ABSTRACT
BACKGROUND: We hypothesized that normal and pathological vessel walls display a differential pattern of secreted proteins. We have recently set up the conditions for comparing secretomes from carotid atherosclerotic plaques and control arteries using a proteomic approach to assess whether differentially secreted proteins could represent markers for atherosclerosis. METHODS AND RESULTS: Normal endartery segments and different regions of endarterectomy pieces (noncomplicated/complicated plaques) were incubated in protein-free medium, and the released proteins were analyzed by 2D electrophoresis (2-DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27). Surprisingly, compared with control arteries, HSP27 release was drastically decreased in atherosclerotic plaques and barely detectable in complicated plaque supernatants. HSP27 was expressed primarily by intact vascular cells of normal arteries and carotid plaques (immunohistochemistry). Plasma detection of soluble HSP27 showed that circulating HSP27 levels are significantly decreased in the blood of patients with carotid stenosis relative to healthy subjects (0.19 [0.1 to 1.95] versus 83 [71.8 to 87.8]) ng/mL, P<0.0001). CONCLUSIONS: HSP27 secretion is decreased in complicated atherosclerotic plaques, and sHSP27 plasma levels are decreased in atherosclerotic patients compared with healthy subjects. Plasma sHSP27 levels could be a potential index of atherosclerosis, although further validation is needed in large patient cohorts.
Subject(s)
Carotid Artery Diseases/physiopathology , Heat-Shock Proteins/metabolism , Neoplasm Proteins/metabolism , Proteomics , Aged , Carotid Arteries/metabolism , Carotid Arteries/pathology , Carotid Artery Diseases/blood , Carotid Artery Diseases/surgery , Chromatography, Affinity , Culture Media, Conditioned/chemistry , Endarterectomy, Carotid , Female , Femoral Artery/metabolism , Femoral Artery/pathology , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/blood , Heat-Shock Proteins/deficiency , Humans , Male , Middle Aged , Molecular Chaperones , Neoplasm Proteins/blood , Neoplasm Proteins/deficiency , Organ Culture Techniques , Phosphorylation , Protein Isoforms/analysis , Protein Processing, Post-Translational , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.
