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Abstract Background Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs. Findings Ribozymes, loaded on a cationic polymer-based vehicle, were delivered into cultured VSMCs. A significant impairment of the activation mechanisms triggered by PDGF-BB was demonstrated since cell migration decreased after treatments. In order to functionally validate the effects of PDGFR-β partial knock down we focused on the phosphorylation status of two proteins, protein disulfide isomerase-A3 (PDI-A3) and heat shock protein-60 (HSP-60), previously identified as indicative of VSMC phenotypic switch after PDGF-BB stimulation. Interestingly, while PDI-A3 phosphorylation was counteracted by the ribozyme administration indicating that PDI-A3 is a factor downstream the receptor signalling cascade, the HSP-60 phosphorylation status was greatly increased by the ribozyme administration. Conclusion These contradictory observations suggested that PDGF-BB might trigger different parallel pathways that could be modulated by alternative isoforms of the receptors for the growth factor. In conclusion the knock down strategy here described enables to discriminate between two tightly intermingled pathways. Moreover it opens new attractive perspectives in functional investigations where combined gene knock down and proteomic technologies would allow the identification of key factors and pathways involved in VSMC-linked pathological disorders.

Abstract Background The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before. Results This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude. Conclusions In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.

Performing cell biology experiments in space imposes the use of hardware that essentially allows fluid exchange in a contained environment. Given the technical and logistical peculiarities, the limited opportunities and the high cost of access to space, a great effort during mission preparation of scientific studies is devoted to preventing loss of the experiment. The European Space Agency (ESA) requires, at the end of the preparation phase, the execution of an Experiment Sequence Test (EST), a dry-run version of the space experiment to check all procedures. At conclusion of the EST of our experiment ‘ENDO’ (ESA ILSRA-2009-1026), we found pitting corrosion of metal parts and biofilm formation within the cell-culture devices. The subsequent chemical (spectral assays), instrumental (OGP SmartScope) and microbiological (MALDI-TOF, 16S rRNA gene sequencing) investigations allowed the identification in contaminated material of Paenibacillus glucanolyticus, a ubiquitous, aerobic, facultative anaerobic, endospore forming, acid-producing, Gram-positive microorganism. A concurrence of P. glucanolyticus contamination and galvanic corrosion determined massive fouling, rust precipitation and damage to cells and cell-culture devices being, to our knowledge, the association between this microbe and corrosion never reported before in literature. As a consequence of the episode a critical procedure of experiment set up, i.e. hardware sterilization, was modified. The ENDO experiment was successfully launched to the International Space Station on September 2nd 2015 and returned to the PI laboratory on September 13th, with all cell culture samples in optimal condition. [ABSTRACT FROM AUTHOR]

With the disclosure of the human genome a new era for bio-medicine has arisen, characterized by the challenge to investigate pathogenic mechanisms, studying simultaneously metabolites, DNA, RNA, and proteins. As a result, the "omics" revolution boomed, giving birth to a new medicine named "omics-based medicine". Among the other "omics", proteomics has been widely used in medicine, since it can produce a more "holistic" overview of a disease and provide a "constellation" of possible specific markers, a molecular fingerprinting that defines the clinical condition of an individual. Endpoint of this comprehensive and detailed analysis is the "diagnostic-omics", i.e. the achievement of personalized diagnoses with obvious benefits for prevention and therapy and this goal can be reached only with a perfect integration between clinicians and proteomists. To impact on the possible key factors involved in the pathological processes, oligonucleotide-based knock-down strategies can be helpful. They exploit omics-derived molecular tools (antisense, siRNA, ribozymes, decoys, and aptamers) that can be used to inhibit, at transcriptional or post-transcriptional levels, the events leading to protein synthesis, thus decreasing its expression. The identification of the pivotal mechanisms involved in diseases using global, "scenic" approaches such as the "omics" ones, and the subsequent validation and detailed description of the processes by specific molecular tools, can result in a more preventive, predictive and personalized medicine.