Mechanical properties of human atherosclerotic intima tissue

Progression and rupture of atherosclerotic plaques in coronary and carotid arteries are the key processes underlying myocardial infarctions and strokes. Biomechanical stress analyses to compute mechanical stresses in a plaque can potentially be used to assess plaque vulnerability. The stress analyses strongly rely on accurate representation of the mechanical properties of the plaque components.     

Sialidase activity in normal and atherosclerotic human aortic intima

Sialidase activity has been determined in homogenates of human aortic intima by measuring the amount of GM1 formed during the incubation of ganglioside GD1a with the tissue homogenates. Areas with atherosclerotic lesions as well as adjacent areas without histological evidence of atherosclerosis were taken for comparison. The rate of GM1 formation from GD1a in the presence of homogenates of the atherosclerotic intima was 20 pmol/h per mg protein. Homogenates of the unaffected intima did not desialylate GD1a. Sialidase activity of the atherosclerotic intima was linear for 1.5 h at GD1a content up to 1.5 nmol and at homogenate protein up to 1 g. NH₄Cl and NeuAc2en, inhibitors of lysosomal function and plasma membrane-bound sialidase, respectively, reduced sialidase activity of homogenates of the atherosclerotic intima by 94%. The results indicate that atherosclerotic lesions and unaffected intima differ in their activity and specificity of sialidases that cleave gangliosides.     

The proteomic alterations of Thermoanaerobacter tengcongensis cultured at different temperatures

Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living conditions in temperatures ranging from 50 to 80 degrees C. In this comprehensive analysis, we present a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant changes in spot volume at three culture temperatures, 55, 75, and 80 degrees C, respectively; furthermore, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic images of the bacterial proteins, extracted from two culture temperatures (55 and 75 degrees C), had similar patterns; however, the bacteria cultured at 80 degrees C had dramatically decreased their spot volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups: specific expression at certain temperatures and consistent changes of expression responsive to temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the bacteria cultured at 55 degrees C. Whereas, two chaperonins, GroES and GroEL, were found to show a consistent increase during the elevated temperatures with the determinations, either by MS or Western blot. The proteomic information, thus expedites our understanding of the molecular mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.     

Fibronectin distribution in human aortic intima and atherosclerotic lesions: concentration of soluble and collagenase-releasable fractions

Fibronectin is associated with cell attachment and migration and interacts with fibrin, collagen and glycosaminoglycans; thus, it may be a factor in the focal proliferation of smooth muscle cells and collagen in atherosclerosis. We have measured, by rocket immunoelectrophoresis, the concentrations of soluble and collagenase-releasable fibronectin in normal human aortic intima and different types of atherosclerotic lesions. Soluble fibronectin concentration showed no significant difference between normal intima and lesions, but was 6-8-times higher than expected on the basis of plasma concentration and molecular mass. The concentration free in the interstitial fluid was about 3-times the expected level, suggesting that it originates from local synthesis as well as plasma insudation. In tissue, about half the fibronectin appeared to be reversibly associated with tissue components. Incubation with collagenase released fibronectin equal to twice the soluble fraction from normal intima and early proliferative lesions. In more advanced plaques that were accumulating lipid, the amount released was significantly higher (P less than 0.05) and more than 3-times the soluble fraction, suggesting that it might be involved in lipid accumulation. However, there was no correlation between release of fibronectin and bound low-density lipoprotein.     

Stimulation of cholesterol ester synthesis in macrophages by lipoproteins from normal and atherosclerotic human aorta intima

A buffer extract from homogenized human aorta was applied to a Bio-Gel A-15m column, and two cholesterol-containing peaks were resolved. Both fractions of aortic lipoproteins present in the extracts from normal and atherosclerotic intima and stimulated cholesteryl ester (CE) synthesis in J774 mouse macrophages caused unregulated loading with CE. The Vmax of CE formation in the presence of both fractions correlated with the degree of intimal atherosclerosis. An excess of both fractions did not inhibit the uptake of malondialdehyde-treated low density lipoproteins by macrophages; their interaction with the cells was not inhibited either by fucoidin or by dextran sulfate. The uptake of labeled LDL by human fibroblasts was markedly decreased with excess of both fractions. Aortic lipoprotein-mediated CE synthesis (for both fractions) was completely blocked by EDTA in fibroblasts, being decreased by 50% in macrophages.     

A proteomic assessment of muscle contractile alterations during unloading and reloading

Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.     

A proteomic analysis of human bile

We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified 87 unique proteins, including several novel proteins as well as known proteins whose functions are unknown. A large majority of the identified proteins have not been previously described in bile. Using lectin affinity chromatography and enzymatically labeling of asparagine residues carrying glycan moieties by (18)O, we have identified a total of 33 glycosylation sites. The strategy described in this study should be generally applicable for a detailed proteomic analysis of most body fluids. In combination with "tagging" approaches for differential proteomics, our method could be used for identification of cancer biomarkers from any body fluid.     

Circulating human monocytes in the acute coronary syndrome express a characteristic proteomic profile

We examined the proteome of circulating monocytes of patients with acute coronary syndrome at different times in comparison to that of patients with stable coronary artery disease. On admission, the expression of 18 spot proteins was altered, 10 of which were totally absent. This pattern changed progressively, and at 6 months, there were no differences with the monocyte proteome of stable patients.     

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.     

A proteomic view at T cell costimulation

The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+) CD69(-) resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.     

A proteomic glimpse into human ureter proteome

Urine has evolved as one of the most important biofluids in clinical proteomics due to its noninvasive sampling and its stability. Yet, it is used in clinical diagnostics of several disorders by detecting changes in its components including urinary protein/polypeptide profile. Despite the fact that majority of proteins detected in urine are primarily originated from the urogenital (UG) tract, determining its precise source within the UG tract remains elusive. In this article, we performed a comprehensive analysis of ureter proteome to assemble the first unbiased ureter dataset. Next, we compared these data to urine, urinary exosome, and kidney mass spectrometric datasets. Our result concluded that among 2217 nonredundant ureter proteins, 751 protein candidates (33.8%) were detected in urine as urinary protein/polypeptide or exosomal protein. On the other hand, comparing ureter protein hits (48) that are not shown in corresponding databases to urinary bladder and prostate human protein atlas databases pinpointed 21 proteins that might be unique to ureter tissue. In conclusion, this finding offers future perspectives for possible identification of ureter disease‐associated biomarkers such as ureter carcinoma. In addition, the ureter proteomic dataset published in this article will provide a valuable resource for researchers working in the field of urology and urine biomarker discovery. All MS data have been deposited in the ProteomeXchange with identifier PXD002620 ( http://proteomecentral.proteomexchange.org/dataset/PXD002620 ).     

A proteomic study on human osteoblastic cells proliferation and differentiation

Changes in expression profiles for 17 proteins were ascertained in human mature osteoblasts compared to pre-osteoblasts (differentiation markers). A differential approach was used to highlight proteomic changes between human osteosarcoma cells and mature osteoblasts, showing a relative over-expression of 8 proteins (proliferation and tumor indicators), as well as under-expression of proteins also found down-regulated in pre-osteoblasts (specific markers of osteoblast differentiation). Our findings confirmed the differences between cell lines and primary human cell cultures and suggested caution on the use of osteosarcoma to study anti-osteoporotic drugs in humans.     

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome,global protein secretion,and T cells

Introduction: The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses.

Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses.

Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.     

p-hydroxyphenylacetaldehyde, an aldehyde generated by myeloperoxidase, modifies phospholipid amino groups of low density lipoprotein in human atherosclerotic intima

Oxidation of low density lipoprotein (LDL) may be of critical importance in the pathogenesis of atherosclerosis. Recent studies suggest that oxidized phospholipids render LDL atherogenic. However, both the structures and the physiologically relevant pathways for the formation of modified phospholipids in oxidized LDL remain poorly understood. We previously showed that p-hydroxyphenylacetaldehyde (pHA) is the major product of L-tyrosine oxidation by the myeloperoxidase/hydrogen peroxide/chloride system of phagocytes. In the current studies, we demonstrate that this reactive aldehyde targets the aminophospholipids of LDL in vitro and in vivo. Activated human neutrophils generated pHA-ethanolamine, the reduced adduct of pHA with the amino group of phosphatidylethanolamine, on LDL phospholipids by a reaction that required myeloperoxidase, H(2)O(2), and L-tyrosine. The cellular system could be replaced by HOCl and L-tyrosine but not by a wide variety of other oxidation systems, indicating that pHA-ethanolamine is a specific marker for covalent modification of aminophospholipids by myeloperoxidase. To determine whether aldehydes modify aminophospholipids in vivo, we quantified levels of pHA-ethanolamine in acid hydrolysates of reduced lipid extracts through isotope dilution gas chromatography/mass spectrometry. Circulating LDL contained undetectable levels of pHA-modified phospholipid (<0.1 mmol/mol). In contrast, the concentration of pHA-ethanolamine in LDL isolated from human atherosclerotic lesions was strikingly elevated (4.5 mmol/mol). Collectively, these results demonstrate a novel, myeloperoxidase-based mechanism for modifying the amino group of LDL phospholipids. They also offer the first evidence that myeloperoxidase may damage LDL lipids in vivo, raising the possibility that aldehyde-modified aminophospholipids play a role in inflammation and vascular disease.