The researchers identified four proteins that are expressed in atherosclerosis legions: pigment epithelium derived factor (PEDF), periostin, milk fat globule-EGF-factor (MFG-E8) and annexin-1, which were all validated using immunohistochemistry (IHC) staining techniques to confirm their presence in human plaques.
Atherosclerosis is a blood vessel disease that manifests itself in a variety of pathological ways, from the simple thickening of the blood vessels to the catastrophic arterial blockages which lead to heart attacks.
The disease has been cited by various sources as being the leading cause of death in the US and Western Europe.
The researchers, led by Dr David Han of the University of Connecticut School of Medicine, said that to gain a better understanding of the progression of atherosclerosis progression it is crucial to define the proteins expressed in the vessel wall.
The researchers used tandem LC-MS/MS (liquid chromatography - tandem mass spectrometry) based direct tissue proteomics (DTP) techniques to identify 806 proteins present in atherosclerotic plaques.
The study, to be published in an upcoming edition of Molecular and Cellular Proteomics, found that while paraformaldehyde-fixed sections could be analysed using the DTP method, frozen sections allowed the identification of twice as many proteins.
According to the authors, while the manuscript was in preparation, PDEF was shown by Baba et al to be expressed in atherosclerotic plaques in the aorta and coronary arteries as well as during embryogenesis.
They believe that this suggests PDEF participates in extracellular remodelling and may be involved with controlling calcification, inflammatory cells migration and proliferation as well as the formation of new blood vessels.
But while the technique is a powerful tool for exploring the pathogenesis of atherosclerosis, I has its limitations. The researchers noted that the identification of cytokines and growth factors was not possible using DTP analysis.
The authors suggested three reasons for this, firstly that the molecules were present in concentrations below the detection limit of the technique, secondly that they may be post-translationally modified impacting the peptide pool available for identification and thirdly that they are mostly involved in plaque inflammation and the plaques studied were at early or stable stages.
However, by using a recombinant isotopic labelling MS technique known as absolute quantification by abundance (AQUA) they were able to detect low abundance cytokines and growth factors.
These included TGF-beta and SDF-1alpha, confirming that these factors are present in the vessel wall, but are undetectable by the less sensitive DTP method.
