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INTRODUCTION: The application of new proteomics methods may help to identify new diagnostic/predictive molecular markers in an attempt to improve the clinical management of atherosclerosis. Areas covered: Technological advances in proteomics have enhanced its sensitivity and multiplexing capacity, as well as the possibility of studying protein interactions and tissue structure. These advances will help us better understand the molecular mechanisms at play in atherosclerosis as a biological system. Moreover, this should help identify new predictive/diagnostic biomarkers and therapeutic targets that may facilitate effective risk stratification and early diagnosis, with the ensuing rapid implementation of treatment. This review provides a comprehensive overview of the novel methods in proteomics, including state-of-the-art techniques, novel biological samples and applications for the study of atherosclerosis. Expert commentary: Collaboration between clinicians and researchers is crucial to further validate and introduce new molecular markers to manage atherosclerosis that are identified using the most up to date proteomic approaches.
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Aterosclerose/diagnóstico , Proteômica/métodos , Aterosclerose/etiologia , Humanos , Modelos Biológicos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Fatores de RiscoRESUMO
BACKGROUND: Calcific aortic stenosis (CAS) is the most common heart valve disease in the elderly, representing an important economic and social burden in developed countries. Currently, there is no way to predict either the onset or progression of CAS, emphasizing the need to identify useful biomarkers for this condition. METHODS: We performed a multi-proteomic analysis on different kinds of samples from CAS patients and healthy donors: tissue, secretome and plasma. The results were validated in an independent cohort of subjects by immunohistochemistry, western blotting and selected reaction monitoring. RESULTS: Alpha 1 antichymotrypsin (AACT) abundance was altered in the CAS samples, as confirmed in the validation phase. The significant changes observed in the amounts of this protein strongly suggest that it could be involved in the molecular mechanisms underlying CAS. In addition, our results suggest there is enhanced release of AACT into the extracellular fluids when the disease commences. CONCLUSIONS: The significant increase of AACT in CAS patients suggests it fulfils an important role in the physiopathology of this disease. These results permit us to propose that AACT may serve as a potential marker for the diagnosis of CAS, with considerable clinical value.
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One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.
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Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Proteínas da Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Peptídeo Hidrolases/metabolismo , Idoso , Estenose da Valva Aórtica/sangue , Western Blotting , Feminino , Humanos , Marcação por Isótopo , Masculino , Espectrometria de Massas , Mapas de Interação de Proteínas , Proteoma/classificação , Proteoma/metabolismo , Proteômica , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu-Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it.
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Estenose da Valva Aórtica/metabolismo , Valvas Cardíacas/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/enzimologia , Estenose da Valva Aórtica/patologia , Western Blotting , Estudos de Casos e Controles , Proteínas do Citoesqueleto/metabolismo , Feminino , Valvas Cardíacas/enzimologia , Valvas Cardíacas/patologia , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente Principal , Proteômica , Eletroforese em Gel Diferencial BidimensionalRESUMO
Aortic stenosis is the most frequent valvular disease in developed countries. It progresses from mild fibrocalcific leaflet changes to a more severe leaflet calcification at the end stages of the disease. Unfortunately, symptoms of aortic stenosis are unspecific and only appear when it is too late, complicating patients' management. The global impact of aortic stenosis is increasing due to the growing elderly population. The disease supposes a great challenge because of the multiple comorbidities of these patients. Nowadays, the only effective treatment is valve replacement, which has a high cost in both social and economic terms. For that reason, it is crucial to find potential diagnostic, prognostic and therapeutic indicators that could help us to detect this disease in its earliest stages. In this article, we comprehensively review several key observations and translational studies related to protein markers that are promising for being implemented in the clinical field as well as a discussion about the role of precision medicine in aortic stenosis.
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Despite promising advances in the medical management of spinal cord injury (SCI), there is still no available effective therapy to repair the neurological damage in patients who experience this life-transforming condition. Recently, we performed a phase II/III placebo-controlled randomized trial of safety and efficacy of growth hormone (GH) treatment in incomplete chronic traumatic spinal cord injury. The main findings were that the combined treatment of GH plus rehabilitation treatment is feasible and safe, and that GH but not placebo slightly improves the SCI individual motor score. Moreover, we found that an intensive and long-lasting rehabilitation program per se increases the functional outcome of SCI individuals. To understand the possible mechanisms of the improvement due to GH treatment (motor score) and due to rehabilitation (functional outcome), we used a proteomic approach. Here, we used a multiple proteomic strategy to search for recovery biomarkers in blood plasma with the potential to predict response to somatropin treatment and to delayed intensive rehabilitation. Forty-six patients were recruited and followed for a minimum period of 1 year. Patients were classified into two groups based on their treatment: recombinant somatropin (0.4 mg) or placebo. Both groups received rehabilitation treatment. Our strategy allowed us to perform one of the deepest plasma proteomic analyses thus far, which revealed two proteomic signatures with predictive value: (i) response to recombinant somatropin treatment and (ii) response to rehabilitation. The proteins implicated in these signatures are related to homeostasis, inflammation, and coagulation functions. These findings open novel possibilities to assess and therapeutically manage patients with SCI, which could have a positive impact on their clinical response.
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Objective: Severe pressure ulcers (PUs) do not respond to conservative wound therapy and need surgical repair. To better understand the pathogenesis and to advance on new therapeutic options, we focused on the proteomic analysis of PU, which offers substantial opportunities to identify significant changes in protein abundance during the course of PU formation in an unbiased manner. Approach: To better define the protein pattern of this pathology, we performed a proteomic approach in which we compare severe PU tissue from spinal cord injury (SCI) patients with control tissue from the same patients. Results: We found 76 proteins with difference in abundance. Of these, 10 proteins were verified as proteins that define the pathology: antithrombin-III, alpha-1-antitrypsin, kininogen-1, alpha-2-macroglobulin, fibronectin, apolipoprotein A-I, collagen alpha-1 (XII) chain, haptoglobin, apolipoprotein B-100, and complement factor B. Innovation: This is the first study to analyze differential abundance protein of PU tissue from SCI patients using high-throughput protein identification and quantification by tandem mass tags followed by liquid chromatography tandem mass spectrometry. Conclusion: Differential abundance proteins are mainly involved in tissue regeneration. These proteins might be considered as future therapeutic options to enhance the physiological response and permit cellular repair of damaged tissue.
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Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Úlcera por Pressão/metabolismo , Traumatismos da Medula Espinal/complicações , Cicatrização , Adulto , Idoso , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Úlcera por Pressão/etiologia , Úlcera por Pressão/patologia , Proteômica , Fatores de Risco , Traumatismos da Medula Espinal/patologia , Adulto JovemRESUMO
Several models are available to calculate the risk of developing cardiovascular complications in mid-life. The estimation of lifetime risk in the long-term remains an unmet clinical need. We previously identified new molecular plasma signatures for cardiovascular risk stratification in a young population (30-50-years old). The aim of the present study was to determine if the specific signature found in young population changes with age. Proteomic analysis was performed in plasma samples obtained from different age groups, middle-age (50-70-years old, n = 63) and elderly (>70-years old, n = 61), which, in turn were classified into 3 subgroups according to cardiovascular risk. Our previous results in a young population clearly showed two different proteomic signatures. Building on these findings, targeted-mass spectrometry and turbidimetry analyses were used to test these signatures in middle-age and elderly populations. This strategy identified three common proteomic signatures between young and adult patients related to cardiovascular stratification, organ damage and risk prediction. Furthermore, receiver operating characteristic analysis revealed the potential value of these novel markers for lifetime risk stratification. Our results provide new insight into altered molecular mechanisms in the pathogenesis of cardiovascular disease and, more importantly, identify novel protein panels that can stratify patients throughout life. SIGNIFICANCE: Our results revealed three common proteomic signatures between young and adult patients related to cardiovascular stratification, organ damage and risk prediction. The results obtained provide a deeper insight into the pathogenesis of CV diseases and allow the identification of novel protein panels to stratify patients according to CV risk throughout life. While current estimators calculate the risk of having a CV event considering age as the most important factor to CV disease, our results represent an alternative to traditional CV risk factors, allowing the stratification of CV risk regardless of the age. Using a combination of traditional markers and established algorithms with these findings as a future preventive strategy, could facilitate an adequate assessment of CV risk.
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Doenças Cardiovasculares , Adulto , Idoso , Biomarcadores , Doenças Cardiovasculares/diagnóstico , Humanos , Pessoa de Meia-Idade , Plasma , Proteômica , Fatores de RiscoRESUMO
Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehension of the molecular mechanisms can yield very useful information in clinics for prevention, diagnosis and recovery monitoring. Proteomics represents an ideal methodology for this purpose, as proteins constitute the effectors of the different biological processes running during pathogenesis. To date, studies in atherosclerosis have been mainly focused on the search for plasma biomarkers. However, tissue proteomics allows going deeper into tissue secretomes, arterial layers or particular cells of interest, which, in turn, constitutes a more direct approximation to in vivo operating mechanisms. The aim of this review is to report latest advances in tissue proteomics in atherosclerosis and related diseases (e.g., aortic stenosis and ischemic injury).
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Aterosclerose/metabolismo , Doenças Cardiovasculares/metabolismo , Proteômica , Animais , Aterosclerose/patologia , Doenças Cardiovasculares/patologia , HumanosRESUMO
The global incidence of calcific aortic stenosis (CAS) is increasing owing, in part, to a growing elderly population. The condition poses a great challenge to public health, because of the multiple comorbidities of these older patients. Using a rabbit model of CAS, we sought to characterize protein alterations associated with calcified valve tissue that can be ultimately measured in plasma as non-invasive biomarkers of CAS. Aortic valves from healthy and mild stenotic rabbits were analyzed by two-dimensional difference gel electrophoresis, and selected reaction monitoring was used to directly measure the differentially expressed proteins in plasma from the same rabbits to corroborate their potential as diagnostic indicators. Similar analyses were performed in plasma from human subjects, to examine the suitability of these diagnostic indicators for transfer to the clinical setting. Eight proteins were found to be differentially expressed in CAS tissue, but only three were also altered in plasma samples from rabbits and humans: transitional endoplasmic reticulum ATPase, tropomyosin α-1 chain and L-lactate dehydrogenase B chain. Results of receiver operating characteristic curves showed the discriminative power of the scores, which increased when the three proteins were analyzed as a panel. Our study shows that a molecular panel comprising three proteins related to osteoblastic differentiation could have utility as a serum CAS indicator and/or therapeutic target.
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Estenose da Valva Aórtica/patologia , Valva Aórtica/patologia , Calcinose/patologia , Idoso , Animais , Estenose da Valva Aórtica/sangue , Biomarcadores/sangue , Calcinose/sangue , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Proteômica , Curva ROC , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The evaluation of cardiovascular (CV) risk is based on equations derived from epidemiological data in individuals beyond the limits of middle age such as the Framingham and SCORE risk assessments. Lifetime Risk calculator (QRisk®), estimates CV risk throughout a subjects' lifetime, allowing those. A more aggressive and earlier intervention to be identified and offered protection from the consequences of CV and renal disease. The search for molecular profiles in young people that allow a correct stratification of CV risk would be of great interest to adopt preventive therapeutic measures in individuals at high CV risk. To improve the selection of subjects susceptible to intervention with aged between 30-50 years, we have employed a multiple proteomic strategy to search for new markers of early CV disease or reported CV events and to evaluate their relationship with Lifetime Risk. Blood samples from 71 patients were classified into 3 groups according to their CV risk (healthy, with CV risk factors and with a previously reported CV event subjects) and they were analyzed using a high through quantitative proteomics approach. This strategy allowed three different proteomic signatures to be defined, two of which were related to CV stratification and the third one involved markers of organ damage.
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Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/classificação , Sistema Cardiovascular/metabolismo , Medição de Risco/métodos , Adulto , Doenças Cardiovasculares/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Fatores de RiscoRESUMO
Degenerative aortic stenosis (AS) is the most common worldwide cause of valve replacement. The aortic valve is a thin, complex, layered connective tissue with compartmentalized extracellular matrix (ECM) produced by specialized cell types, which directs blood flow in one direction through the heart. There is evidence suggesting remodeling of such ECM during aortic stenosis development. Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms. Aortic valve samples were collected from 18 patients which underwent aortic valve replacement (50% males, mean age of 74 years) and 18 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by 2D-LC MS/MS iTRAQ methodology. The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses. These findings are substantiated by our previous results demonstrating differential ECM protein expression. The present study has demonstrated a differential ECM protein pattern in individuals with AS, therefore supporting previous evidence of a dynamic ECM remodeling in human aortic valves during AS development.
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Estenose da Valva Aórtica/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteômica , Idoso , Estenose da Valva Aórtica/patologia , Matriz Extracelular/patologia , Feminino , Humanos , MasculinoRESUMO
Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In order to identify proteins that are involved in AS, we designed a simple method for protein extraction, digestion, labeling, identification, and differential protein expression analysis of both normal and stenotic aortic valve to identify biomarkers of this pathology, which may facilitate early diagnosis and treatment.
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Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Expressão Gênica , Proteoma/metabolismo , Coloração e Rotulagem/métodos , Valva Aórtica/química , Valva Aórtica/patologia , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos , Mapeamento de Peptídeos , Proteólise , Proteoma/genética , Espectrometria de Massas em TandemRESUMO
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.
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Aterosclerose/metabolismo , Vasos Coronários/metabolismo , Matriz Extracelular/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/patologia , Células Cultivadas , Cromatografia Líquida , Vasos Coronários/química , Vasos Coronários/patologia , Eletroforese em Gel Bidimensional , Matriz Extracelular/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
INTRODUCTION AND OBJECTIVES: For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. METHODS: The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. RESULTS: We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. CONCLUSIONS: We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.
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Estenose da Valva Aórtica/genética , Protocolos Clínicos , Proteômica , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible and effective method to carry out proteomic analysis of stenotic human valves by conventional 2-DE and 2D-DIGE, minimizing the interference due to high calcium concentrations. Furthermore, the protocol permits the aortic stenosis proteome to be analysed, advancing our knowledge in this area. SUMMARY: Until recently, aortic stenosis (AS) was considered a passive process secondary to calcium deposition in the aortic valves. However, it has recently been highlighted that the risk factors associated with the development of calcified AS in the elderly are similar to those of coronary artery disease. Furthermore, degenerative AS shares histological characteristics with atherosclerotic plaques, leading to the suggestion that calcified aortic valve disease is a chronic inflammatory process similar to atherosclerosis. Nevertheless, certain data does not fit with this theory making it necessary to further study this pathology. The aim of this study is to develop an effective protein extraction protocol for aortic stenosis valves such that proteomic analyses can be performed on these structures. In the present work we have defined a rapid, reproducible and effective method to extract proteins and that is compatible with 2-DE, 2D-DIGE and MS techniques. Defining the protein profile of this tissue is an important and challenging task that will help to understand the mechanisms of physiological/pathological processes in aortic stenosis valves.
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Introducción y objetivos. Durante años, la estenosisaórtica (EA) degenerativa ha sido considerada como un proceso pasivo secundario al depósito de calcio en la válvula aórtica. Aunque no se conoce su etiología, diversos autores han señalado que comparte los mismos factores de riesgo que la enfermedad arterial coronaria. Además, se han encontrado similitudes histológicas en la válvula aórtica estenótica y la placa de ateroma, lo que ha llevado a la hipótesis de que la EA degenerativa es un proceso inflamatorio similar a la aterosclerosis. No obstante, existen algunos datos discordantes con esta teoría, lo que hace necesario profundizar en el conocimiento de esta patología. El principal objetivo de este trabajo es la puesta a punto de un protocolo de extracción proteínica eficaz paraválvulas estenóticas aórticas y válvulas aórticas sanas, compatible con la realización de estudios proteómicos. Métodos. Para el desarrollo del objetivo planteado, sehan utilizado diferentes abordajes proteómicos: electroforesis bidimensional, espectrometría de masas y técnicas complementarias. Resultados. Hemos puesto a punto una metodología, sencilla, reproducible y desarrollada en el laboratorio, que permite el análisis proteómico de la válvula aórtica humana, así como la identificación de las proteínas quelas componen. Conclusiones. Obtención de un método de extracción de proteínas sencillo, reproducible y compatible con la espectrometría de masas, que permite el análisis a gran escala del proteoma de válvulas con estenosis aórtica. Además, esta metodología aumentará de forma significativa nuestro conocimiento del proteoma valvular (AU)
Introduction and objectives. For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. Methods. The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. Results. We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. Conclusions. We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry outlarge-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome (AU)