Aroa S. Maroto

A Proteomic Focus on the Alterations Occurring at the Human Atherosclerotic Coronary Intima

Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries.

Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis☆

AIMS Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development. METHODS AND RESULTS Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5. CONCLUSION The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer.

UNLABELLED Fatal events derived from coronary atherosclerosis are the major cause of mortality in the developed countries. Proteomic analysis of the atherosclerotic coronary artery has been mainly carried out with whole tissue extracts, making it difficult to distinguish the alterations present in every region of the plaque. For this reason, we have recently described proteins altered in the human coronary intima layer as a consequence of the atherosclerotic disease. In order to complement this work, we aimed here to analyze proteomic alterations occurring within the human coronary media layer. Media layers from human atherosclerotic and preatherosclerotic coronary arteries were isolated by laser microdissection and compared by means of two-dimensional differential in-gel electrophoresis (2D-DIGE). Twelve proteins were found altered, 5 of which were cytoskeleton proteins decreased in the atherosclerotic coronary media. Among these, 4 proteins (filamin A, gelsolin, vinculin and vimentin) were further analyzed by immunohistochemistry and its alteration validated. Such cytoskeleton deregulation evidence, at the molecular level, explains how medial vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. Moreover, an oxidative stress response within the media, leaded by superoxide dismutase 3 and glycolysis activation, may have been triggered by atherosclerosis development. BIOLOGICAL SIGNIFICANCE Although atherosclerosis is mainly a disease of the intima layer, the media plays an important role in the initiation of the pathology, as a source of vascular smooth muscle cells (VSMCs), which migrate into the intima and may additionally be affected by intima layer degeneration through pathogenesis. In fact, intimal thickening has been related to a mechanical compression of the media layer, resulting on a significant thinning of the latter in the atherosclerotic carotid and coronary arteries, which may provoke alterations at a molecular level. Here we provide the first differential proteomic analysis of atherosclerotic coronary media layer, reporting important alterations of this sub-proteome with pathogenesis. It is important to remark a cytoskeleton deregulation observed at the molecular level within VSMCs, which may be explained by a contractile to synthetic phenotype switch. Moreover, atherosclerosis seems to trigger an oxidative stress response within the coronary media layer.

An optimum method designed for 2-D DIGE analysis of human arterial intima and media layers isolated by laser microdissection.

The formation and progression of atherosclerotic lesions involve complex mechanisms which are still not fully understood. A variety of cell types from the distinct arterial layers are implicated in the whole process from lipid accumulation within the vascular wall to plaque development and final rupture. In the present work, we employ the combination of laser microdissection and pressure catapulting and 2‐D DIGE saturation labeling to investigate the human intima and media sub‐proteomes isolated from atherosclerotic (coronary and aorta) or non‐atherosclerotic vessels (preatherosclerotic coronary arteries). Laser microdissection and pressure catapulting allows the specific isolation of regions of interest. In turn, DIGE saturation labeling overcomes the limitation of extensive microdissection times to recover the protein amount required to perform comparative 2‐DE, particularly when dealing with tissue regions rich in myofilament proteins, which result in low protein recovery. The compatibility and optimum performance of both techniques were investigated in detail, paying special attention to tissue staining and protein solubilization. Since scarce amount of protein obtained from microdissected tissue made it impossible to directly perform protein identification from 2‐DE spots by MS, we performed in‐solution digestion followed by LC‐MS/MS analysis of total protein extracts from intima and media in order to get an overall picture of protein composition. Proteins so identified confirm the nature of the isolated regions. Finally, similar spot resolution on 2‐D DIGE gels was obtained for the different human artery types (coronary, aorta) and studied layers (intima, media), setting the basis for future clinical comparative studies.

Molecular anatomy of ascending aorta in atherosclerosis by MS Imaging: Specific lipid and protein patterns reflect pathology

The molecular anatomy of healthy and atherosclerotic tissue is pursued here to identify ongoing molecular changes in atherosclerosis development. Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. Mass spectrometry imaging (MSI) is a novel unexplored ex vivo imaging approach in CVD able to provide in-tissue molecular maps. A rabbit model of early atherosclerosis was developed and high-spatial-resolution MALDI-MSI was applied to comparatively analyze histologically-based arterial regions of interest from control and early atherosclerotic aortas. Specific protocols were applied to identify lipids and proteins significantly altered in response to atherosclerosis. Observed protein alterations were confirmed by immunohistochemistry in rabbit tissue, and additionally in human aortas. Molecular features specifically defining different arterial regions were identified. Localized in the intima, increased expression of SFA and lysolipids and intimal spatial organization showing accumulation of PI, PG and SM point to endothelial dysfunction and triggered inflammatory response. TG, PA, SM and PE-Cer were identified specifically located in calcified regions. Thymosin β4 (TMSB4X) protein was upregulated in intima versus media layer and also in response to atherosclerosis. This overexpression and localization was confirmed in human aortas. In conclusion, molecular histology by MS Imaging identifies spatial organization of arterial tissue in response to atherosclerosis.

KLK1 and ZG16B proteins and arginine–proline metabolism identified as novel targets to monitor atherosclerosis, acute coronary syndrome and recovery

We pursued here the identification of specific signatures of proteins and metabolites in urine which respond to atherosclerosis development, acute event and/or recovery. An animal model (rabbit) of atherosclerosis was developed and molecules responding to atherosclerosis silent development were identified. Those molecules were investigated in human urine from patients suffering an acute coronary syndrome (ACS), at onset and discharge. Kallikrein1 (KLK1) and zymogen granule protein16B (ZG16B) proteins, and l-alanine, l-arabitol, scyllo-inositol, 2-hydroxyphenilacetic acid, 3-hydroxybutyric acid and N-acetylneuraminic acid metabolites were found altered in response to atherosclerosis progression and the acute event, composing a molecular panel related to cardiovascular risk. KLK1 and ZG16B together with 3-hydroxybutyric acid, putrescine and 1-methylhydantoin responded at onset but also showed normalized levels at discharge, constituting a molecular panel to monitor recovery. The observed decreased of KLK1 is in alignment with the protective mechanism of the kallikrein–kinin system. The connection between KLK1 and ZG16B shown by pathway analysis explains reduced levels of toll-like receptor 2 described in atherosclerosis. Metabolomic analysis revealed arginine and proline metabolism, glutathione metabolism and degradation of ketone bodies as the three main pathways altered. In conclusion, two novel urinary panels of proteins and metabolites are here for the first time shown related to atherosclerosis, ACS and patient’s recovery.

A role for the membrane proteome in human chronic kidney disease erythrocytes.

The molecular basis of the reduced half-life of chronic kidney disease (CKD) erythrocytes is unclear. The erythrocyte membrane plays a key role in the erythrocyte mechanical properties and survival. The aim of the present work is to uncover erythrocyte membrane proteins whose expression could be altered in CKD. The erythrocyte membrane subproteome was analyzed by a non-biased approach where the whole set of proteins was simultaneously investigated by 2D fluorescence difference gel electrophoresis without preselection of potential targets. Proteins significantly altered in CKD were identified by mass spectrometry (MS) and results validation was performed by Western blot and confocal microscopy. Nine differentially expressed spots among healthy individuals, non-dialyzed CKD and erythropoietin/dialysis-treated CKD patients were identified by MS/MS corresponding to 5 proteins (beta-adducin, HSP71/72, tropomodulin-1, ezrin, and radixin). Ezrin and radixin were higher in dialyzed CKD patients than in the other 2 groups. Beta-adducin was increased in CKD patients (dialyzed or not). Three spots were normalized in patients on the dialysis/erythropoietin combination compared with non-dialyzed CKD. Among these, a spot corresponding to tropomodulin 1, was found to be of higher abundance in non-dialyzed CKD patients compared with controls or dialyzed CKD. In conclusion, this study identifies changes in erythrocyte membrane proteins in CKD, which may be relevant for the pathogenesis of red cell abnormalities in uremia.

Watermelon Profilin: Characterization of a Major Allergen as a Model for Plant-Derived Food Profilins

Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.

Molecular and immunological characterization of the first allergenic lipocalin in hamster: the major allergen from Siberian hamster (Phodopus sungorus).

Background: Exposure to uncommon pets such as a Siberian hamster has been described as a new risk factor for the development of allergic disease. Results: For the first time, we have cloned and characterized an allergen from Siberian hamster. Conclusion: The recombinant allergen shows a similar pattern of IgE binding and biological activity compared with salivary gland extract. Significance: The recombinant form will be useful for clinical applications. The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster.

Detection of major food allergens in amniotic fluid: initial allergenic encounter during pregnancy.

Ingestion of food allergens present in maternal milk during breastfeeding has been hypothesized as a gateway to sensitization to food; however, this process could develop during pregnancy, as the maternal–fetal interface develops a Th2‐ and Treg‐mediated environment to protect the fetus. We hypothesized that in these surroundings, unborn children are exposed to food allergens contained in the mothers diet, possibly giving rise to first sensitization.