional studies in vitro (angiogenesis, “wound” healing and chemotaxis, cell viability, adhesion, and apoptosis) were conducted in both primary MMECs and ECs of patients with monoclonal gammopathies (MGUS) of undetermined significance (MGEC) or healthy human umbilical vein endothelial cells (HUVEC). Real-time reverse transcriptase PCR, Western blotting, and differential proteomic analysis were used to correlate morphologic and biological EC features with the lenalidomide effects at the gene and protein levels. Results: Lenalidomide exerted a relevant antiangiogenic effect in vivo at 1.75 μmol/L, a dose reached in interstitial fluids of patients treated with 25 mg/d. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of MGECs or control HUVECs, and had no effect on MMEC viability, apoptosis, or fibronectin- and vitronectin-mediated adhesion. Lenalidomide-treated MMECs showed changes in VEGF/VEGFR2 signaling pathway and several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. Conclusions: This study provides information on the molecular mechanisms associated with the antimigratory and antiangiogenic effects of lenalidomide in primary MMECs, thus giving new avenues for effective endothelium-targeted therapies in MM. Clin Cancer Res; 17(7); 1935–46. ©2011 AACR.

Cystic fibrosis transmembrane conductance regulator (CFTR) expression in human platelets: impact on mediators and mechanisms of the inflammatory response

Inflammatory lung disease is a primary cause of morbidity and mortality in cystic fibrosis (CF). Mechanisms of unresolved acute inflammation in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in nonrespiratory cells is emerging. Here we examined CFTR expression and function in human platelets (PLTs) and found that they express a biologically active CFTR. CFTR blockade gave an ∼50% reduction in lipoxin A4 (LXA4) formation during PLT/polymorphonuclear leukocytes (PMN) coincubations by inhibiting the lipoxin synthase activity of PLT 12‐lipoxygenase. PLTs from CF patients generated ∼40% less LXA4 compared to healthy subject PLTs. CFTR inhibition increased PLT‐dependent PMN viability (33.0±5.7 vs. 61.2±8.2%; P=0.033), suppressed nitric oxide generation (0.23±0.04 vs. 0.11±0.002 pmol/108 PLTs; P= 0.004), while reducing AKT (1.02±0.12 vs. 0.71±0.007 U; P=0.04), and increasing p38 MAPK phosphorylation (0.650±0.09 vs. 1.04±0.24 U; P=0.03). Taken together, these findings indicate that PLTs from CF patients are affected by the molecular defect of CFTR. Moreover, this CF PLT abnormality may explain the failure of resolution in CF.—Mattoscio, D., Evangelista, V., De Cristofaro, R., Recchiuti, A., Pandolfi, A., Di Silvestre, S., Manarini, S., Martelli, N., Rocca, B., Petrucci, B., Angelini, D.F., Battistini, L., Robuffo, I., Pensabene, T., Pieroni, L., Furnari, M.L., Pardo, F., Quattrucci, S., Lancellotti, S., Davi, G., Romano, M. Cystic fibrosis transmembrane conductance regulator (CFTR) expression in human platelets: impact on mediators and mechanisms of the inflammatory response. FASEB J. 24, 3970–3980 (2010). www.fasebj.org

New insights into neuroblastoma cisplatin resistance: A comparative proteomic and meta-mining investigation

Neuroblastoma is one of the most aggressive solid tumors in the childhood. Therapy resistance to anticancer drugs represents the major limitation to the effectiveness of clinical treatment. To better understand the mechanisms underlying cisplatin resistance, we performed a comparative proteomic study of the human neuroblastoma cell line SH-SY5Y and its cisplatin resistant counterpart by both the classical 2-DE electrophoresis coupled to mass spectrometry and the more innovative label-free nLC-MS(E). The differentially expressed proteins were classified by bioinformatic tools according to their biological functions and their involvement in several cellular processes. Moreover, a meta-mining investigation of protein ontologies was also performed on available data from previously published proteomics studies to highlight the modulation of significant cellular pathways, which may regulate the sensitivity of neuroblastoma to cisplatin. In particular, we hypothesized a major role of the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. Confocal microscopy experiments, enzyme assay, and Western blotting of proteins regulated by Nrf2 provided evidences that this pathway, playing a protective role in normal cells, may represent a potential novel target to control cisplatin resistance in neuroblastoma.

Higher pain perception and lack of recovery from neuropathic pain in females: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice.

Summary Neuropathic pain as well as reactive gliosis are shorter lasting in male than in female mice, and are associated with faster nerve regeneration. ABSTRACT In experimental and clinical pain studies, the sex of subjects was rarely taken into account, even if nociceptive inputs appear to be processed and modulated by partially distinct neural mechanisms in each sex. In this study we analysed, in male and female mice, behavioural and neuronal responses in developing, maintaining, and recovering from neuropathic pain. Experiments were carried out in adult CD1 mice by using Chronic Constriction Injury (CCI) as neuropathic pain model. We investigated the temporal trend of mechanical nociceptive threshold together with functional recovery of the injured paw, and the immunofluorescence staining of proteins associated with nerve injury and repair and with spinal gliosis, 7 and 121 days after CCI. A proteomic analysis on proteins extracted from sciatic nerves was also performed. Male mice showed a gradual decrease of CCI‐induced allodynia, the complete recovery occurring 81 days after the sciatic nerve ligation. On the contrary, in female mice, allodynia was still present 121 days after CCI. Sex‐dependent differences also resulted from immunofluorescence experiments: in sciatic nerve, the expression of P0 and Neu200 is greater in neuropathic males than in neuropathic females, suggesting faster nerve regeneration. Proteomic analysis confirmed sex‐related differences of proteins associated with nerve regenerative processes. In addition, the reactive gliosis induced by CCI at day 7, as revealed by colocalization of glial fibrillary acidic protein (astrocytes) and CD11b (microglia) with phosphorylated p38, disappeared 121 days after CCI in male but not in female mice. These results may have important therapeutic implications for the treatment of neuropathic pain.

Schwann cell autophagy counteracts the onset and chronification of neuropathic pain

Summary Rapamycin enhances autophagic flux of Schwann cells, reduces prooxidant and proinflammatory proteins, and prevents neuropathic pain; autophagy deficiency induces exacerbated pain and delays recovery. ABSTRACT Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long‐lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3‐methyladenine administration or as occurs in activating molecule in Beclin‐1‐regulated autophagy transgenic mice (Ambra1+/gt), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox‐sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

Exosomal clusterin, identified in the pericardial fluid, improves myocardial performance following MI through epicardial activation, enhanced arteriogenesis and reduced apoptosis.

BACKGROUND We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair. METHODS AND RESULTS Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit(+)/α-SMA(+), 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts. CONCLUSIONS Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis.

Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis

Impaired dopamine homeostasis is an early event in the pathogenesis of Parkinsons disease. Generation of intracellular reactive oxygen species consequent to dopamine oxidation leads to mitochondrial dysfunction and eventually cell death. Alterations in the mitochondrial proteome due to dopamine exposure were investigated in the SH-SY5Y human neuroblastoma cell line. The combination of two orthogonal proteomic approaches, two-dimensional electrophoresis and shotgun proteomics (proteomeXchange dataset PXD000838), was used to highlight the specific pathways perturbed by the increase of intracellular dopamine, in comparison with those perturbed by a specific mitochondrial toxin (4-methylphenylpyridinium, MPP(+)), a neurotoxin causing Parkinsonism-like symptoms in animal models. Proteins altered by MPP(+) did not completely overlap with those affected by dopamine treatment. In particular, the MPP(+) target complex I component NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 was not affected by dopamine together with 26 other proteins. The comparison of proteomics approaches highlighted the fragmentation of some mitochondrial proteins, suggesting an alteration of the mitochondrial protease activity. Pathway and disease association analysis of the proteins affected by dopamine revealed the overrepresentation of the Parkinsons disease and the parkin-ubiquitin proteasomal system pathways and of gene ontologies associated with generation of precursor metabolites and energy, response to topologically incorrect proteins and programmed cell death. These alterations may be globally interpreted in part as the result of a direct effect of dopamine on mitochondria (e.g. alteration of the mitochondrial protease activity) and in part as the effect on mitochondria of a general activation of cellular processes (e.g. regulation of programmed cell death).

Proteomic analysis of protein adsorption capacity of different haemodialysis membranes

Protein-adsorptive properties are a key feature of membranes used for haemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membranes performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with chemical feature of the membrane surfaces. Dialyzers composed of either cellulose triacetate (Sureflux 50 L, effective surface area 0.5 m(2); Nipro Corporation, Japan) or the polysulfone-based helixone (FX40, effective surface area 0.4 m(2); Fresenius Medical Care AG, Germany) materials were employed to develop an ex vivo apparatus to study protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by a proteomic approach. The profiling strategy was based on 2D-electrophoresis separation of desorbed protein coupled to MALDI-TOF/TOF analysis. The total protein adsorption was not significantly different between the two materials. An average of 179 protein spots was visualised for helixone membranes while a map of retained proteins of cellulose triacetate membranes was made up of 239 protein spots. The cellulose triacetate material showed a higher binding capacity for albumin and apolipoprotein. In fact, a number of different protein spots belonging to the gene transcript of albumin were visible in the cellulose triacetate map. In contrast, helixone bound only a small proportion of albumin, while proved to be particularly active in retaining protein associated with the coagulation cascade, such as the fibrinogen isoforms. Our data indicate that proteomic techniques are a useful approach for the investigation of proteins surface-adsorbed onto haemodialysis membranes, and may provide a molecular base for the interpretation of the efficacy and safety of anticoagulation treatment during renal replacement therapy.