Publications by authors named "Marta Martin-Lorenzo"

37 Publications

Machine learning for grading and prognosis of esophageal dysplasia using mass spectrometry and histological imaging.

Comput Biol Med 2021 Oct 4;138:104918. Epub 2021 Oct 4.

Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, 6229 ER Maastricht, The Netherlands; Department of Radiology and Nuclear Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre+, 6202 AZ, Maastricht, The Netherlands.

Background: Barrett's esophagus (BE) is a precursor lesion of esophageal adenocarcinoma and may progress from non-dysplastic through low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and cancer. Grading BE is of crucial prognostic value and is currently based on the subjective evaluation of biopsies. This study aims to investigate the potential of machine learning (ML) using spatially resolved molecular data from mass spectrometry imaging (MSI) and histological data from microscopic hematoxylin and eosin (H&E)-stained imaging for computer-aided diagnosis and prognosis of BE.

Methods: Biopsies from 57 patients were considered, divided into non-dysplastic (n = 15), LGD non-progressive (n = 14), LGD progressive (n = 14), and HGD (n = 14). MSI experiments were conducted at 50 × 50 μm spatial resolution per pixel corresponding to a tile size of 96x96 pixels in the co-registered H&E images, making a total of 144,823 tiles for the whole dataset.

Results: ML models were trained to distinguish epithelial tissue from stroma with area-under-the-curve (AUC) values of 0.89 (MSI) and 0.95 (H&E)) and dysplastic grade (AUC of 0.97 (MSI) and 0.85 (H&E)) on a tile level, and low-grade progressors from non-progressors on a patient level (accuracies of 0.72 (MSI) and 0.48 (H&E)).

Conclusions: In summary, while the H&E-based classifier was best at distinguishing tissue types, the MSI-based model was more accurate at distinguishing dysplastic grades and patients at progression risk, which demonstrates the complementarity of both approaches. Data are available via ProteomeXchange with identifier PXD028949.
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http://dx.doi.org/10.1016/j.compbiomed.2021.104918DOI Listing
October 2021

Metabolic Alterations Identified in Urine, Plasma and Aortic Smooth Muscle Cells Reflect Cardiovascular Risk in Patients with Programmed Coronary Artery Bypass Grafting.

Antioxidants (Basel) 2021 Aug 27;10(9). Epub 2021 Aug 27.

Immunoallergy and Proteomics Laboratory, Department of Immunology, IIS-Fundacion Jimenez Diaz, UAM, 28040 Madrid, Spain.

Atherosclerosis is the predominant pathology associated to premature deaths due to cardiovascular disease. However, early intervention based on a personalized diagnosis of cardiovascular risk is very limited. We have previously identified metabolic alterations during atherosclerosis development in a rabbit model and in subjects suffering from an acute coronary syndrome. Here we aim to identify specific metabolic signatures which may set the basis for novel tools aiding cardiovascular risk diagnosis in clinical practice. In a cohort of subjects with programmed coronary artery bypass grafting (CABG), we have performed liquid chromatography and targeted mass spectrometry analysis in urine and plasma. The role of vascular smooth muscle cells from human aorta (HA-VSMCs) was also investigated by analyzing the intra and extracellular metabolites in response to a pro-atherosclerotic stimulus. Statistically significant variation was considered if value < 0.05 (Mann-Whitney test). Urinary trimethylamine N-oxide (TMAO), arabitol and spermidine showed higher levels in the CVrisk group compared with a control group; while glutamine and pantothenate showed lower levels. The same trend was found for plasma TMAO and glutamine. Plasma choline, acetylcholine and valine were also decreased in CVrisk group, while pyruvate was found increased. In the secretome of HA-VSMCs, TMAO, pantothenate, glycerophosphocholine, glutathion, spermidine and acetylcholine increased after pro-atherosclerotic stimulus, while secreted glutamine decreased. At intracellular level, TMAO, pantothenate and glycerophosphocholine increased with stimulation. Observed metabolic deregulations pointed to an inflammatory response together with a deregulation of oxidative stress counteraction.
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http://dx.doi.org/10.3390/antiox10091369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8466954PMC
August 2021

TCA Cycle and Fatty Acids Oxidation Reflect Early Cardiorenal Damage in Normoalbuminuric Subjects with Controlled Hypertension.

Antioxidants (Basel) 2021 Jul 9;10(7). Epub 2021 Jul 9.

Immunology Department, IIS-Fundación Jiménez Díaz-UAM, 28040 Madrid, Spain.

Moderately increased albuminuria, defined by an albumin to creatinine ratio (ACR) > 30 mg/g, is an indicator of subclinical organ damage associated with a higher risk of cardiovascular and renal disease. Normoalbuminuric subjects are considered at no cardiorenal risk in clinical practice, and molecular changes underlying early development are unclear. To decipher subjacent mechanisms, we stratified the normoalbuminuria condition. A total of 37 hypertensive patients under chronic renin-angiotensin system (RAS) suppression with ACR values in the normoalbuminuria range were included and classified as control (C) (ACR < 10 mg/g) and high-normal (HN) (ACR = 10-30 mg/g). Target metabolomic analysis was carried out by liquid chromatography and mass spectrometry to investigate the role of the cardiorenal risk urinary metabolites previously identified. Besides this, urinary free fatty acids (FFAs), fatty acid binding protein 1 (FABP1) and nephrin were analyzed by colorimetric and ELISA assays. A Mann-Whitney test was applied, ROC curves were calculated and Spearman correlation analysis was carried out. Nine metabolites showed significantly altered abundance in HN versus C, and urinary FFAs and FABP1 increased in HN group, pointing to dysregulation in the tricarboxylic acid cycle (TCA) cycle and fatty acids β-oxidation. We showed here how cardiorenal metabolites associate with albuminuria, already in the normoalbuminuric range, evidencing early renal damage at a tubular level and suggesting increased β-oxidation to potentially counteract fatty acids overload in the HN range.
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http://dx.doi.org/10.3390/antiox10071100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301016PMC
July 2021

Early renal and vascular damage within the normoalbuminuria condition.

J Hypertens 2021 Nov;39(11):2220-2231

Immunoallergy and Proteomics Laboratory, Department of Immunology, IIS-Fundación Jiménez Díaz, UAM.

Objective: A continuous association between albuminuria and cardiorenal risk exists further below moderately increased albuminuria ranges. If only based in albumin to creatinine ratio (ACR) higher than 30 mg/g, a significant percentage of individuals may be out of the scope for therapeutic management. Despite epidemiological outcomes, the identification of biochemical changes linked to early albuminuria is underexplored, and normoalbuminuric individuals are usually considered at no risk in clinical practice. Here, we aimed to identify early molecular alterations behind albuminuria development.

Methods: Hypertensive patients under renin-angiotensin system (RAS) suppression were classified as control, (ACR < 10 mg/g) or high-normal (ACR = 10-30 mg/g). Urinary protein alterations were quantified and confirmed by untargeted and targeted mass spectrometry. Coordinated protein responses with biological significance in albuminuria development were investigated. Immunohistochemistry assays were performed in human kidney and arterial tissue to in situ evaluate the associated damage.

Results: A total of 2663 identified proteins reflect inflammation, immune response, ion transport and lipids metabolism (P value ≤ 0.01). A1AT, VTDB and KNG1 varied in high-normal individuals (P value < 0.05), correlated with ACR and associated with the high-normal condition (odds ratio of 20.76, 6.00 and 7.04 were found, respectively (P value < 0.001)). After 12 months, protein variations persist and aggravate in progressors to moderately increased albuminuria. At tissue level, differential protein expression was found in kidney from individuals with moderately increased albuminuria and atherosclerotic aortas for the three proteins, confirming their capacity to reflect subclinical organ damage.

Conclusion: Early renal and vascular damage is molecularly evidenced within the normoalbuminuria condition.
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http://dx.doi.org/10.1097/HJH.0000000000002936DOI Listing
November 2021

Proteomic and Biological Analysis of an Human Endothelial System in Response to Drug Anaphylaxis.

Front Immunol 2021 25;12:692569. Epub 2021 Jun 25.

Allergy and Inmunology Department, Instituto de Investigaciones Sanitarias (ISS)-Fundación Jiménez Díaz, Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Anaphylaxis is a life-threatening systemic hypersensitivity reaction. During anaphylaxis, mediator release by effector cells causes endothelial barrier breakdown, increasing vascular permeability and leakage of fluids, which may lead to tissue edema. Although endothelial cells (ECs) are key players in this context, scant attention has been paid to the molecular analysis of the vascular system, and further analyses of this cell type are necessary, especially in humans. The protein expression pattern of human microvascular ECs was analyzed in response to sera from anaphylactic patients (EC-anaphylaxis) and sera from non-allergic subjects (EC-control) after 2 hours of contact. Firstly, a differential quantitative proteomic analysis of the protein extracts was performed by mass spectrometry using an isobaric labeling method. Second, the coordinated behavior of the identified proteins was analyzed using systems biology analysis (SBA). The proteome of the EC-anaphylaxis system showed 7,707 proteins, of which 1,069 were found to be significantly altered between the EC-control and EC-anaphylaxis groups (p-value < 0.05). Among them, a subproteome of 47 proteins presented a high rate of change (|ΔZq| ≥ 3). This panel offers an endothelial snapshot of the anaphylactic reaction. Those proteins with the highest individual changes in abundance were hemoglobin subunits and structural support proteins. The interacting network analysis of this altered subproteome revealed that the coagulation and complement systems are the main biological processes altered in the EC-anaphylactic system. The comprehensive SBA resulted in 5,512 functional subcategories (biological processes), 57 of which were significantly altered between EC-control and EC-anaphylaxis. The complement system, once again, was observed as the main process altered in the EC system created with serum from anaphylactic patients. Findings of the current study further our understanding of the underlying pathophysiological mechanisms operating in anaphylactic reactions. New target proteins and relevant signaling pathways operating in the endothelial-serum system have been identified. Interestingly, our results offer a protein overview of the micro-EC-anaphylaxis environment. The relevance of the coagulation, fibrinolytic, contact and complement systems in human anaphylaxis is described. Additionally, the untargeted high-throughput analysis used here is a novel approach that reveals new pathways in the study of the endothelial niche in anaphylaxis.
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http://dx.doi.org/10.3389/fimmu.2021.692569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269062PMC
June 2021

Urinary Spermidine Predicts and Associates with In-Hospital Acute Kidney Injury after Cardiac Surgery.

Antioxidants (Basel) 2021 Jun 2;10(6). Epub 2021 Jun 2.

Department of Immunology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz-UAM, 28040 Madrid, Spain.

Acute Kidney Injury (AKI) affects up to 30% of the patients who undergo cardiac surgery (CVS) and is related to higher mortality. We aim to investigate molecular features associated with in-hospital AKI development and determine the predictive value of these features when analyzed preoperatively. This is a case-control study. From an initial cohort of 110 recruited subjects, a total of 60 patients undergoing cardiac surgery were included: 20 (33%) developed in-hospital AKI (CVS-AKI) and 40 did not (controls, CVS-C). Pre- and post-surgery samples were collected and a prospective study was carried out. A total of 312 serum samples and 258 urine samples were analyzed by nuclear magnetic resonance, mass spectrometry and ELISA. Six features predicted AKI development in pre-surgery samples: urinary kidney functional loss marker kidney injury molecule-1 (uKIM-1), 2-hydroxybutyric acid, 2-hydroxyphenylacetic acid, hippuric acid, phosphoethanolamine and spermidine. Two of them stood out as powerful predictors. Pre-surgery uKIM-1 levels were increased in CVS-AKI vs. CVS-C (AUC = 0.721, -value = 0.0392) and associated strongly with the outcome (OR = 5.333, -value = 0.0264). Spermidine showed higher concentration in CVS-AKI (-value < 0.0001, AUC = 0.970) and had a strong association with the outcome (OR = 69.75, -value < 0.0001). uKIM-1 and particularly spermidine predict in-hospital AKI associated with CVS in preoperative samples. These findings may aid in preventing postoperative AKI and improve prognosis of CVS.
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http://dx.doi.org/10.3390/antiox10060896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229689PMC
June 2021

Non-syndromic thoracic aortic aneurysm: cellular and molecular insights.

J Pathol 2021 Jul 24;254(3):229-238. Epub 2021 May 24.

Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain.

Thoracic aortic aneurysm (TAA) develops silently and asymptomatically and is a major cause of mortality. TAA prevalence is greatly underestimated, it is usually diagnosed incidentally, and its treatment consists mainly of prophylactic surgery based on the aortic diameter. The lack of effective drugs and biological markers to identify and stratify TAAs by risk before visible symptoms results from scant knowledge of its pathophysiological mechanisms. Here we integrate the structural impairment affecting non-syndromic non-familial TAA with the main cellular and molecular changes described so far and consider how these changes are interconnected through specific pathways. The ultimate goal is to define much-needed novel markers of TAA, and so the potential of previously identified molecules to aid in early diagnosis/prognosis is also discussed. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8251829PMC
July 2021

Spatial differentiation of metabolism in prostate cancer tissue by MALDI-TOF MSI.

Cancer Metab 2021 Jan 29;9(1). Epub 2021 Jan 29.

Department of Circulation and Medical Imaging, NTNU-Norwegian University of Science and Technology, Trondheim, Norway.

Background: Prostate cancer tissues are inherently heterogeneous, which presents a challenge for metabolic profiling using traditional bulk analysis methods that produce an averaged profile. The aim of this study was therefore to spatially detect metabolites and lipids on prostate tissue sections by using mass spectrometry imaging (MSI), a method that facilitates molecular imaging of heterogeneous tissue sections, which can subsequently be related to the histology of the same section.

Methods: Here, we simultaneously obtained metabolic and lipidomic profiles in different prostate tissue types using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI. Both positive and negative ion mode were applied to analyze consecutive sections from 45 fresh-frozen human prostate tissue samples (N = 15 patients). Mass identification was performed with tandem MS.

Results: Pairwise comparisons of cancer, non-cancer epithelium, and stroma revealed several metabolic differences between the tissue types. We detected increased levels of metabolites crucial for lipid metabolism in cancer, including metabolites involved in the carnitine shuttle, which facilitates fatty acid oxidation, and building blocks needed for lipid synthesis. Metabolites associated with healthy prostate functions, including citrate, aspartate, zinc, and spermine had lower levels in cancer compared to non-cancer epithelium. Profiling of stroma revealed higher levels of important energy metabolites, such as ADP, ATP, and glucose, and higher levels of the antioxidant taurine compared to cancer and non-cancer epithelium.

Conclusions: This study shows that specific tissue compartments within prostate cancer samples have distinct metabolic profiles and pinpoint the advantage of methodology providing spatial information compared to bulk analysis. We identified several differential metabolites and lipids that have potential to be developed further as diagnostic and prognostic biomarkers for prostate cancer. Spatial and rapid detection of cancer-related analytes showcases MALDI-TOF MSI as a promising and innovative diagnostic tool for the clinic.
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http://dx.doi.org/10.1186/s40170-021-00242-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847144PMC
January 2021

Analysis of urinary exosomal metabolites identifies cardiovascular risk signatures with added value to urine analysis.

BMC Biol 2020 12 14;18(1):192. Epub 2020 Dec 14.

Immunology Department, IIS-Fundacion Jimenez Diaz-UAM, Madrid, Spain.

Background: Subclinical atherosclerosis may result in fatal cardiovascular (CV) events, but the underlying mechanisms and molecular players leading to disease are not entirely understood. Thus, novel approaches capable of identifying the factors involved in pathological progression and providing a better understanding of the subjacent mechanisms are needed. Extracellular vesicles (EVs) have been shown to have numerous biological functions, and their metabolome has recently generated interest as a source of novel biomarkers. The metabolic content of the exosomes has been so far unexplored in cardiovascular disease (CVD), and here, we developed an analytical strategy aimed at probing urinary exosomal metabolite content and its association to CV risk.

Results: Direct analysis of the exosomes without metabolite extraction was evaluated by high-resolution magic angle spinning (H HR-MAS). Other two methodologies for the analysis of exosomal metabolites by H NMR were set up, based on methanol or organic solvents sequential extraction. The three methods were compared in terms of the number of detected signals and signal to noise ratio (S/N). The methanol method was applied to identify altered metabolites in the urinary exosomes of subjects with programmed coronary artery by-pass grafting (CABG) versus a control group. Target mass spectrometry (MS) was also performed for differential analysis. The clinical performance of exosomal metabolites of interest in CVD was investigated, and the added value of the exosomes compared to urine analysis was evaluated. Based on S/N ratio, simplicity, reproducibility, and quality of the spectrum, the methanol method was chosen for the study in CVD. A cardiometabolic signature composed by 4-aminohippuric acid, N-1-methylnicotinamide, and citric acid was identified in urinary exosomes. Directly in urine, 4-aminohippuric acid and citric acid do not show variation between groups and changes in N-1-methylnicotinamide are less pronounced, proving the added value of exosomes.

Conclusions: We set up a novel methodology to analyze metabolic alterations in urinary exosomes and identified a cardiometabolic signature in these microvesicles. This study constitutes the first evidence of a role for the exosomal metabolism in CVD and demonstrates the possibility to evaluate the urinary exosomal metabolic content by NMR and MS.
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http://dx.doi.org/10.1186/s12915-020-00924-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737341PMC
December 2020

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

J Mol Med (Berl) 2020 11 11;98(11):1603-1613. Epub 2020 Sep 11.

Department of Immunology, Immunoallergy and Proteomics Laboratory, IIS-Fundación Jiménez Díaz, UAM, Avenida Reyes Católicos 2, 28040, Madrid, Spain.

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.
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http://dx.doi.org/10.1007/s00109-020-01976-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591416PMC
November 2020

Atheroma-Specific Lipids in and Mice Using 2D and 3D Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

J Am Soc Mass Spectrom 2020 Sep 12;31(9):1825-1832. Epub 2020 Aug 12.

Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, 6200 MD Maastricht, The Netherlands.

Atherosclerosis is the major contributor to cardiovascular diseases. It is a spatially and temporally complex inflammatory disease, in which intravascular accumulation of a plethora of lipids is considered to play a crucial role. To date, both the composition and local distribution of the involved lipids have not been thoroughly mapped yet. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) enables analyzing and visualizing hundreds of lipid molecules within the plaque while preserving each lipid's specific location. In this study, we aim to identify and verify aortic plaque-specific lipids with high-spatial-resolution 2D and 3D MALDI-MSI common to high-fat-diet-fed low-density lipoprotein receptor deficient () mice and chow-fed apolipoprotein E deficient () mice, the two most widely used animal models for atherosclerosis. A total of 11 lipids were found to be significantly and specifically colocalized to the plaques in both mouse models. These were identified and belong to one sphingomyelin (SM), three lysophosphatidic acids (LPA), four lysophosphatidylcholines (LPC), two lysophosphatidylethanolamines (LPE), and one lysophosphatidylinositol (LPI). While these lysolipids and SM 34:0;2 were characteristic of the atherosclerotic aorta plaque itself, LPI 18:0 was mainly localized in the necrotic core of the plaque.
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http://dx.doi.org/10.1021/jasms.0c00070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472746PMC
September 2020

Differential metabolic profile associated with the condition of normoalbuminuria in the hypertensive population.

Nefrologia (Engl Ed) 2020 Jul - Aug;40(4):440-445. Epub 2020 Mar 3.

Laboratorio de Inmunoalergia y Proteómica, Departamento de Inmunología, IIS-Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, España; REDINREN. Electronic address:

Background And Aim: Albuminuria is an indicator of sub-clinical organ damage and a marker of cardiovascular risk and renal disease. A percentage of hypertensive patients develop albuminuria despite being under chronic suppression of the renin-angiotensin system (RAS). We previously identified urinary metabolites associated with the development of albuminuria. In this study, we searched for metabolic alterations which reflect different levels within the condition of normoalbuminuria.

Patients, Materials And Methods: Urine from 48 hypertensive patients under chronic RAS suppression was analysed. They were classified according to the albumin/creatinine ratio (ACR) into 3groups: Normoalbuminuria (<10mg/g); high-normal (10-30mg/g in men, or 20-40mg/g in women); and moderately high albuminuria (microalbuminuria, 30-200mg/g or 40-300mg/g, respectively). The metabolome was analysed by mass spectrometry and a correlation analysis was performed between altered metabolite levels and ACR.

Results: Oxaloacetate, 3-ureidopropionate, guanidoacetate and malate show significant variation between the normo and micro groups. Additionally, these metabolites are able to differentiate between patients in the normo and high-normal range. A significant correlation between metabolites and ACR was found. Observed variations point to alterations in the energy metabolism already in patients with albuminuria in the high-normal range.

Conclusions: The association between the molecular panel consisting of 3-ureidopropionate, oxaloacetate, malate and guanidoacetate and different levels of albuminuria is confirmed. A metabolic fingerprint was also identified showing variations within the condition of normoalbuminuria allowing an earlier molecular stratification of patients.
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http://dx.doi.org/10.1016/j.nefro.2019.10.007DOI Listing
September 2021

Precise co-registration of mass spectrometry imaging, histology, and laser microdissection-based omics.

Anal Bioanal Chem 2019 Sep 1;411(22):5647-5653. Epub 2019 Jul 1.

Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, Universiteitssingel 50, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

Mass spectrometry imaging (MSI) is an analytical technique for the unlabeled and multiplex imaging of molecules in biological tissue sections. It therefore enables the spatial and molecular annotations of tissues complementary to histology. It has already been shown that MSI can guide subsequent material isolation technologies such as laser microdissection (LMD) to enable a more in-depth molecular characterization of MSI-highlighted tissue regions. However, with MSI now reaching spatial resolutions at the single-cell scale, there is a need for a precise co-registration between MSI and the LMD. As proof-of-principle, MSI of lipids was performed on a breast cancer tissue followed by a segmentation of the data to detect molecularly distinct segments within its tumor areas. After image processing of the segmentation results, the coordinates of the MSI-detected segments were passed to the LMD system by three co-registration steps. The errors of each co-registration step were quantified and the total error was found to be less than 13 μm. With this link established, MSI data can now accurately guide LMD to excise MSI-defined regions of interest for subsequent extract-based analyses. In our example, the excised tissue material was then subjected to ultrasensitive microproteomics in order to determine predominant molecular mechanisms in each of the MSI-highlighted intratumor segments. This work shows how the strengths of MSI, histology, and extract-based omics can be combined to enable a more comprehensive molecular characterization of in situ biological processes.
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http://dx.doi.org/10.1007/s00216-019-01983-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704276PMC
September 2019

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

Hypertension 2019 04;73(4):794-802

From the Laboratory of Immunoallergy and Proteomics, Department of Immunology (M.M.-L., P.J.M., A.S.-H., G.A.-L.), IIS-Fundación Jiménez Díaz-UAM, Madrid, Spain.

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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.12242DOI Listing
April 2019

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

Atherosclerosis 2019 03 14;282:67-74. Epub 2019 Jan 14.

Immunoallergy and Proteomics Laboratory, Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain; REDINREN, Madrid, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.01.003DOI Listing
March 2019

Integrative Clustering in Mass Spectrometry Imaging for Enhanced Patient Stratification.

Proteomics Clin Appl 2019 01 4;13(1):e1800137. Epub 2019 Jan 4.

Maastricht MultiModal Molecular Imaging institute (M4I), Maastricht University, 6229 ER, Maastricht, The Netherlands.

Scope: In biomedical research, mass spectrometry imaging (MSI) can obtain spatially-resolved molecular information from tissue sections. Especially matrix-assisted laser desorption/ionization (MALDI) MSI offers, depending on the type of matrix, the detection of a broad variety of molecules ranging from metabolites to proteins, thereby facilitating the collection of multilevel molecular data. Lately, integrative clustering techniques have been developed that make use of the complementary information of multilevel molecular data in order to better stratify patient cohorts, but which have not yet been applied in the field of MSI.

Materials And Methods: In this study, the potential of integrative clustering is investigated for multilevel molecular MSI data to subdivide cancer patients into different prognostic groups. Metabolomic and peptidomic data are obtained by MALDI-MSI from a tissue microarray containing material of 46 esophageal cancer patients. The integrative clustering methods Similarity Network Fusion, iCluster, and moCluster are applied and compared to non-integrated clustering.

Conclusion: The results show that the combination of multilevel molecular data increases the capability of integrative algorithms to detect patient subgroups with different clinical outcome, compared to the single level or concatenated data. This underlines the potential of multilevel molecular data from the same subject using MSI for subsequent integrative clustering.
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http://dx.doi.org/10.1002/prca.201800137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590511PMC
January 2019

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

Dis Model Mech 2018 06 19;11(6). Epub 2018 Jun 19.

Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM, 45071 Toledo, Spain

The global incidence of calcific aortic stenosis (CAS) is increasing owing, in part, to a growing elderly population. The condition poses a great challenge to public health, because of the multiple comorbidities of these older patients. Using a rabbit model of CAS, we sought to characterize protein alterations associated with calcified valve tissue that can be ultimately measured in plasma as non-invasive biomarkers of CAS. Aortic valves from healthy and mild stenotic rabbits were analyzed by two-dimensional difference gel electrophoresis, and selected reaction monitoring was used to directly measure the differentially expressed proteins in plasma from the same rabbits to corroborate their potential as diagnostic indicators. Similar analyses were performed in plasma from human subjects, to examine the suitability of these diagnostic indicators for transfer to the clinical setting. Eight proteins were found to be differentially expressed in CAS tissue, but only three were also altered in plasma samples from rabbits and humans: transitional endoplasmic reticulum ATPase, tropomyosin α-1 chain and L-lactate dehydrogenase B chain. Results of receiver operating characteristic curves showed the discriminative power of the scores, which increased when the three proteins were analyzed as a panel. Our study shows that a molecular panel comprising three proteins related to osteoblastic differentiation could have utility as a serum CAS indicator and/or therapeutic target.
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http://dx.doi.org/10.1242/dmm.033423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031362PMC
June 2018

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

Sci Rep 2018 Mar 2;8(1):4154. Epub 2018 Mar 2.

Departament of Immunology, IIS-Fundacion JimenezDiaz, REDinREN, Madrid, Spain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-22185-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834532PMC
March 2018

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

J Mol Med (Berl) 2017 12 4;95(12):1399-1409. Epub 2017 Oct 4.

Department of Immunology, IIS-Fundacion Jimenez Diaz-UAM, REDinREN, Madrid, Spain.

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-β-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 HS 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 HS availability to AKI.
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http://dx.doi.org/10.1007/s00109-017-1594-5DOI Listing
December 2017

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

Hypertension 2017 11 5;70(5):1049-1056. Epub 2017 Sep 5.

From the Department of Immunology, IIS-Fundacion Jimenez Diaz, REDinREN, Madrid, Spain (M.M.-L., P.J.M., F.V., G.A.-L.); Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos SESCAM, Toledo, Spain (M.B.-M., M.G.B.); Hypertension Unit, Instituto de Investigación imas12, Hospital Universitario 12 de Octubre, Madrid, Spain (G.R.-H., J.C.P., J.S., L.M.R.); Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, United Kingdom (F.d.l.C.); Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain (F.V.); and Universidad Europea, Madrid, Spain (L.M.R.).

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 (<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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.09819DOI Listing
November 2017

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

Sci Rep 2017 08 21;7(1):8894. Epub 2017 Aug 21.

Departament of Immunology, IIS-Fundacion JimenezDiaz, REDinREN, Madrid, Spain.

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.
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http://dx.doi.org/10.1038/s41598-017-09042-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566220PMC
August 2017

Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria.

Oncotarget 2017 07;8(27):44217-44231

Department of Immunology, IIS-Fundacion Jimenez Diaz, REDinREN, Madrid, Spain.

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.
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http://dx.doi.org/10.18632/oncotarget.17787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546475PMC
July 2017

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

Transl Res 2016 12 15;178:25-37.e7. Epub 2016 Jul 15.

Departamento de Inmunologia, IIS-Fundacion Jimenez Diaz, UAM, Madrid, Spain. Electronic address:

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 β-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.
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http://dx.doi.org/10.1016/j.trsl.2016.07.003DOI Listing
December 2016

Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: predictors of patient prognosis?

Sci Rep 2016 Jan 21;6:19667. Epub 2016 Jan 21.

Department of Immunology, IIS-Fundacion Jimenez Diaz-UAM, REDinREN, Madrid, Spain.

Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients' urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient's recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation.
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http://dx.doi.org/10.1038/srep19667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726181PMC
January 2016

Urinary alpha-1 antitrypsin and CD59 glycoprotein predict albuminuria development in hypertensive patients under chronic renin-angiotensin system suppression.

Cardiovasc Diabetol 2016 Jan 16;15. Epub 2016 Jan 16.

Departamento de Inmunologia, Laboratorio de Inmunoalergia y Proteomica, IIS-Fundacion Jimenez Diaz, UAM, REDinREN, Avda Reyes Catolicos 2, 28040, Madrid, Spain.

Background: Hypertension is a multi-factorial disease of increasing prevalence and a major risk factor for cardiovascular mortality even in the presence of adequate treatment. Progression of cardiovascular disease (CVD) occurs frequently during chronic renin-angiotensin-system (RAS) suppression, and albuminuria is a marker of CV risk. High prevalence of albuminuria in treated hypertensive patients has been demonstrated, but there are no available markers able to predict evolution. The aim of this study was the identification of novel indicators of albuminuria progression measurable in urine of diabetic and non-diabetic patients.

Methods: 1143 hypertensive patients under chronic treatment were followed for a minimum period of 3 years. Among them, 105 diabetic and non-diabetic patients were selected and classified in three groups according to albuminuria development during follow-up: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Differential urine analysis was performed by 2D gel electrophoresis (2D-DIGE) and further confirmed by liquid chromatography-mass spectrometry. Non-parametric statistical tests were applied.

Results: CD59 glycoprotein and alpha-1 antitrypsin (AAT) resulted already altered in patients developing albuminuria de novo, with a similar response in those with maintained albuminuria. A prospective study in a sub-group of normoalbuminuric patients who were clinically followed up for at least 1 year from urine sampling, revealed CD59 and AAT proteins significantly varied in the urine collected from normoalbuminurics who will negatively progress, serving as predictors of future albuminuria development.

Conclusions: CD59 and AAT proteins are significantly altered in hypertensive patients developing albuminuria. Interestingly, CD59 and AAT are able to predict, in normoalbuminuric individuals, who will develop albuminuria in the future, being potential predictors of vascular damage and CV risk. These findings contribute to early identify patients at risk of developing albuminuria even when this classical predictor is still in the normal range, constituting a novel strategy towards a prompt and more efficient therapeutic intervention with better outcome.
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http://dx.doi.org/10.1186/s12933-016-0331-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715311PMC
January 2016

Cytoskeleton deregulation and impairment in amino acids and energy metabolism in early atherosclerosis at aortic tissue with reflection in plasma.

Biochim Biophys Acta 2016 04 7;1862(4):725-732. Epub 2015 Dec 7.

Department of Immunology, IIS-Fundacion Jimenez Diaz, UAM, Madrid, Spain.

Background: Cardiovascular disease (CVD) is the leading cause of death globally, being atherosclerosis the main cause. Main risk factors are known and current effort is very much dedicated to improve prevention. However, the asymptomatic and silent course of atherosclerosis hampers an accurate and individualized risk evaluation.

Objectives: Here we investigate subjacent molecular changes taking place in arterial tissue which can be ultimately translated in a measurable fingerprint in plasma.

Methods: First, we applied a combined approach to find out main molecular alterations at protein and metabolite level in response to early atherosclerosis development in a rabbit model. A potential reflection of all these alterations observed in aortic tissue was investigated in rabbit plasma and further analyzed in a translational study in human plasma from 62 individuals.

Results: Data link the structural remodeling taking place in atherosclerotic arteries in terms of loss of contractile properties and favored cellular migration, with an up-regulation of integrin linked kinase, tropomyosin isoform 2 and capping protein gelsolin-like, and a down-regulation of vinculin. A molecular response to oxidative stress is evidenced, involving changes in the glucose metabolism enzymes pyruvate kinase (PKM) and phosphoglycerate kinase (PGK), and pyruvate. Up-regulation of aspartate connects different changes observed in amino acid metabolism and, additionally, alterations in the phosphatidylcholine route of the glycerophospholipid metabolism were found.

Conclusions: A specific molecular marker panel composed by PKM, valine and pyruvate is shown here linked to cardiovascular risk.
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http://dx.doi.org/10.1016/j.bbadis.2015.12.006DOI Listing
April 2016

Molecular histology of arteries: mass spectrometry imaging as a novel ex vivo tool to investigate atherosclerosis.

Expert Rev Proteomics 2016 4;13(1):69-81. Epub 2015 Dec 4.

a Department of Immunology , IIS-Fundacion Jimenez Diaz, UAM , Madrid , Spain.

Atherosclerosis is usually the underlying cause of a fatal event such as myocardial infarction or ictus. The atherome plaque develops silently and asymptomatically within the arterial intima layer. In this context, the possibility to analyze the molecular content of arterial tissue while preserving each molecule's specific localization is of great interest as it may reveal further insights into the physiopathological changes taking place. Mass spectrometry imaging (MSI) enables the spatially resolved molecular analysis of proteins, peptides, metabolites, lipids and drugs directly in tissue, with a resolution sufficient to reveal molecular features specific to distinct arterial structures. MSI represents a novel ex vivo imaging tool still underexplored in cardiovascular diseases. This review focuses on the MSI technique applied to cardiovascular disease and covers the main contributions to date, ongoing efforts, the main challenges and current limitations of MSI.
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http://dx.doi.org/10.1586/14789450.2016.1116944DOI Listing
October 2016

KLK1 and ZG16B proteins and arginine-proline metabolism identified as novel targets to monitor atherosclerosis, acute coronary syndrome and recovery.

Metabolomics 2015;11(5):1056-1067. Epub 2014 Dec 14.

Department of Immunology, IIS-Fundacion Jimenez Diaz, UAM, REDinREN, Avenida Reyes Católicos 2, 28040 Madrid, Spain.

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 -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.
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http://dx.doi.org/10.1007/s11306-014-0761-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573654PMC
December 2014

Lipid and protein maps defining arterial layers in atherosclerotic aorta.

Data Brief 2015 Sep 24;4:328-31. Epub 2015 Jun 24.

Department of Immunology, IIS-Fundacion Jimenez Diaz, UAM, REDinREN, Madrid, Spain.

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].
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http://dx.doi.org/10.1016/j.dib.2015.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510571PMC
September 2015

Molecular anatomy of ascending aorta in atherosclerosis by MS Imaging: Specific lipid and protein patterns reflect pathology.

J Proteomics 2015 Aug 12;126:245-51. Epub 2015 Jun 12.

Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDinREN, Madrid, Spain. Electronic address:

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 β4 (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.
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http://dx.doi.org/10.1016/j.jprot.2015.06.005DOI Listing
August 2015
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