Abstracts
computational approaches to the experimental lab, as it converts data into concepts and information into understanding. doi:10.1016/j.vph.2011.08.105
L.15.3 Integration of miRNA and proteomic screening for cardiovascular disease Manuel Mayr British Heart Foundation Centre, King's College London, London, UK E-mail address:
[email protected] The recent discovery that microRNAs (miRNAs) are present in blood sparked interest in their use as potential biomarkers. These circulating microRNAs are not cell associated but seem to escape degradation by endogenous RNase activity by residing in microvesicles, exosomes, microparticles and apoptotic bodies, although recently the formation of protein–miRNA complexes has also been proposed. While research on circulating miRNAs is still in its infancy, high analytical standards in statistics and study design are a prerequisite to obtain robust data and avoid repeating the mistakes of the early genetic association studies. Otherwise, studies tend to get published because of their novelty despite low numbers, poorly matched cases and controls and no multivariate adjustment for conventional risk factors. Research on circulating miRNAs can only progress by bringing more statistical rigour to bear in this field and by evaluating changes of individual miRNAs in the context of the overall miRNA network. We have previously performed the first prospective population-based study on circulating miRNAs and demonstrated that loss of endothelial miR-126 is associated with peripheral vascular complications in patients with type 2 diabetes (Zampetaki et al., Circ Res, 2010). A proteomics approach was employed to identify potential targets of miR-126. Endothelial cells transfected with the precursor of miR-126 showed reduced secretion of plasminogen-activator inhibitor-1, the key inhibitor of endogenous fibrinolysis. Thus, in subjects with type 2 diabetes differential co-expression patterns of circulating miRNAs occur around endothelial-enriched miR-126. This biomarker association has a potential mechanistic underpinning in the observed regulatory effect of miR-126 on fibrinolysis.
doi:10.1016/j.vph.2011.08.106
O.15.1 Extracellular matrix composition and remodelling in human abdominal aortic aneurysms: A proteomics approach Athanasios Didangelos, Xiaoke Yin, Angelika Saje, Alberto Smith, Qinqbo Xu, Marjan Jahangiri, Manuel Mayr Cardiovascular Division. King's College London, London, UK E-mail addresses:
[email protected] (A. Didangelos),
[email protected] (M. Mayr) Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-LC-MS/MS and labelfree quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein
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(ACLP), periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.
Reference 1. A. Didangelos, A., X. Yin, X., K. Mandal, K., A. Saje, A., A. Smith, A., Q. Xu, Q., M. Jahangiri, M., M. Mayr, M., 2011. Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approachMol Cell Proteomics. May 18. PubMed PMID: 21593211. doi:10.1016/j.vph.2011.08.107
O.15.2 Proteomic analysis of iPS and embryonic stem cells identifies alternate vascular cell differentiation properties Andriana Margariti, John Paul Kirton, Xiaoke Yin, Eirini Karamariti, Manuel Mayr, Yanhua Hu, Lingfang Zeng, Qingbo Xu Cardiovascular Division, King's College London BHF Centre, London, UK E-mail address:
[email protected] Background: The generation of induced pluripotent stem (iPS) cells from somatic cells can be a useful tool for regenerative medicine. The artificial nature of iPS cells raises concerns whether iPS and embryonic stem (ES) cells, are molecularly and functionally comparable, e.g. vascular lineage differentiation. Methods and Results: In this study we generated iPS cells using a genomic integration free method, and using the advance of proteomics, we aimed to elucidate any differences in the protein profile between iPS and ES cells, in terms of pluripotency and differentiation potential to vascular cell lineages. The results demonstrated that 180 proteins were differentially expressed, in which 66% showed a decreased expression pattern in iPS cells. Functional classification analysis revealed that these proteins were associated with mRNA processing, energy metabolism, cytoskeleton, proliferation, and differentiation. Further experiments demonstrated that iPS cells have a decreased proliferation capacity when compared to ES cells. Importantly, iPS cells also displayed a greater potential to differentiate into smooth muscle and endothelial cell lineages, when compared to ES cells. iPS and ES cells were seeded on collagen IVcoated dishes and cultured with differentiation media, α-MEM supplemented with 10% serum, for indicated time points. iPS cells displayed a differentiation morphology as early as day 3 in comparison to ES cells, and real-time PCR data confirmed that iPS express smooth muscle cell markers such as SMA, Calponin, and SM22 in higher levels. When vascular endothelial growth factor (VEGF) was added to differentiation media, iPS cells expressed endothelial markers such as CD144, FLK-1 and vWF as early as day 5 on differentiation, while ES cells showed limited expression of these markers during the early stages of differentiation. Importantly, the expression level of proteins related to cell differentiation such as the protein quaking was higher in iPS cells in comparison to ES cells. Interestingly, iPS cells showed a higher expression of quaking in comparison to ES cells when vascular cell differentiation was induced. Further experiments in iPS cells showed that knockdown of quaking by siRNA resulting in suppression of SMC markers during vascular cell differentiation. Conclusion: We identified differentially regulated protein expression between iPS and ESC cells and the pathways they influence, which allow us to better understand the reprogramming process and
