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.

Urinary metabolic signatures reflect cardiovascular risk in the young, middle-aged, and elderly populations.

The predictive value of traditional cardiovascular risk estimators is limited, and young and elderly populations are particularly underrepresented. We aimed to investigate the urine metabolome and its association with cardiovascular risk to identify novel markers that might complement current estimators based on age. Urine samples were collected from 234 subjects categorized into three age-grouped cohorts: 30-50 years (cohort I, young), 50-70 years (cohort II, middle-aged), and > 70 years (cohort III, elderly). Each cohort was further classified into three groups: (a) control, (b) individuals with cardiovascular risk factors, and (c) those who had a previous cardiovascular event. Novel urinary metabolites linked to cardiovascular risk were identified by nuclear magnetic resonance in cohort I and then evaluated by target mass spectrometry quantification in all cohorts. A previously identified metabolic fingerprint associated with atherosclerosis was also analyzed and its potential risk estimation investigated in the three aged cohorts. Three different metabolic signatures were identified according to age: 2-hydroxybutyrate, gamma-aminobutyric acid, hypoxanthine, guanidoacetate, oxaloacetate, and serine in young adults; citrate, cyclohexanol, glutamine, lysine, pantothenate, pipecolate, threonine, and tyramine shared by middle-aged and elderly adults; and trimethylamine N-oxide and glucuronate associated with cardiovascular risk in all three cohorts. The urinary metabolome contains a metabolic signature of cardiovascular risk that differs across age groups. These signatures might serve to complement existing algorithms and improve the accuracy of cardiovascular risk prediction for personalized prevention. KEY MESSAGES: • Cardiovascular risk in the young and elderly is underestimated. • The urinary metabolome reflects cardiovascular risk across all age groups. • Six metabolites constitute a metabolic signature of cardiovascular risk in young adults. • Middle-aged and elderly adults share a cardiovascular risk metabolic signature. • TMAO and glucuronate levels reflect cardiovascular risk across all age groups.

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.

Urine Haptoglobin and Haptoglobin-Related Protein Predict Response to Spironolactone in Patients With Resistant Hypertension.

Resistant hypertension prevalence is progressively increasing, and prolonged exposure to suboptimal blood pressure control results in higher cardiovascular risk and end-organ damage. Among various antihypertensive agents, spironolactone seems the most effective choice to treat resistant hypertension once triple therapy including a diuretic fails. However success in blood pressure control is not guaranteed, adverse effects are not negligible, and no clinical tools are available to predict patient's response. Complementary to our previous study of resistant hypertension metabolism, here we investigated urinary proteome changes with potential capacity to predict response to spironolactone. Twenty-nine resistant hypertensives were included. A prospective study was conducted and basal urine was collected before spironolactone administration. Patients were classified in responders or nonresponders in terms of blood pressure control. Protein quantitation was performed by liquid chromatography-mass spectrometry; ELISA and target mass spectrometry analysis were performed for confirmation. Among 3310 identified proteins, HP (haptoglobin) and HPR (haptoglobin-related protein) showed the most significant variations, with increased levels in nonresponders compared with responders before drug administration (variation rate, 5.98 and 7.83, respectively). Protein-coordinated responses were also evaluated by functional enrichment analysis, finding oxidative stress, chronic inflammatory response, blood coagulation, complement activation, and regulation of focal adhesions as physiopathological mechanisms in resistant hypertension. In conclusion, protein changes able to predict patients' response to spironolactone in basal urine were here identified for the first time. These data, once further confirmed, will support clinical decisions on patients' management while contributing to optimize the rate of control of resistant hypertensives with spironolactone.

Identification of six cardiovascular risk biomarkers in the young population: A promising tool for early prevention.

BACKGROUND AND AIMS: The predictive value of traditional CV risk calculators is limited. Novel indicators of CVD progression are needed particularly in the young population. The main aim of this study was the identification of a molecular profile with added value to classical CV risk estimation. METHODS: Eighty-one subjects (30-50 years) were classified in 3 groups according to their CV risk: healthy subjects; individuals with CV risk factors; and those who had suffered a previous CV event. The urine proteome was quantitatively analyzed and significantly altered proteins were identified between patients' groups, either related to CV risk or established organ damage. Target-MS and ELISA were used for confirmation in independent patients' cohorts. Systems Biology Analysis (SBA) was carried out to identify functional categories behind CVD. RESULTS: 4309 proteins were identified, 75 of them differentially expressed. ADX, ECP, FETUB, GDF15, GUAD and NOTCH1 compose a fingerprint positively correlating with lifetime risk estimate (LTR QRISK). Best performance ROC curve was obtained when ECP, GDF15 and GUAD were combined (AUC = 0.96). SBA revealed oxidative stress response, dilated cardiomyopathy, signaling by Wnt and proteasome, as main functional processes related to CV risk. CONCLUSIONS: A novel urinary protein signature is shown, which correlates with CV risk estimation in young individuals. Pending further confirmation, this six-protein-panel could help in CV risk assessment.

Molecular evidence of field cancerization initiated by diabetes in colon cancer patients.

The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. While several clinical studies show a higher incidence of CC and a lower survival rate in diabetics, others report no association. Our own experience indicates that diabetes does not seem to worsen the prognosis once the tumor is present. Despite this controversy, there are no wide-spectrum molecular studies that delve into the impact of T2DM-related mechanisms in colon carcinogenesis. Here, we present a transcriptomic and proteomic profiling of paired tumor and normal colon mucosa samples in a cohort of 42 CC patients, 23 of which have T2DM. We used gene set enrichment and network approaches to extract relevant pathways in diabetics, referenced them to current knowledge, and tested them using in vitro techniques. Through our transcriptomics approach, we identified an unexpected overlap of pathways overrepresented in diabetics compared to nondiabetics, in both tumor and normal mucosa, including diabetes-related metabolic and signaling processes. Proteomic approaches highlighted several cancer-related signaling routes in diabetics found only in normal mucosa, not in tumors. An integration of the transcriptome and proteome analyses suggested the deregulation of key pathways related to colon carcinogenesis which converged on tumor initiation axis TEAD/YAP-TAZ as a potential initiator of the process. In vitro studies confirmed upregulation of this pathway in nontumor colon cells under high-glucose conditions. In conclusion, T2DM associates with deregulation of cancer-related processes in normal colon mucosa adjacent to tissue which has undergone a malignant transformation. These data support that in diabetic patients, the local microenvironment in normal colon mucosa may be a factor driving field cancerization promoting carcinogenesis. Our results set a new framework to study links between diabetes and colon cancer, including a new role of the TEAD/YAP-TAZ complex as a potential driver.

A comprehensive study of calcific aortic stenosis: from rabbit to human samples.

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.

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.

Author Correction: Immune system deregulation in hypertensive patients chronically RAS suppressed developing albuminuria.

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Endothelial Regulator of Calcineurin 1 Promotes Barrier Integrity and Modulates Histamine-Induced Barrier Dysfunction in Anaphylaxis.

Anaphylaxis, the most serious and life-threatening allergic reaction, produces the release of inflammatory mediators by mast cells and basophils. Regulator of calcineurin 1 (Rcan1) is a negative regulator of mast-cell degranulation. The action of mediators leads to vasodilation and an increase in vascular permeability, causing great loss of intravascular volume in a short time. Nevertheless, the molecular basis remains unexplored on the vascular level. We investigated Rcan1 expression induced by histamine, platelet-activating factor (PAF), and epinephrine in primary human vein (HV)-/artery (HA)-derived endothelial cells (ECs) and human dermal microvascular ECs (HMVEC-D). Vascular permeability was analyzed in vitro in human ECs with forced Rcan1 expression using Transwell migration assays and in vivo using Rcan1 knockout mice. Histamine, but neither PAF nor epinephrine, induced Rcan1-4 mRNA and protein expression in primary HV-ECs, HA-ECs, and HMVEC-D through histamine receptor 1 (H1R). These effects were prevented by pharmacological inhibition of calcineurin with cyclosporine A. Moreover, intravenous histamine administration increased Rcan1 expression in lung tissues of mice undergoing experimental anaphylaxis. Functional in vitro assays showed that overexpression of Rcan1 promotes barrier integrity, suggesting a role played by this molecule in vascular permeability. Consistent with these findings, in vivo models of subcutaneous and intravenous histamine-mediated fluid extravasation showed increased response in skin, aorta, and lungs of Rcan1-deficient mice compared with wild-type animals. These findings reveal that endothelial Rcan1 is synthesized in response to histamine through a calcineurin-sensitive pathway and may reduce barrier breakdown, thus contributing to the strengthening of the endothelium and resistance to anaphylaxis. These new insights underscore its potential role as a regulator of sensitivity to anaphylaxis in humans.

Urine metabolomics insight into acute kidney injury point to oxidative stress disruptions in energy generation and H2S availability.

Acute kidney injury (AKI) is one of the main complications in acute care medicine and a risk factor for chronic kidney disease (CKD). AKI incidence has increased; however, its diagnosis has limitations and physiopathological mechanisms are underexplored. We investigated urine samples, aiming to identify major metabolite changes during human AKI evolution. Metabolic signatures found were further explored for a potential link to severity of injury. Twenty-four control subjects and 38 hospitalized patients with AKI were recruited and urine samples were collected at the time of diagnosis, during follow-up and at discharge. Nuclear magnetic resonance (NMR) was used in a first discovery phase for identifying potential metabolic differences. Target metabolites of interest were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS) in an independent group. Underlying metabolic defects were further explored by kidney transcriptomics of murine toxic AKI. Urinary 2-hydroxybutyric acid, pantothenic acid, and hippuric acid were significantly downregulated and urinary N-acetylneuraminic acid, phosphoethanolamine, and serine were upregulated during AKI. Hippuric acid, phosphoethanolamine, and serine showed further downregulation/upregulation depending on the metabolite in acute tubular necrosis (ATN) AKI compared to prerenal AKI. Kidney transcriptomics disclosed decreased expression of cystathionase, cystathionine-ß-synthase, and ethanolamine-phosphate cytidylyltransferase, and increased N-acetylneuraminate synthase as the potentially underlying cause of changes in urinary metabolites. A urinary metabolite panel identified AKI patients and provided insight into intrarenal events. A urine fingerprint made up of six metabolites may be related to pathophysiological changes in oxidative stress, energy generation, and H2S availability associated with AKI. KEY MESSAGES: The urinary metabolome reflects AKI evolution and severity of injury. Kidney transcriptomics revealed enzymatic expression changes. Enzymatic expression changes may be the potentially underlying cause of changes in urine metabolites. Identified metabolite changes link oxidative stress, energy generation, and H2S availability to AKI.

Citric Acid Metabolism in Resistant Hypertension: Underlying Mechanisms and Metabolic Prediction of Treatment Response.

Resistant hypertension (RH) affects 9% to 12% of hypertensive adults. Prolonged exposure to suboptimal blood pressure control results in end-organ damage and cardiovascular risk. Spironolactone is the most effective drug for treatment, but not all patients respond and side effects are not negligible. Little is known on the mechanisms responsible for RH. We aimed to identify metabolic alterations in urine. In addition, a potential capacity of metabolites to predict response to spironolactone was investigated. Urine was collected from 29 patients with RH and from a group of 13 subjects with pseudo-RH. For patients, samples were collected before and after spironolactone administration and were classified in responders (n=19) and nonresponders (n=10). Nuclear magnetic resonance was applied to identify altered metabolites and pathways. Metabolites were confirmed by liquid chromatography-mass spectrometry. Citric acid cycle was the pathway most significantly altered (P<0.0001). Metabolic concentrations were quantified and ranged from ng/mL malate to µg/mL citrate. Citrate and oxaloacetate increased in RH versus pseudoresistant. Together with α-ketoglutarate and malate, they were able to discriminate between responders and nonresponders, being the 4 metabolites increased in nonresponders. Combined as a prediction panel, they showed receiver operating characteristiccurve with area under the curve of 0.96. We show that citric acid cycle and deregulation of reactive oxygen species homeostasis control continue its activation after hypertension was developed. A metabolic panel showing alteration before spironolactone treatment and predicting future response of patients is shown. These molecular indicators will contribute optimizing the rate of control of RH patients with spironolactone.

Immune system deregulation in hypertensive patients chronically RAS suppressed developing albuminuria.

Albuminuria development in hypertensive patients is an indicator of higher cardiovascular (CV) risk and renal damage. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control but it does not prevent from albuminuria development. We pursued the identification of protein indicators in urine behind albuminuria development in hypertensive patients under RAS suppression. Urine was collected from 100 patients classified in three groups according to albuminuria development: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Quantitative analysis was performed in a first discovery cohort by isobaric labeling methodology. Alterations of proteins of interest were confirmed by target mass spectrometry analysis in an independent cohort. A total of 2416 proteins and 1223 functional categories (coordinated protein responses) were identified. Immune response, adhesion of immune and blood cells, and phagocytosis were found significantly altered in patients with albuminuria compared to normoalbuminuric individuals. The complement system C3 increases, while Annexin A1, CD44, S100A8 and S100A9 proteins showed significant diminishment in their urinary levels when albuminuria is present. This study reveals specific links between immune response and controlled hypertension in patients who develop albuminuria, pointing to potential protein targets for novel and future therapeutic interventions.

Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria.

Albuminuria is an indicator of cardiovascular risk and renal damage in hypertensive individuals. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control and prevents development of new-onset-albuminuria. A significant number of patients, however, develop albuminuria despite chronic RAS blockade, and the physiopathological mechanisms are underexplored. Urinary exosomes reflect pathological changes taking place in the kidney. The objective of this work was to examine exosomal protein alterations in hypertensive patients with albuminuria in the presence of chronic RAS suppression, to find novel clues underlying its development. Patients were followed-up for three years and were classified as: a) patients with persistent normoalbuminuria; b) patients developing de novo albuminuria; and c) patients with maintained albuminuria. Exosomal protein alterations between groups were identified by isobaric tag quantitation (iTRAQ). Confirmation was approached by target analysis (SRM). In total, 487 proteins were identified with high confidence. Specifically, 48 proteins showed an altered pattern in response to hypertension and/or albuminuria. Out of them, 21 proteins interact together in three main functional clusters: glycosaminoglycan degradation, coagulation and complement system, and oxidative stress. The identified proteins constitute potential targets for drug development and may help to define therapeutic strategies to evade albuminuria progression in hypertensive patients chronically treated.

Kalirin and CHD7: novel endothelial dysfunction indicators in circulating extracellular vesicles from hypertensive patients with albuminuria.

Despite of the great advances in anti-hypertensive therapies, many patients under Renin-Angiotensin- System (RAS) suppression develop albuminuria, which is a clear indicator of therapeutic inefficiency. Hence, indicators of vascular function are needed to assess patients' condition and help deciding future therapies.Proteomic analysis of circulating extracellular vesicles (EVs) showed two proteins, kalirin and chromodomain-helicase-DNA-binding protein 7 (CHD7), increased in albuminuric patients. A positive correlation of both with the expression of the endothelial activation marker E-selectin was found in EVs. In vitro analysis using TNFα-treated adult human endothelial cells proved their involvement in endothelial cell activation.Hence, we propose protein levels of kalirin and CHD7 in circulating EVs as novel endothelial dysfunction markers to monitor vascular condition in hypertensive patients with albuminuria.

2017 update on the relationship between diabetes and colorectal cancer: epidemiology, potential molecular mechanisms and therapeutic implications.

Worldwide deaths from diabetes mellitus (DM) and colorectal cancer increased by 90% and 57%, respectively, over the past 20 years. The risk of colorectal cancer was estimated to be 27% higher in patients with type 2 DM than in non-diabetic controls. However, there are potential confounders, information from lower income countries is scarce, across the globe there is no correlation between DM prevalence and colorectal cancer incidence and the association has evolved over time, suggesting the impact of additional environmental factors. The clinical relevance of these associations depends on understanding the mechanism involved. Although evidence is limited, insulin use has been associated with increased and metformin with decreased incidence of colorectal cancer. In addition, colorectal cancer shares some cellular and molecular pathways with diabetes target organ damage, exemplified by diabetic kidney disease. These include epithelial cell injury, activation of inflammation and Wnt/ß-catenin pathways and iron homeostasis defects, among others. Indeed, some drugs have undergone clinical trials for both cancer and diabetic kidney disease. Genome-wide association studies have identified diabetes-associated genes (e.g. TCF7L2) that may also contribute to colorectal cancer. We review the epidemiological evidence, potential pathophysiological mechanisms and therapeutic implications of the association between DM and colorectal cancer. Further studies should clarify the worldwide association between DM and colorectal cancer, strengthen the biological plausibility of a cause-and-effect relationship through characterization of the molecular pathways involved, search for specific molecular signatures of colorectal cancer under diabetic conditions, and eventually explore DM-specific strategies to prevent or treat colorectal cancer.

Progression of Renal Insufficiency in Patients with Essential Hypertension Treated with Renin Angiotensin Aldosterone System Blockers: An Electrocardiographic Correlation.

BACKGROUND: There is a frequent association between renal insufficiency and cardiovascular disease in patients with essential hypertension (HTN). The aim of this study was to analyze the relationship between ECG parameters and the progress of renal damage in patients with treated HTN. METHODS: 109 patients with HTN had their microalbuminuria monitored over a 3-year time frame. During the last 3 months of follow-up, an ECG was recorded. Patients were divided into 3 groups according to the deterioration of their renal function: normoalbuminuria during the study period (normo-normo; n = 51); normoalbuminuria developing microalbuminuria (normo-micro; n = 29); and microalbuminuria at baseline (micro-micro; n = 29). RESULTS: There were no differences in presence of left ventricular hypertrophy between the 3 groups. RV6/RV5 >1 was observed more frequently as renal function declined (p = 0.025). The 12-lead QRS-complex voltage-duration product was significantly increased in patients without microalbuminuria at baseline who went on to develop microalbuminuria (p = 0.006). Patients who developed microalbuminuria during follow-up, with positive Cornell voltage criteria, showed a lesser degree of progression of microalbuminuria when compared with the rest of the subgroups (p = 0.044). Furthermore, patients with microalbuminuria at baseline treated with angiotensin receptor blockers and diuretics, and positive Cornell voltage criteria, showed a higher degree of microalbuminuria compared to those with negative Cornell voltage criteria (p = 0.016). CONCLUSIONS: In patients with HTN, we identified some ECG parameters, which predict renal disease progression in patients with HTN, which may permit the identification of patients who are at risk of renal disease progression, despite optimal antihypertensive pharmacotherapy.

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.

Hypertensive patients exhibit an altered metabolism. A specific metabolite signature in urine is able to predict albuminuria progression.

Hypertension (HTN) is increasing in prevalence, and albuminuria is a strong indicator of cardiovascular risk and renal damage progression. Despite blood pressure control with chronic treatment, a relevant subgroup of patients develop albuminuria. However, the biological factors responsible for albuminuria development and progression are underexplored. We aimed to identify key metabolic targets and biological pathways involved in the negative progression of cardiovascular and renal damage in hypertensives undergoing chronic treatment. A series of 1533 patients were followed for 5 years to investigate the evolution of albuminuria. Patients were classified as: (1) patients with persistent normoalbuminuria; (2) patients developing de novo albuminuria; and (3) patients with maintained albuminuria. At the end of follow-up, urine from 30 nonhypertensive subjects (control group) and a representative cohort of 118 patients was collected for metabolomic analysis. Metabolic patterns of interest were identified in a first discovery phase by nuclear magnetic resonance and further confirmed by liquid chromatography-mass spectrometry. Metabolites corresponding to HTN or albuminuria were measured in a prospective study carried out in 35 individuals still in normoalbuminuria, to evaluate their potential as predictors of albuminuria development. Nine metabolites were significantly altered, linking ß-alanine metabolism, arginine and proline metabolism, and tricarboxylic acid cycle. The prospective study revealed a panel composed of guanidinoacetate, glutamate, and pantothenate, which was able to predict development of albuminuria. These metabolic signatures open new possibilities in hypertensive therapy and cardiovascular risk control, providing prompt and more efficient intervention, particularly in patients with worse cardiovascular prognosis.

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

BACKGROUND: Ingestion of food allergens present in maternal milk during breastfeeding has been hypothesized as a gateway to sensitization to food; however, this process could develop during pregnancy, as the maternal-fetal interface develops a Th2- and Treg-mediated environment to protect the fetus. We hypothesized that in these surroundings, unborn children are exposed to food allergens contained in the mother's diet, possibly giving rise to first sensitization. METHODS: The presence of allergens in utero was studied by analyzing amniotic fluid (AF) samples in two different stages of pregnancy: at 15-20 weeks and after delivery at term. An antibody microarray was developed to test for the most common food allergens. The array detects the presence of ten allergens from milk, fruit, egg, fish, nuts, and wheat. RESULTS: AF from 20 pregnant women was collected: eight after delivery at term and 12 from women who underwent diagnostic amniocentesis between weeks 15 and 20 of gestation. The presence of allergens was detected in all samples. Samples from amniocentesis had a higher allergen concentration than samples after delivery at term. CONCLUSIONS: We demonstrated the presence of intact major food allergens in AF samples. This early contact could explain subsequent sensitization to foods never eaten before.