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

Decreased circulating Fas ligand in patients with familial combined hyperlipidemia or carotid atherosclerosis Normalization by atorvastatin

AbstractObjectivesWe sought to study whether patients with familial combined hyperlipidemia (FCH) or carotid atherosclerosis have modified circulating solubilized Fas ligand (sFasL) levels, as well as the potential modifications by atorvastatin. We also examined the effect of atorvastatin on FasL expression and sFasL release in cytokine-stimulated cultured human endothelial cells (ECs).BackgroundIn normal situations, FasL is expressed in most cells, including ECs. Proinflammatory stimuli can downregulate its expression in ECs and facilitate the vascular infiltration of inflammatory cells.MethodsWe have measured sFasL plasma levels (by ELISA) in 58 patients with FCH, 14 normocholesterolemic patients with carotid atherosclerosis, and 15 healthy volunteers. We analyzed FasL expression (by Western blot analysis) and sFasL release in cultured ECs stimulated with tumor necrosis factor (TNF)-alpha.ResultsSolubilized FasL levels were decreased in hyperlipidemic patients (49 pg/ml), as compared with healthy volunteers (123 pg/ml, p < 0.0001). Patients were randomized to atorvastatin (n = 28) or bezafibrate (n = 30) during 12 months. Atorvastatin treatment increased sFasL concentrations (111 pg/ml, p < 0.0001), reaching normal values. However, treatment with bezafibrate only marginally affected sFasL (85 pg/ml, p < 0.05). Solubilized FasL was also diminished in patients with carotid atherosclerosis (39 pg/ml), and intensive treatment with atorvastatin normalized sFasL levels (90 pg/ml, p = 0.02). Finally, atorvastatin prevented the diminution of FasL expression and sFasL release elicited by TNF-alpha in cultured ECs.ConclusionsPatients with FCH or carotid atherosclerosis have decreased circulating sFasL levels, probably indicating endothelial dysfunction, but treatment with atorvastatin restored normal blood levels. These data provide a novel effect of atorvastatin and add support for the well-known anti-inflammatory properties of statins

Searching for biomarkers of aneurysmal disease

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

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis

Background: Tumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling.However, the functional effect ofTWEAK on vascular smoothmuscle cells (VSMCs) is not completely elucidated. Methods: Next generation sequencing-based methodswere performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwellmigration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. Findings: TWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method,we found a functionalmodule involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation. Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. Interpretation: Our data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis.This work was supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias ISCiii/FEDER PI13/00395; PI16/01419; PI17/ 01495) and Spanish Biomedical Research Centre in Cardiovascular Disease (CIBERCV) and Metabolic Diseases and Diabetes (CIBERDEM). PM was supported by ISCIII Miguel Servet Program (CP16/00116). CGM was supported by Fundación Conchita Rábago. NMB and VE were supported by the Spanish Ministry of Economy and Competitiveness (Juan de la Cierva IJCI-2016-29630 and Ramón y Ramón Cajal Program RyC-2013-12880, respectively). JMM has been supported a postdoctoral fellowship fromthe American Diabetes Association (Grant 1-15-MI-03) and a postdoctoral fellowship fromthe American Heart Association

Proteomic approach in the search of new cardiovascular biomarkers

Proteomic approach in the search of new cardiovascular biomarkers With the increasing incidence of cardiovascular diseases worldwide, specifically atherosclerosis and heart failure, the search for novel biomarkers remains a priority. As opposed to complex diagnostic techniques that may not be suitable to be applied to the wider population, biomarkers are useful for population screening. The search for novel biomarkers is based on knowledge of the molecular and cellular processes that take place in the development of a specific disease. Atherosclerosis and heart failure are characterized by a long period of silent disease progression, allowing early diagnosis and the potential of early therapeutic intervention. The use of the so-called proteomic techniques allows not only protein identification but partial characterization, which includes expression and also post-translational modification of these proteins. This allows for the discovery of previously unknown proteins involved in cardiovascular diseases, including some that may be suitable to be used as biomarkers. However, to approach this issue, we have to overcome difficulties such as tissue heterogeneity (vessel wall or myocardium) and the lack of fresh human samples. We discuss the proteomic study of human plaques, secreted proteins by pathologic and normal vessel wall, and left ventricular hypertrophy as potential sources of new biologic markers of cardiovascular disease

Identification of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) as a possible biomarker of subclinical atherosclerosis

OBJECTIVES: Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. METHODS AND RESULTS: We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=-0.4; P<0.001), an index of subclinical atherosclerosis. CONCLUSIONS: These results suggest that sTWEAK could be a potential biomarker of atherosclerosis

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury

Combined Immunoglobulin Free Light Chains Are Novel Predictors of Cardiovascular Events in Patients With Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is characterised by the presence of B cells and immunoglobulins in the aortic wall, mainly in the adventitia. Kappa (κ) and lambda (λ) free light chains (FLCs) are produced from B cells during immunoglobulin synthesis. This study investigated the presence and prognostic value of combined FLCs (cFLCs or summed κ and λ) in patients with AAA. cFLCs were analysed by a turbidimetric specific assay in tissue conditioned media from AAA samples (n = 34) compared with healthy aortas (n = 34) from France and in plasma samples from patients with AAA (n = 434) and age matched controls (n = 104) selected from the Viborg Vascular (VIVA) AAA screening trial in Denmark. t test, logistic regression, and Cox regression were used to test whether plasma cFLCs serve as a marker for AAA presence and whether cFLCs were predictive of death, major adverse cardiovascular events (MACE), or major adverse lower limb events (MALE). Increased cFLC levels were detected in the AAA adventitial layer compared with the AAA medial layer and healthy media layer (13.65 ± 3.17 vs. 6.57 ± 1.01 vs. 0.49 ± 0.09 mg/L, respectively, p < .050). The upper tertile of plasma cFLCs was independently associated with AAA presence after correcting for confounders (odds ratio [OR] 7.596, 95% confidence intervals [CI] 3.117 - 18.513; p < .001). Of 434 patients with AAA, 89 (20.5%) died, 104 (24.0%) suffered MACE, and 63 (14.5%) suffered MALE, during a five year follow up. In univariable analysis, the cFLC upper tertile was associated with a higher risk of death, MACE, and MALE (p < .001 for all). After adjustment for confounders, cFLCs remained an independent predictor of all cause mortality (hazard ratio [HR] 4.310, 95% CI 2.157 - 8.609; p < .001), MACE (HR 2.153, 95% CI 1.218 - 3.804; p = .008), or MALE (HR 3.442, 95% CI 1.548 - 7.652; p = .002) for those in the upper tertile. Increased cFLCs are observed in adventitial tissue of patients with AAA, indicating local activation of B cells. Plasma cFLC levels are an independent predictor of death, MACE, and MALE in patients with AAA.This study was funded by the Spanish MINECO (PID2019- 106814RB-I00 and PGC2018-097019-B-I00), la Caixa Foundation (HR17-00247), CAM (S2017/BMD-3673), and Fondo de Investigaciones Sanitarias ISCiii-FEDER (PI19/ 00128).S