Diabetes Mellitus and Its Implications in Aortic Stenosis Patients.

Aortic stenosis (AS) and diabetes mellitus (DM) are both progressive diseases that if left untreated, result in significant morbidity and mortality. Several studies revealed that the prevalence of DM is substantially higher in patients with AS and, thus, the progression from mild to severe AS is greater in those patients with DM. DM and common comorbidities associated with both diseases, DM and AS, increase patient management complexity and make aortic valve replacement the only effective treatment. For that reason, a better understanding of the pathogenesis underlying both these diseases and the relationships between them is necessary to design more appropriate preventive and therapeutic approaches. In this review, we provided an overview of the main aspects of the relationship between AS and DM, including common comorbidities and risk factors. We also discuss the established treatments/therapies in patients with AS and DM.

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.

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.

The plasma proteomic signature as a strategic tool for early diagnosis of acute coronary syndrome.

BACKGROUND: Acute coronary syndrome is the major cause of death in developed countries. Despite its high prevalence, there is still a strong need for new biomarkers which permit faster and more accurate diagnostics and new therapeutic drugs. The basis for this challenge lay in improving our understanding of the whole atherosclerotic process from atherogenesis to atherothrombosis. In this study, we conducted two different proteomic analyses of peripheral blood plasma from non-ST elevation acute coronary syndrome and ST elevation acute coronary syndrome patients vs healthy controls. RESULTS: Two-dimensional Fluorescence Difference in Gel Electrophoresis and mass spectrometry permitted the identification of 31 proteins with statistical differences (p < 0.05) between experimental groups. Additionally, validation by Western blot and Selected Reaction Monitoring permitted us to confirm the identification of a different and characteristic plasma proteomic signature for NSTEACS and STEACS patients. CONCLUSIONS: We purpose the severity of hypoxia as the cornerstone for explaining the differences observed between both groups.

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.

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.

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.

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.

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.

Cardiovascular Risk Stratification Based on Oxidative Stress for Early Detection of Pathology.

Aims: Current cardiovascular (CV) risk prediction algorithms are able to quantify the individual risk of CV disease. However, CV risk in young adults is underestimated due to the high dependency of age in biomarker-based algorithms. Because oxidative stress is associated with CV disease, we sought to examine CV risk stratification in young adults based on oxidative stress to approach the discovery of new markers for early detection of pathology. Results: Young adults were stratified into (i) healthy controls, (ii) subjects with CV risk factors, and (iii) patients with a reported CV event. Plasma samples were analyzed using FASILOX, a novel approach to interrogate the dynamic thiol redox proteome. We also analyzed irreversible oxidation by targeted searches using the Uniprot database. Irreversible oxidation of cysteine (Cys) residues was greater in patients with reported CV events than in healthy subjects. These results also indicate that oxidation is progressive. Moreover, we found that glutathione reductase and glutaredoxin 1 proteins are differentially expressed between groups and are proteins involved in antioxidant response, which is in line with the impaired redox homeostasis in CV disease. Innovation: This study, for the first time, describes the oxidative stress (reversible and irreversible Cys oxidation) implication in human plasma according to CV risk stratification. Conclusion: The identification of redox targets and the quantification of protein and oxidative changes might help to better understand the role of oxidative stress in CV disease, and aid stratification for CV events beyond traditional prognostic and diagnostic markers. Antioxid. Redox Signal. 35, 602-617.

Patient Management in Aortic Stenosis: Towards Precision Medicine Through Protein Analysis, Imaging and Diagnostic Tests.

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.

Novel molecular plasma signatures on cardiovascular disease can stratify patients throughout life.

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.

Recent advances in atherosclerosis-based proteomics: new biomarkers and a future perspective.

Vascular proteomics is providing two main types of data: proteins that actively participate in vascular pathophysiological processes and novel protein candidates that can potentially serve as useful clinical biomarkers. Although both types of proteins can be identified by similar proteomic strategies and methods, it is important to clearly distinguish biomarkers from mediators of disease. A particular protein, or group of proteins, may participate in a pathogenic process but not serve as an effective biomarker. Alternatively, a useful biomarker may not mediate pathogenic pathways associated with disease (i.e., C-reactive protein). To date, there are no clear successful examples in which discovery proteomics has led to a novel useful clinical biomarker in cardiovascular diseases. Nevertheless, new sources of biomarkers are being explored (i.e., secretomes, circulating cells, exosomes and microparticles), an increasing number of novel proteins involved in atherogenesis are constantly described, and new technologies and analytical strategies (i.e., quantitative proteomics) are being developed to access low abundant proteins. Therefore, this presages a new era of discovery and a further step in the practical application to diagnosis, prognosis and early action by medical treatment of cardiovascular diseases.

Potential role of new molecular plasma signatures on cardiovascular risk stratification in asymptomatic individuals.

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.

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.

iTRAQ proteomic analysis of extracellular matrix remodeling in aortic valve disease.

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.