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

Macrophage Cholesterol Efflux Downregulation Is Not Associated with Abdominal Aortic Aneurysm (AAA) Progression

Recent studies have raised the possibility of a role for lipoproteins, including high-density lipoprotein cholesterol (HDLc), in abdominal aortic aneurysm (AAA). The study was conducted in plasmas from 39 large size AAA patients (aortic diameter > 50 mm), 81 small/medium size AAA patients (aortic diameter between 30 and 50 mm) and 38 control subjects (aortic diameter 5 mm per year) in patients with small/medium size AAA. Moreover, no correlation was found between MCE capacity and the aneurysm growth rate. A multivariate Cox regression analysis revealed a significant association between lower MCE capacity with the need for surgery in all AAA patients. Nevertheless, the significance was lost when only small/medium size AAA patients were included. Our results suggest that MCE, a major HDL functional activity, is not involved in AAA progression

Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm

High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas alpha-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA.We thank Simon Bartlett for language and scientific editing. This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2016-80843-R, BIO2012-37926 and BIO2015-67580-P), Fondo de Investigaciones Sanitarias ISCiii-FEDER (PRB2) (IPT13/0001, ProteoRed, Redes RIC RD12/0042/00038 and RD12/0042/0056, Biobancos RD09/0076/00101 and CA12/00371), Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas (CIBERDEM), and FRIAT. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).S

Targeted gold-coated iron oxide nanoparticles for CD163 detection in atherosclerosis by MRI

CD163 is a membrane receptor expressed by macrophage lineage. Studies performed in atherosclerosis have shown that CD163 expression is increased at inflammatory sites, pointing at the presence of intraplaque hemorrhagic sites or asymptomatic plaques. Hence, imaging of CD163 expressing macrophages is an interesting strategy in order to detect atherosclerotic plaques. We have prepared a targeted probe based on gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody for the specific detection of CD163 by MRI. Firstly, the specificity of the targeted probe was validated in vitro by incubation of the probe with CD163(+) or (−) macrophages. The probe was able to selectively detect CD163(+) macrophages both in human and murine cells. Subsequently, the targeted probe was injected in 16 weeks old apoE deficient mice developing atherosclerotic lesions and the pararenal abdominal aorta was imaged by MRI. The accumulation of probe in the site of interest increased over time and the signal intensity decreased significantly 48 hours after the injection. Hence, we have developed a highly sensitive targeted probe capable of detecting CD163-expressing macrophages that could provide useful information about the state of the atheromatous lesionsThis work was funded by Spanish Government through a Plan Nacional (CTQ2011–27268), FEDER funds through the Fondo de Investigación Sanitaria (PI10/00072, PI13/00051, PI13/00395, PI13/00802, PI14/00883 and PI14/00386), CIBERDEM group, RETICS RD12/0042/0038, Programa Miguel Servet (CP10/00479) and cvREMOD CENIT project (CEN-20091044), the Basque Government through Etortek 2011 (IE11–301), and Fundacion Lilly, Spanish Society of Atherosclerosis, Spanish Society of Nephrology and Fundacion Renal Iñigo Alvarez de Toled

TWEAK promotes peritoneal inflammation

Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r = 0.491, p = 0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD.This work was supported by FIS PS09/00447, PI08/1564, PI10/00234, MS12/03262, FEDER funds ISCIII-RETIC REDinREN/RD06/0016, RD12/0021, Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378). Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laı´n-Entralgo/CM) to AO, Programa Estabilizacio´n Investigadores to LB-C, Miguel Servet to ABS, Sara Borrell to BS, MDSN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Aki associated with macroscopic glomerular hematuria: Clinical and pathophysiologic consequences

Hematuria is a common finding in various glomerular diseases. This article reviews the clinical data on glomerular hematuria and kidney injury, as well as the pathophysiology of hematuria-associated renal damage. Although glomerular hematuria has been considered a clinical manifestation of glomerular diseases without real consequences on renal function and long-term prognosis, many studies performed have shown a relationship between macroscopic glomerular hematuria and AKI and have suggested that macroscopic hematuria-associated AKI is related to adverse long-term outcomes. Thus, up to 25% of patients with macroscopic hematuria– associated AKI do not recover baseline renal function. Oral anticoagulation has been associated with glomerular macrohematuria–related kidney injury. Several pathophysiologic mechanisms may account for the tubular injury found on renal biopsy specimens. Mechanical obstruction by red blood cell casts was thought to play a role. More recent evidence points to cytotoxic effects of oxidative stress induced by hemoglobin, heme, or iron released from red blood cells. These mechanisms of injury may be shared with hemoglobinuria or myoglobinuria-induced AKI. Heme oxygenase catalyzes the conversion of heme to biliverdin and is protective in animal models of heme toxicity. CD163, the recently identified scavenger receptor for extracellular hemoglobin, promotes the activation of anti-inflammatory pathways, opening the gates for novel therapeutic approachesThis work was supported by FIS (Programa Miguel Servet) to J.A.M.; ISCIII and FEDER funds CP04/00060, PS09/00447, Sociedad Espa~nola de Nefrologia, ISCIII-RETIC REDinREN/RD06/ 0016, Comunidad de Madrid/FRACM/S-BIO0283/2006, Programa Intensificación Actividad Investigadora (ISCIII/) to A.O.; FIS 10/ 02668 and AITER (Asociación para el Estudio y Tratamiento de las Enfermedades Renales) to E.G. and M.P.; and ISCIII-Redes RECAVA (RD06/0014/0035) and ISCIII funds PI10/00072 and Fundacion Lilly to J.E

Galectin-1 prevents pathological vascular remodeling in atherosclerosis and abdominal aortic aneurysm

Pathological vascular remodeling is the underlying cause of atherosclerosis and abdominal aortic aneurysm (AAA). Here, we analyzed the role of galectin-1 (Gal-1), a β-galactoside-binding protein, as a therapeutic target for atherosclerosis and AAA. Mice lacking Gal-1 (Lgals1 −/− ) developed severe atherosclerosis induced by pAAV/D377Y-mPCSK9 adenovirus and displayed higher lipid levels and lower expression of contractile markers of vascular smooth muscle cells (VSMCs) in plaques than wild-type mice. Proteomic analysis of Lgals1 −/− aortas showed changes in markers of VSMC phenotypic switch and altered composition of mitochondrial proteins. Mechanistically, Gal-1 silencing resulted in increased foam cell formation and mitochondrial dysfunction in VSMCs, while treatment with recombinant Gal-1 (rGal-1) prevented these effects. Furthermore, rGal-1 treatment attenuated atherosclerosis and elastase-induced AAA, leading to higher contractile VSMCs in aortic tissues. Gal-1 expression decreased in human atheroma and AAA compared to control tissue. Thus, Gal-1-driven circuits emerge as potential therapeutic strategies in atherosclerosis and AAA. Galectin-1 plays an essential role in prevention of atherosclerosis and abdominal aortic aneurysm

TWEAK/Fn14 axis: a promising target for the treatment of cardiovascular diseases.

Cardiovascular diseases (CVD) are the first cause of mortality in Western countries. CVD include several pathologies such as coronary heart disease, stroke or cerebrovascular accident, congestive heart failure, peripheral arterial disease and aortic aneurysm, among others. Interaction between members of the tumor necrosis factor (TNF) superfamily and their receptors elicits several biological actions that could participate in CVD. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its functional receptor, fibroblast growth factor-inducible molecule 14 (Fn14), are two proteins belonging to the TNF superfamily that activate NF-κB by both canonical and non-canonical pathways and regulate several cell functions such as proliferation, migration, differentiation, cell death, inflammation, and angiogenesis. TWEAK/Fn14 axis plays a beneficial role in tissue repair after acute injury. However, persistent TWEAK/Fn14 activation mediated by blocking experiments or overexpression experiments in animal models has shown an important role of this axis in the pathological remodeling underlying CVD. In this review, we summarize the role of TWEAK/Fn14 pathway in the development of CVD, focusing on atherosclerosis and stroke and the molecular mechanisms by which TWEAK/Fn14 interaction participates in these pathologies. We also review the role of the soluble form of TWEAK as a biomarker for the diagnosis and prognosis of CVD. Finally, we highlight the results obtained with other members of the TNF superfamily that also activate canonical and non-canonical NF-κB pathway

Papel de los isoprenoides de la vía de síntesis del colesterol en el control de la muerte celular|bimplicaciones en la lesión aterosclerótica

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-12-2001La enfermedad cardiovascular es la 1' causa de mortalidad de los paises occidentales. La formación de la placa ataoscler6tica es debida a un proceso inflamatorio y proliferativo en el que están implicadas tanto células residmtes m la pared v d a r (células de miisailo liso y cc\lulas mdoteliales) amo células inflamatorias (monocitos/macr6fasos y linfocitos T). El número de células de la pared vascular esta implicado m el desarrollo y progresión de las lesiones ateroscleróticas. Existen diferentes $dores de riesgo cudiovascular entre los que se mcuenhm los lipidos. Los f8miacos hipolipemiantes y, m concreto, los inhibidons de la HMG-COA reductasa (estatinas), han demostrado una alta eficacia en reducir la mortalidad debida a eventos monarios. Sm embergo, los efectos beneficiosos de estos f8miaas no pueden SR explicados únicamente por su acción hipolipaniante y deben de taia acciones independientes de la sintesis de colesterol. Hemos observado que el tratamiento con diferentes inhibidores de la HMG-COA reductasa da lugar a la muerte celular por apoptosis de células de miisculo liso vascular m cultivo. Este efecto esta relacionado con la inhibición de la sintesis de isoprmoides y, en particular, con la inhibición de la isoprenilación de proteínas G pequeAas y con la disnmución de la expresión de la proteina proapopt&ica ücl-2, dando lugar a la activación de la via mitocaidrial de mueie celular. También, hemos eshuiido la presencia de Fas y de su ligando (LFas) m la lesión ataoscler&ica humana y hemos observado que estas dos proteinas proapopt&icas se expresan preferentemente en la una con mayor predisposición a la raiaa, los hombros. En un modelo de ataoszlerosis experimental hemos observado que el tratamimto con un inhibidor de la HMG-CoA reductasa, atorvastatina, reduce la presaicia de Fas y de LFas en la lesión. Además, el tratamimto con atorvastatina disnmuye la expresión de LFas en células T activadas in vitro, efecto relacionado mn la disminución de la sintesis de isqñaioides y con la inhibición de la isoprmilación proteica, jugando un papel principal la proteína O pequdía RhoA. Esta reducción se correlaciona con la disminución de la citotoxicidad de estas dlulas sobre células diana sensibles a LFas. Por lo tanto, los inhibidores de la HMG-COA reductasa, a través de la inhibición de la isoprenilacih proteica, sai capaces de regular la muerte celular de diferentes células presentes en la lesión ateroscIer&ica. La celularidad de la lesión vascular parece ser clave m el desarrollo y progresión de la placa de aterana y su control puede ayudar a mantena la ectabilidad de las lesiones ataoscleróticas.Cardiovamilar disease is the main cause of mortality in Westem countries. The athaosclerotic plaque formation is due to an inflammatory and proliferative pr- involving residmt cells in vamilar wall (smooth muscle mlls and mdothelial cells) as well as inflammatory cells (monocyies/mrurophages and T lymphocytes). The number of cells in the vascular wall is involved in the development and progression of atherosclerotic lesions. There are several cardiovascular risk factm and ammg them the lipids have a prehinant role. Hypolipemic dmgs su& as the HMG-COA redudase inhibitors (statins) have shown to be eñcacious in reducing morfnliíy due to cardiovaseular evmts. Howeva, the beneñcial effeds of these dnigs can not only be explained by thek hypolipemic acticm brt also by sane adion indcpendeit of diolesterol syníhesis. We have okrved that aeahnmt of cultured vascular smooth muscle cells with differait HMG-COA reductase inhibitors induces cell death by apoptosis. This effect is related to the inhibition of the isoprenoid synthesis and particularly with the inhibition of the isoprenylation of small G proteins and with the decrease in the proaPoprotic ücl-2 protein expression, l d m g to mitochondrial cell death activation. We have also studied the presence of Fas and its ligand (Fasi.) in the human athaosclerotic lesion and we have obsaved that these two latter proapoptotic patesis are mamly expresstd in areas with maja predisposition to niptilyr Iike the shwldas. In a model of experimental athaosclaosis we have obsaved that treatment with an HMG-CoA reductase inhibita, atorvasiatin, reduces the presence of Fas and FasL m the lesion. Furthmae, beahnent with a t d dar esss LFas expnssion m T cdls in vitro. This afst is related to isoprmoids synthesis and the inhibitim of protein isoprmylation whae the snall G protem RhoA plays an importaat role. This reduction is mlated with the daresse of cyiotoUciíy of &ese cells m targei cells saisitive to FasL. Thus, the HMG-CoA red- inhibitarg thrwgh the inhibitim ofprotein isoprmylation are eble to regulate the apoptosis of different cells prwent in the athaosclerotic lesion. The cellulariiy of the vascular lesion ~ a n tso have a key role in íhe development and progressicm of the athaanatous plaque and its control may help to maintain the stability of the athaosclerotic lesions

Proteomic strategies in the search of new biomarkers in atherothrombosis

Extensive research has focused on the identification of novel plasma biomarkers to improve our ability to predict cardiovascular events in atherothrombosis. However, classical techniques can only assess a limited number of proteins at a time. Given that plasma contains more than 900,000 proteins, this approach will be extremely time-consuming. Novel proteomic approaches make it possible to compare the expression of hundreds of proteins in several samples in a single experiment. The classical approach consists of separation of proteins on a 2-dimensional gel followed by protein identification with mass spectrometry, although new complementary gel-free techniques are emerging. We can thus compare protein expression in an atherosclerotic plaque with that in a normal artery or study plasma proteins in patients with atherothrombosis as compared with healthy subjects. For such approaches, it is not necessary to study the published data to select potential biomarkers. However, because the number of patients that can be studied with most of these techniques is limited, what is really important is the design of the studies, assessing carefully what kind of patients should be included to obtain valid conclusions. Clinicians should thus play a key role in this design along with the basic scientist. In this article, we review several proteomic strategies carried out by our group and others, and we make a call for collaboration between clinicians and experts in proteomics. This collaboration could greatly increase the likelihood of identifying new prognostic biomarker panels in atherothrombosis and other cardiovascular disorders.This work was supported by SAF (2007/63648 and 2007/60896), CAM (S2006/GEN-0247), FIS (PI050451, PS09/01405 and CP04/ 00060), European Network (HEALTH F2-2008-200647), EUROSALUD (EUS2008-03565), Fundacion Ramon Areces, Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Red RECAVA (RD06/0014/0035), Fundacion Española del Corazon, Sociedad Española de Arteriosclerosis, Mutua Madrileña Automovilista and Pfizer. The CNIC is supported by the Ministerio de Ciencia e Innovacion and the Fundacion ProCNIC.S