The scaffold nucleoporins SAR1 and SAR3 are essential for proper meiotic progression in Arabidopsis thaliana.

Nuclear Pore Complexes (NPCs) are embedded in the nuclear envelope (NE), regulating macromolecule transport and physically interacting with chromatin. The NE undergoes dramatic breakdown and reformation during plant cell division. In addition, this structure has a specific meiotic function, anchoring and positioning telomeres to facilitate the pairing of homologous chromosomes. To elucidate a possible function of the structural components of the NPCs in meiosis, we have characterized several Arabidopsis lines with mutations in genes encoding nucleoporins belonging to the outer ring complex. Plants defective for either SUPPRESSOR OF AUXIN RESISTANCE1 (SAR1, also called NUP160) or SAR3 (NUP96) present condensation abnormalities and SPO11-dependent chromosome fragmentation in a fraction of meiocytes, which is increased in the double mutant sar1 sar3. We also observed these meiotic defects in mutants deficient in the outer ring complex protein HOS1, but not in mutants affected in other components of this complex. Furthermore, our findings may suggest defects in the structure of NPCs in sar1 and a potential link between the meiotic role of this nucleoporin and a component of the RUBylation pathway. These results provide the first insights in plants into the role of nucleoporins in meiotic chromosome behavior.

Effects of Growth Hormone Treatment and Rehabilitation in Incomplete Chronic Traumatic Spinal Cord Injury: Insight from Proteome Analysis.

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.

Comprehensive Proteomic Profiling of Pressure Ulcers in Patients with Spinal Cord Injury Identifies a Specific Protein Pattern of Pathology.

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.

A clinical perspective on the utility of alpha 1 antichymotrypsin for the early diagnosis of calcific aortic stenosis.

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.

Proteomic characterization of human coronary thrombus in patients with ST-segment elevation acute myocardial infarction.

Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombosis and results in total occlusion of the coronary artery. To date, characterization of the coronary thrombus proteome in STEMI patients has not been yet accomplished. Therefore, we aimed to perform an in-depth proteomic characterization of the human coronary thrombus by means of three different approaches: 2-DE followed by mass spectrometry (MALDI MS/MS), 1-DE combined either with liquid chromatography coupled to mass spectrometry in a MALDI TOF/TOF (LC-MALDI-MS/MS), or in a LTQ-Orbitrap (LC-ESI-MS/MS). This approach allowed us to identify a total of 708 proteins in the thrombus. Expression in coronary thrombi (n=20) of 14 proteins was verified, and the expression of fibrin and 6 cell markers (platelets, monocytes, neutrophils, eosinophils, T-cells and B-cells) quantified by selected reaction monitoring (SRM). A positive correlation of 5 proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) with CD41 was found, pointing out the potential activation of a focal adhesion pathway within thrombus platelets. DIDO1 protein was found to correlate negatively with thrombus fibrin, and was found up-regulated in the plasma of these STEMI patients, which may constitute a starting point for further analyses in the search for biomarkers of thrombosis. BIOLOGICAL SIGNIFICANCE: The proteomic characterization of the human coronary thrombus may contribute to a better understanding of the mechanisms involved in acute coronary syndrome, and thus pave the road for the identification of new therapeutic targets that may help addressing this and other thrombotic diseases. A novel methodology to characterize thrombus composition and expression of a sub-group of proteins is hereby described, which allowed linking protein expression with cellular and ECM matrix composition of the thrombus. Five proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) co-express within the human coronary thrombus with CD41, pointing out the potential activation of a focal adhesion pathway within thrombus platelets during thrombus formation. Besides, the protein death-inducer obliterator 1, found to be expressed within the human coronary thrombus, has been proved to increase in the plasma of STEMI patients, which constitutes an important starting point for further analyses in the search for biomarkers of thrombosis.

Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

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.

Secretome of human aortic valves.

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Between different proteomics studies, the secretome is a valuable tool in the search for biomarkers locally released by a studied tissue and remains particularly important while working with vascular tissues, since the secreted proteins would be probably shed to the blood. In this chapter, we described a method to validate the origin of secreted proteins in human aortic valves, demonstrating their synthesis and release by the tissue and ruling out blood origin.

A comparative study of immunodepletion and equalization methods for aortic stenosis human plasma.

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. Until now, aortic stenosis (AS) was thought to result from aging and "wear and tear" of the aortic valve, but nowadays, it is known that it presents the same risk factors as atherosclerosis and cardiovascular diseases.A proteomic analysis of plasma could permit to identify the changes in protein expression induced by AS in this biological sample. However, the characterization of human plasma proteome is a very complicated task, due to the wide dynamic range of concentration that separates the most abundant proteins and the less common ones (10-12 orders of magnitude). For this reason, plasma analysis requires pre-fractionation methods, and several such techniques are currently used to deplete albumin and other abundant plasma proteins.In this work we describe two different and optimized protocols to decrease the plasma proteome complexity for proteomic analysis. With this, comprehensive and systematic characterization of the plasma proteome in the healthy and diseased aortic stenosis (AS) state will greatly facilitate the development of "useful" biomarkers for early disease detection, clinical diagnosis, and therapy.

Potential blood biomarkers for stroke.

Stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Despite advances in research during the last decade, prevention and treatment strategies still suffer from significant limitations, and therefore new theoretical and technical approaches are required. Technological advances in the proteomic and metabolomic areas, during recent years, have permitted a more effective search for novel biomarkers and therapeutic targets that may allow for effective risk stratification and early diagnosis with subsequent rapid treatment. This review provides a comprehensive overview of the latest candidate proteins and metabolites proposed as new potential biomarkers in stroke.

Proteomic profile of human aortic stenosis: insights into the degenerative process.

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.

Inside human aortic stenosis: a proteomic analysis of plasma.

Valvular aortic stenosis (AS) produces a slowly progressive obstruction in left ventricular outflow track. For this reason, aortic valve replacement is warranted when the valvular stenosis is hemodinamically significant, becoming the most common worldwide cause of aortic valve surgery. Recent epidemiologic studies have revealed an association between degenerative AS and cardiovascular risk factors for atherosclerosis, althought reducing the exposure to such factors and statin therapies both fail to delay or reverse the pathology. Hence, a deeper understanding of the pathophysiology of this disease is required to identify appropriate preventive measures. A proteomic analysis of plasma will permit to know and identify the changes in protein expression induced by AS in this tissue. Using two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry (MS), we compared the crude (not pre-fractioned) and pre-fractioned plasma from AS patients and control subjects. We sought to identify plasma proteins whose expression is modified in AS. In addition we investigated if crude plasma presented some alterations in the more abundant proteins since to date, has never been studied before. We also further investigated the link between this disease and atherosclerosis with a view to identifying new potential markers and therapeutic targets.

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.

Valvular aortic stenosis: a proteomic insight.

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.

Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves.

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.

Obtención de un protocolo óptimo para el análisis proteómico de válvulas aórticas humanas sanas y estenóticas / Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves

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)

Tissue proteomics in atherosclerosis: elucidating the molecular mechanisms of cardiovascular diseases.

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).