BackgroundThe 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.ResultsThis 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.ConclusionsIn 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.
Vascular restenosis is affecting 30-40% of patients treated by percutaneous coronary angioplasty (PTCA). The advent of stenting reduced but not abolished restenosis. The introduction of drug eluting stent (DES) further reduced restenosis, but impaired endothelization exposed to intracoronary thrombosis as late adverse event. It is widely accepted that the endothelial denudation and coronary wall damages expose Vascular Smooth Muscle Cells (VSMC) to multiple growth factors and plasma circulating agents thus activating migration and proliferative pathways leading to restenosis. Among the major players of this processes, phosphorylated Elk-1, forming the Elk-1/SRF transcription complex, controls the expression of a different set of genes responsible for cell proliferation. Therefore, it is feasible that gene-specific oligonucleotide therapy targeting VSMC migration and proliferation genes can be a promising therapeutic approach. While a plethora of vehicles is suitably working in static in vitro cultures, methods for in vivo delivery of oligonucleotides are still under investigation. Recently, we have patented a novel erythrocyte-based drug delivery system with high capability to fuse with targeted cells thus improving drug bioavailability at the site of action. Here, the potential of these engineered porcine erythrocytes to deliver a synthetic DNA Elk-1 decoy inside syngenic porcine VSMC was tested. The results of this study indicate that Elk-1 decoy is actually able to inhibit cell proliferation and migration of VSMC. Our data also suggest that erythrocyte-based carriers are more efficient in delivering these oligonucleotides in comparison to conventional vehicles. As a consequence, a lower dose of Elk-1 decoy, delivered by engineered erythrocytes, was sufficient to inhibit cell growth and migration. This approach represents the translational step to reach in vivo experiments in pigs after PTCA and/or stent implantation where oligonucleotide drugs will be site-specific administered by using erythrocyte-based carriers to prevent restenosis.
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