[The impact of COVID-19 in patients with severe aortic stenosis: Artificial intelligence research]. / Impacto del COVID-19 en pacientes con estenosis aórtica severa: análisis basado en inteligencia artificial.

Introduction: Untreated, severe, symptomatic aortic stenosis is associated with an ominous diagnosis without intervention. This study aims to determine the impact of the COVID-19 pandemic on the mortality of patients with severe stenosis during the first wave and compare it with the same period last year. Methods: All patients who went to the hospitals in an Spanish region during the first wave, and in the same period of previous year, were analyzed using artificial intelligence-based software, evaluating the mortality of patients with severe aortic stenosis with and without COVID-19 during the pandemic and the pre-COVID era. Mortality of the 3 groups was compared. Regarding cardiac surgeries was a tendency to decrease (P = .07) in patients without COVID-19 between the pandemic and the previous period was observed. A significant decrease of surgeries between patients with COVID-19 and without COVID-19 was shown. Results: Data showed 13.82% less admitted patients during the first wave. A total of 1,112 of them had aortic stenosis and 5.48% were COVID-19 positive. Mortality was higher (P = .01), in COVID-19 negative during the pandemic (4.37%) versus those in the pre-COVID-19 era (2.57%); it was also in the COVID-19 positive group (11.47%), versus COVID-19 negative (4.37%) during the first wave (P = .01). Conclusions: The study revealed a decrease in patients who went to the hospital and an excess of mortality in patients with severe aortic stenosis without infection during the first wave, compared to the same period last year; and also, in COVID-19 positive patients versus COVID-19 negative.

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.

Albumin Redox Modifications Promote Cell Calcification Reflecting the Impact of Oxidative Status on Aortic Valve Disease and Atherosclerosis.

Calcific aortic valve disease (CAVD) and coronary artery disease (CAD) are related cardiovascular diseases in which common mechanisms lead to tissue calcification. Oxidative stress plays a key role in these diseases and there is also evidence that the redox state of serum albumin exerts a significant influence on these conditions. To further explore this issue, we used multimarker scores (OxyScore and AntioxyScore) to assess the global oxidative status in patients with CAVD, with and without CAD, also evaluating their plasma thiol levels. In addition, valvular interstitial cells were treated with reduced, oxidized, and native albumin to study how this protein and its modifications affect cell calcification. The differences we found suggest that oxidative status is distinct in CAVD and CAD, with differences in redox markers and thiol levels. Importantly, the in vitro interstitial cell model revealed that modified albumin affects cell calcification, accelerating this process. Hence, we show here the importance of the redox system in the development of CAVD, emphasizing the relevance of multimarker scores, while also offering evidence of how the redox state of albumin influences vascular calcification. These data highlight the relevance of understanding the overall redox processes involved in these diseases, opening the door to new studies on antioxidants as potential therapies for these patients.

Global Oxidative Status Is Linked to Calcific Aortic Stenosis: The Differences Due to Diabetes Mellitus and the Effects of Metformin.

Calcific aortic stenosis (CAS) and type 2 diabetes mellitus (T2DM) are related and often concomitant pathologies, accompanied by common comorbidities such as hypertension or dyslipidemia. Oxidative stress is one of the mechanisms that trigger CAS, and it can drive the vascular complications in T2DM. Metformin can inhibit oxidative stress, yet its effects have not been studied in the context of CAS. Here, we assessed the global oxidative status in plasma from patients with CAS, both alone and with T2DM (and under treatment with metformin), using multimarker scores of systemic oxidative damage (OxyScore) and antioxidant defense (AntioxyScore). The OxyScore was determined by measuring carbonyls, oxidized LDL (oxLDL), 8-hydroxy-20-deoxyguanosine (8-OHdG), and xanthine oxidase (XOD) activity. In contrast, the AntioxyScore was determined through the catalase (CAT) and superoxide dismutase (SOD) activity, as well as the total antioxidant capacity (TAC). Patients with CAS displayed enhanced oxidative stress compared to control subjects, probably exceeding their antioxidant capacity. Interestingly, patients with CAS and T2DM displayed less oxidative stress, possibly due to the benefits of their pharmacological therapy (metformin). Thus, reducing oxidative stress or enhancing antioxidant capacity through specific therapies could be a good strategy to manage CAS, focusing on personalized medicine.

Diabetes mellitus and aortic stenosis head to head: toward personalized medicine in patients with both pathologies.

Diabetes mellitus (DM) and calcific aortic stenosis (CAS) are common morbidities in the elderly, which are both chronic, progressive and often concomitant diseases. Several studies revealed that DM increases the risk of developing severe CAS, yet clear information about the relationship between both these diseases and the influence of DM on the progression of CAS is currently lacking. To evaluate the effect of DM on aortic valves and on the process of calcification, and to achieve better patient management in daily clinical practice, we analysed calcified and noncalcified valve tissue from patients with severe CAS, with or without DM. A proteomic strategy using isobaric tags was adopted and the plasma concentrations of nine proteins were studied using 3 orthogonal methods and in a separate cell model. The differentially expressed proteins identified are implicated in biological processes like endopeptidase activity, lipid metabolism, coagulation, and fibrinolysis. The results obtained provide evidence that DM provokes changes in the proteome of aortic valves, affecting valve calcification. This finding may help enhance our understanding of the pathogenesis of CAS and how DM affects the evolution of this condition, an important step in identifying targets to personalize the treatment of these patients.

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.

The impact of COVID-19 in patients with severe aortic stenosis: Artificial intelligence research.

INTRODUCTION: Untreated, severe, symptomatic aortic stenosis is associated with an ominous diagnosis without intervention. This study aims to determine the impact of the COVID-19 pandemic on the mortality of patients with severe stenosis during the first wave and compare it with the same period last year. METHODS: All patients who went to the hospitals in a spanish region during the first wave, and in the same period of previous year, were analysed using Artificial Intelligence-based software, evaluating the mortality of patients with severe aortic stenosis with and without COVID-19 during the pandemic and the pre-COVID era. Mortality of the three groups were compared. Regarding cardiac surgeries was a tendency to decrease (p = .07) in patients without COVID-19 between the pandemic and the previous period was observed. A significant decrease of surgeries between patients with COVID-19 and without COVID-19 was shown. RESULTS: Data showed 13.82% less admitted patients during the first wave. 1112 of them, had aortic stenosis and 5.48% were COVID-19 positive. Mortality was higher (p = .01), in COVID-19 negative during the pandemic (4.37%) versus those in the pre-COVID19 era (2.57%); it was also in the COVID-19 positive group (11.47%), versus covid-19 negative (4.37%) during the first wave (p = .01). CONCLUSIONS: The study revealed a decrease in patients who went to the hospital and an excess of mortality in patients with severe AD without infection during the first wave, compared to the same period last year; and also, in COVID-19 positive patients versus COVID-19 negative.

Prioritization of Candidate Biomarkers for Degenerative Aortic Stenosis through a Systems Biology-Based In-Silico Approach.

Degenerative aortic stenosis is the most common valve disease in the elderly and is usually confirmed at an advanced stage when the only treatment is surgery. This work is focused on the study of previously defined biomarkers through systems biology and artificial neuronal networks to understand their potential role within aortic stenosis. The goal was generating a molecular panel of biomarkers to ensure an accurate diagnosis, risk stratification, and follow-up of aortic stenosis patients. We used in silico studies to combine and re-analyze the results of our previous studies and, with information from multiple databases, established a mathematical model. After this, we prioritized two proteins related to endoplasmic reticulum stress, thrombospondin-1 and endoplasmin, which have not been previously validated as markers for aortic stenosis, and analyzed them in a cell model and in plasma from human subjects. Large-scale bioinformatics tools allow us to extract the most significant results after using high throughput analytical techniques. Our results could help to prevent the development of aortic stenosis and open the possibility of a future strategy based on more specific therapies.

The Influence of Coronary Artery Disease in the Development of Aortic Stenosis and the Importance of the Albumin Redox State.

Calcific aortic valve and coronary artery diseases are related cardiovascular pathologies in which common processes lead to the calcification of the corresponding affected tissue. Among the mechanisms involved in calcification, the oxidative stress that drives the oxidation of sulfur-containing amino acids such ascysteines is of particular interest. However, there are important differences between calcific aortic valve disease and coronary artery disease, particularly in terms of the reactive oxygen substances and enzymes involved. To evaluate what effect coronary artery disease has on aortic valves, we analyzed valve tissue from patients with severe calcific aortic stenosis with and without coronary artery disease. Proteins and peptides with oxidized cysteines sites were quantified, leading to the identification of 16 proteins with different levels of expression between the two conditions studied, as well as differences in the redox state of the tissue. We also identified two specific sites of cysteine oxidation in albumin that have not been described previously. These results provide evidence that coronary artery disease affects valve calcification, modifying the molecular profile of aortic valve tissue. In addition, the redox proteome is also altered when these conditions coincide, notably affecting human serum albumin.

Post-COVID Complications after Pressure Ulcer Surgery in Patients with Spinal Cord Injury Associate with Creatine Kinase Upregulation in Adipose Tissue.

The risk of complications following surgical procedures is significantly increased in patients with SARS-CoV-2 infection. However, the mechanisms underlying these correlations are not fully known. Spinal cord injury (SCI) patients who underwent reconstructive surgery for pressure ulcers (PUs) before and during the COVID-19 pandemic were included in this study. The patient's postoperative progression was registered, and the subcutaneous white adipose tissue (s-WAT) surrounding the ulcers was analyzed by proteomic and immunohistochemical assays to identify the molecular/cellular signatures of impaired recovery. Patients with SCI and a COVID-19-positive diagnosis showed worse recovery and severe postoperative complications, requiring reintervention. Several proteins were upregulated in the adipose tissue of these patients. Among them, CKMT2 and CKM stood out, and CKM increased for up to 60 days after the COVID-19 diagnosis. Moreover, CKMT2 and CKM were largely found in MGCs within the s-WAT of COVID patients. Some of these proteins presented post-translational modifications and were targeted by autoantibodies in the serum of COVID patients. Overall, our results indicate that CKMT2, CKM, and the presence of MGCs in the adipose tissue surrounding PUs in post-COVID patients could be predictive biomarkers of postsurgical complications. These results suggest that the inflammatory response in adipose tissue may underlie the defective repair seen after surgery.

Single Low-Volume Center Experience with Frozen Elephant Trunk in Acute Type A Aortic Dissections.

BACKGROUND: Acute Type A aortic dissection (AAAD) is a surgical emergency. In patients with arch and descending aorta involvement (DeBakey Type I), a total aortic arch replacement with frozen elephant trunk (FET) could favor false lumen thrombosis and improve long-term results. The authors hereby present their experience with this technique in a single low-volume center, to assess whether the technique is feasible to treat such disease. METHODS: From January 2011 to December 2016, 43 patients with AAAD were operated on in the authors' institution, which carries out 300 to 350 annual procedures. Among these, 12 patients with an intimal tear in the aortic arch and/or proximal descending aorta received a FET procedure (10 males, age 57 years). Concomitant procedures were aortic valve replacement (42%), Bentall (25%), and aortic valve repair (17%). RESULTS: Cardiopulmonary bypass, cardiac arrest, and circulatory arrest times were 235 ± 43, 171 ± 33, and 75 ± 20 minutes, respectively. The operative mortality was 16.7% (n = 2). Stroke and re-thoracotomy for bleeding occurred in 8% (n = 1) and 8% (n = 1), respectively. There was no spinal cord injury. Follow-up was 36.1 months. During follow-up, no patients died or required a reoperation on the downstream aorta. CONCLUSION: Although all patients were operated on in a low-volume center, the results with FET in AAAD are acceptable. Even though this technique demands high technical skills, it is a promising approach in patients with acute aortic dissection.

MALDI-Imaging Mass Spectrometry: a step forward in the anatomopathological characterization of stenotic aortic valve tissue.

Aortic stenosis (AS) is the most common form of valve disease. Once symptoms develop, there is an inexorable deterioration with a poor prognosis; currently there are no therapies capable of modifying disease progression, and aortic valve replacement is the only available treatment. Our goal is to study the progression of calcification by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) and get new insights at molecular level that could help in the understanding of this disease. In this work, we analyzed consecutive slices from aortic valve tissue by MALDI-IMS, to establish the spatial distribution of proteins and peptides directly from the surface of the histological sections. The analysis showed different structures corresponding to regions observed in conventional histology, including large calcification areas and zones rich in collagen and elastic fibers. Peptide extraction from the tissue, followed by liquid chromatography mass spectrometry analysis, provided the identification of collagen VI α-3 and NDRG2 proteins which correlated with the masses obtained by MALDI-IMS and were confirmed by immunohistochemistry. These results highlighted the molecular mechanism implied in AS using MALDI-IMS, a novel technique never used before in this pathology. In addition, we can define specific regions proving a complementary resolution of the molecular histology.

Patients with calcific aortic stenosis exhibit systemic molecular evidence of ischemia, enhanced coagulation, oxidative stress and impaired cholesterol transport.

BACKGROUND: The most common valve diseases are calcific aortic stenosis (AS) and aortic regurgitation (AR). The former is characterized by thickening of valve leaflets followed by progressive calcification, which produces progressive aortic valve (AV) narrowing, increased pressure afterload on the left ventricle (LV) and subsequent LV hypertrophy. On the other hand, AR is due to malcoaptation of the valve leaflets with resultant diastolic reflux of blood from aorta back to the LV producing volume and pressure overload and progressive LV dilatation. In order to isolate the molecular mechanisms taking place during AS, we have used an integrated "-omic" approach to compare plasma samples from AS and from AR patients used as controls. The final purpose of this work is to find molecular changes in response to the calcification of the AV, diminishing the effects of the AV dysfunction. METHODS AND RESULTS: Using two-dimensional difference gel electrophoresis (2D-DIGE) and gas chromatography coupled to mass spectrometry (GC-MS) in a cohort of 6 subjects, we have found differences in 24 protein spots and 19 metabolites, respectively. Among them, 7 proteins and 3 metabolites have been verificated by orthogonal techniques (SRM or turbidimetry): fibrinogen beta and gamma chain, vitronectin, apolipoprotein C-II, antithrombin III, haptoglobin, succinic acid, pyroglutamic acid and alanine. Classification according to their main function showed alterations related to coagulation, inflammation, oxidative stress, response to ischemia and lipid metabolism, defining 4 different molecular panels that characterize AS with high specificity and sensitivity. CONCLUSION: These results may facilitate management of these patients by making faster diagnostics of the disease and better understand these pathways for regulating its progression.

[Neurologic complications in the immediate postoperative period after cardiac surgery. Role of brain magnetic resonance imaging]. / Complicaciones neurológicas en el postoperatorio inmediato de la cirugía cardíaca. Aportación de la resonancia magnética cerebral.

INTRODUCTION AND OBJECTIVES: Neurologic complications still cause significant morbidity and mortality in the immediate postoperative period following cardiac surgery. Our understanding of the pathogenesis, prevention, and management of these lesions is constantly developing. MATERIAL AND METHOD: We describe neurologic complications and their course in a cardiac surgery cohort and analyze the value of brain magnetic resonance imaging (MRI), using T1-weighted, T2-weighted, and FLAIR sequences, in patients with postoperative stroke or encephalopathy in whom CT scanning revealed no abnormalities explaining their clinical condition. RESULTS: In 688 patients studied postoperatively, we observed 57 neurologic complications (8.3%): 25 strokes, 24 encephalopathies, 5 seizure disorders, 2 brain deaths, and 1 intracranial hemorrhage. Initial CT scanning failed to show significant findings in 70%. 18 patients underwent brain MRI. In all but 1 of the 11 with stroke, MRI showed areas of acute or subacute infarction (i.e., hyperintensity in FLAIR or T2-weighted sequences) in different locations, mainly in a watershed distribution. In 3 of the 4 patients with mild-to-moderate encephalopathy, MRI showed lesions similar to those previously described for stroke. In the remaining 3 patients, who had severe encephalopathy, MRI showed diffuse cortical necrosis. CONCLUSIONS: The incidence of neurologic complications in the postoperative period following cardiac surgery is significant. In a high percentage of patients, brain CT scanning may not show pathologic findings. In selected patients, MRI could help identify areas of infarction not detected by CT. These images could improve clinicians' understanding of the pathogenic, pathophysiologic, clinical, and prognostic characteristics of such neurologic complications.

Lipid and protein maps defining arterial layers in atherosclerotic aorta.

Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. The molecular anatomy of healthy and atherosclerotic tissue is pursued to identify ongoing molecular changes in atherosclerosis development. Mass Spectrometry Imaging (MSI) accounts with the unique advantage of analyzing proteins and metabolites (lipids) while preserving their original localization; thus two dimensional maps can be obtained. Main molecular alterations were investigated in a rabbit model in response to early development of atherosclerosis. Aortic arterial layers (intima and media) and calcified regions were investigated in detail by MALDI-MSI and proteins and lipids specifically defining those areas of interest were identified. These data further complement main findings previously published in J Proteomics (M. Martin-Lorenzo et al., J. Proteomics. (In press); M. Martin-Lorenzo et al., J. Proteomics 108 (2014) 465-468.) [1,2].

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.

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 ß4 (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.

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.

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.