Identificación de biomarcadores diagnósticos y terapéuticos de aneurismas de aorta abdominal mediante nuevas aproximaciones proteómicas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina, Departamento de Farmacología. Tesis realizada en el Laboratorio de Nefrología Experimental y Patología Vascular de la Fundación Jiménez Díaz. Fecha de lectura: 14 de Diciembre de 2012

Proteomic analysis of PMN identifies catalase as a protein downregulated in AAA patients. Potential implication of oxidative stress in the early phases of AAA progression

Comunicaciones a congreso

Identification of Peroxiredoxin-1 as a novel biomarker of abdominal aortic aneurysm

Comunicaciones a congreso

Identification of novel biomarkers of abdominal aortic aneurysms by 2D-DIGE and MALDI-MS from AAA-thrombus-conditioned media

In the search for novel biomarkers, noncandidate-based proteomic strategies open up new opportunities to gain a deeper insight into disease processes regarding their molecular mechanisms, the risk factors involved, and the monitoring of disease progression. To carry out these complex analyses, the combined use of gel electrophoresis with mass spectrometry (MS) represents a powerful choice. In addition, the introduction of protein dye labeling has notably improved the reliability of differential expression studies by increasing the statistical significance of the protein candidates. Here, we describe a strategy where different layers (luminal/abluminal) from the intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) patients were incubated in protein-free medium. Then, the levels of the proteins released were compared by two-dimensional differential in-gel electrophoresis (2D-DIGE) and the proteins of interest identified by MS. We consider that the use of tissue-conditioned media could offer a substantial advantage in the analytical study of biological fluids, as they provide a source of proteins to be released to the bloodstream, which could serve as potential circulating biomarkers.This chapter has been supported by the EC, FAD project (FP-7, HEALTH F2-2008-200647), the Spanish MICIN (SAF2010/21852), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035), EUS2008-03565, and Fundacion Pro CNIC.S

Effect of parathyroid-hormone-related protein on human platelet activation

A B S T R A C T Evidence suggests that PTHrP [PTH (parathyroid hormone)-related protein] can act as an inflammatory mediator in several pathological settings including cardiovascular disease. The aim of the present study was to determine whether PTHrP might be involved in human platelet activation. We used a turbidimetric method to determine platelet aggregation. The expression of PTH1R (PTH type 1 receptor) in human platelets was analysed by Western blot and flow cytometry analyses. PTHrP-(1-36) (10 −7 mol/l) by itself failed to modify the activation of platelets. However, it significantly enhanced ADP-induced platelet activation, and also increased the ability of other agonists (thrombin, collagen and arachidonic acid) to induce platelet aggregation. H89 (10 −6 mol/l) and 25 × 10 −6 mol/l Rp-cAMPS (adenosine 3 ,5 -cyclic monophosphorothioate Rp-isomer), two protein kinase A inhibitors, and 25 × 10 −9 mol/l bisindolylmaleimide I, a protein kinase C inhibitor, partially decreased the enhancing effect of PTHrP-(1-36) on ADP-induced platelet activation. Meanwhile, 10 −6 mol/l PTHrP-(7-34), a PTH1R antagonist, as well as 10 −5 mol/l PD098059, a MAPK (mitogen-activated protein kinase) inhibitor, or a farnesyltransferase inhibitor abolished this effect of PTHrP-(1-36). Moreover, 10 −7 mol/l PTHrP-(1-36) increased (2-fold over control) MAPK activation in human platelets. PTH1R was detected in platelets, and the number of platelets expressing it on their surface in patients during AMI (acute myocardial infarction) was not different from that in a group of patients with similar cardiovascular risk factors without AMI. Western blot analysis showed that total PTH1R protein levels were markedly higher in platelets from control than those from AMI patients. PTH1R was found in plasma, where its levels were increased in AMI patients compared with controls. In conclusion, human platelets express the PTH1R. PTHrP can interact with this receptor to enhance human platelet activation induced by several agonists through a MAPK-dependent mechanism

Cell Stress Proteins in Atherothrombosis

Cell stress proteins (CSPs) are a large and heterogenous family of proteins, sharing two main characteristics: their levels and/or location are modified under stress and most of them can exert a chaperon function inside the cells. Nonetheless, they are also involved in the modulation of several mechanisms, both at the intracellular and the extracellular compartments. There are more than 100 proteins belonging to the CSPs family, among them the thioredoxin (TRX) system, which is the focus of the present paper. TRX system is composed of several proteins such as TRX and peroxiredoxin (PRDX), two thiol-containing enzymes that are key players in redox homeostasis due to their ability to scavenge potential harmful reactive oxygen species. In addition to their main role as antioxidants, recent data highlights their function in several processes such as cell signalling, immune inflammatory responses, or apoptosis, all of them key mechanisms involved in atherothrombosis. Moreover, since TRX and PRDX are present in the pathological vascular wall and can be secreted under prooxidative conditions to the circulation, several studies have addressed their role as diagnostic, prognostic, and therapeutic biomarkers of cardiovascular diseases (CVDs)

Increased plasma levels of NGAL, a marker of neutrophil activation, in patients with abdominal aortic aneurysm

Objective: \ud Neutrophil gelatinase-associated lipocalin (NGAL) plasma concentrations have been associated with cardiovascular diseases. We aimed to assess the association of NGAL with abdominal aortic aneurysm (AAA).\ud \ud Methods:\ud NGAL concentrations were analyzed by Western blotting in conditioned medium of polymorphonuclear neutrophils (PMNs) from AAA patients (n = 22) and controls (n = 11), and also in aortic biopsies from AAA patients and healthy controls (n = 10). Plasma NGAL concentrations were measured by ELISA in three groups of subjects from France (n = 60), Spain (n = 75) and Australia (n = 100) and associated with AAA presence and growth.\ud \ud Results: \ud PMNs isolated from AAA patients secreted significantly greater amounts of NGAL than PMNs from controls. Luminal thrombus released large amounts of NGAL compared to abluminal AAA thrombus, AAA wall and healthy aortic media. Plasma NGAL concentrations were significantly higher in patients with AAA than controls from France [115 (78–200) vs. 94 (72–114) ng/ml, p < 0.001]. NGAL plasma concentrations in AAA patients from Spain correlated with other markers of thrombus activity (plasmin–antiplasmin complexes and D-dimer). Furthermore, a positive correlation between plasma NGAL and retrospective AAA growth (rho = 0.4, p = 0.01) was observed, which remained significant after adjusting for other risk factors. Plasma NGAL was only weakly associated with prospective growth in both Spanish and Australian patients.\ud \ud Conclusions: \ud NGAL is released by PMNs and by the luminal part of AAA thrombus. NGAL plasma levels were increased in AAA patients compared with healthy subjects and correlated with retrospective AAA growth. Further studies in larger subjects groups are needed to confirm the association between NGAL and AAA presence and growth

Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression

OBJECTIVE: Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression. We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis. METHODS AND RESULTS: PMNs from 8 AAA patients (4 large AAA >5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H(2)O(2) and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (n=47) and small (n=56) AAA patients compared with controls (n=34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H(2)O(2) levels were observed in AAA thrombus-conditioned medium compared with the media layer. CONCLUSIONS: Diminished catalase levels in circulating PMNs and plasma are observed in AAA patients, supporting an important role of oxidative stress in AAA evolution.This work was supported by the European Commission, Fighting Aneurysmal Disease project (FP-7, HEALTH F2-2008-200647), the Spanish Ministerio de Ciencia e Innovacion (SAF2010/21852), Fundacion Ramon Areces, Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035), EUS2008-03565, and Fundacion Pro CNIC.S