Begoña Muñoz-García

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.

Intensive treatment with atorvastatin reduces inflammation in mononuclear cells and human atherosclerotic lesions in one month.

Background and Purpose— To investigate the effect of short-term high-dose atorvastatin on blood and plaque inflammation in patients with carotid stenosis. Methods— Twenty patients undergoing carotid endarterectomy without previous statin treatment were randomized to receive either atorvastatin 80 mg/d (n=11) or no statins (n=9) for 1 month. We studied inflammatory mediators in plasma (enzyme-linked immunosorbent assay), peripheral blood mononuclear cells (PBMCs; quantitative RT-PCR and EMSA) and plaques (immunohistochemistry and Southwestern histochemistry). Results— Atorvastatin significantly decreased total and low-density lipoprotein cholesterol and prostaglandin E2 plasma levels. PBMCs from treated patients showed impaired NF-&kgr;B activation and MCP-1 and COX-2 mRNA expression. Carotid atherosclerotic plaques demonstrated a significant reduction in macrophage infiltration, activated NF-&kgr;B, and COX-2 and MCP-1 expression. Conclusions— Intensive treatment with atorvastatin decreases inflammatory activity of PBMCs and carotid atherosclerotic plaques in 1 month. These data strongly suggest that the antiinflammatory effect of high doses of statins in humans can be seen very early.

The CD163-expressing macrophages recognize and internalize TWEAK Potential consequences in atherosclerosis

BACKGROUND CD163 is a new potential scavenger receptor of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) which elicits diverse biologic actions involved in atherosclerosis. We have analyzed the importance of TWEAK-CD163 interaction in atherosclerosis. METHODS TWEAK and CD163 interaction was studied in cultured human macrophages. Moreover, TWEAK and CD163 expression was analyzed in carotid atherosclerotic plaques (immunohistochemistry) and plasma (ELISA). We have also assessed their potential association with intima/media thickness (IMT) in asymptomatic subjects. RESULTS In vitro studies revealed that CD163-expressing macrophages can bind and internalize TWEAK protein exogenously added from supernatants. Accordingly, we observed an inverse correlation between the expression of CD163 and TWEAK (r=-0.51; p=0.008) in the shoulder region of atherosclerotic plaques obtained from 25 patients undergoing carotid endarterectomy. The same trend was observed when we analyzed the plasma concentration of both proteins in 90 subjects free from clinical cardiovascular disease (r=-0.25; p=0.016) in which carotid ultrasonography was performed to determine IMT. In these subjects, we found a positive correlation between sCD163 and IMT (r=0.36; p<0.001) and between sCD163-sTWEAK ratio and IMT (r=0.51; p<0.001). This association remained significant after adjusting for traditional cardiovascular risk factors and inflammatory markers explaining 39% (sCD163) or 48% (sCD163-sTWEAK ratio) of IMT variance. CONCLUSIONS Our results suggest that TWEAK-CD163 interaction takes place in vivo, probably decreasing TWEAK plasma concentration. Furthermore, we have observed that CD163-TWEAK plasma ratio is a potential biomarker of clinical and subclinical atherosclerosis.

Fn14 Is Upregulated in Cytokine-Stimulated Vascular Smooth Muscle Cells and Is Expressed in Human Carotid Atherosclerotic Plaques Modulation by Atorvastatin

Background and Purpose— Interaction between different members of the tumor necrosis factor superfamily and their receptors elicits diverse biologic actions that are implicated in the pathogenesis of atherosclerosis. We have analyzed the expression of Fn14 and its ligand TWEAK in carotid atherosclerotic plaques and its potential modulation by atorvastatin in vivo. Furthermore, we have studied whether proinflammatory cytokines regulate Fn14 expression in human aortic smooth muscle cells (hASMCs) in culture as well as the potential regulation by atorvastatin treatment. Methods— Fn14 and TWEAK expression was analyzed in human carotid atherosclerotic plaques. Furthermore, Fn14 expression was studied in hASMCs in culture. Results— Fn14 and TWEAK are expressed in macrophages and smooth muscle cells in carotid atherosclerotic plaques. Proinflammatory cytokines (interleukin-1β and interferon-γ) upregulate Fn14 expression in hASMCs. This effect was prevented by atorvastatin treatment and reversed by mevalonate and geranylgeranyl pyrophosphate. Geranylgeranyl transferase inhibitor, toxin B (Rac and Rho inhibitor), C3 exoenzyme (Rho inhibitor), and Y-27632 (Rho kinase inhibitor) also decreased Fn14 expression, implicating the Rho/Rho kinase pathway in the regulation of Fn14 expression. Finally, atorvastatin treatment reduced Fn14 expression in vivo. Conclusions— TWEAK and Fn14 are expressed in atherosclerotic plaques and could be novel mediators of atherosclerosis. Atorvastatin diminishes Fn14 expression in vitro and in vivo providing novel information of the beneficial properties of statins.

Suppressors of Cytokine Signaling Modulate JAK/STAT-Mediated Cell Responses During Atherosclerosis

Objective—Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction, mainly Janus kinase/signal transducers and activators of transcription (JAK/STAT). We investigated the effects of SOCS modulation on the JAK/STAT-dependent responses in vascular cells, and their implication in atherosclerotic plaque development. Methods and Results—Immunohistochemistry in human plaques revealed a high expression of SOCS1 and SOCS3 by vascular smooth muscle cells (VSMCs) and macrophages in the inflammatory region of the shoulders, when compared to the fibrous area. SOCS were also increased in aortic lesions from apoE−/− mice. In cultured VSMCs, endothelial cells, and monocytes, SOCS1 and SOCS3 were transiently induced by proinflammatory cytokines, proatherogenic lipoproteins, and immune molecules. Furthermore, overexpression of SOCS suppressed STAT activation and reduced inflammatory gene expression and cell growth, whereas SOCS knockdown increased these cell responses. In vivo, antisense oligodeoxynucleotides targeting SOCS3 exacerbated the atherosclerotic process in apoE−/− mice by increasing the size, leukocyte content, and chemokine expression in the lesions. Conclusions—SOCS expressed in atherosclerotic lesions are key regulators of vascular cell responses. Activation of this endogenous antiinflammatory pathway might be of interest in the treatment of atherosclerosis.

NF-κB Activation and Fas Ligand Overexpression in Blood and Plaques of Patients With Carotid Atherosclerosis Potential Implication in Plaque Instability

Background and Purpose— Apoptosis is present in human atherosclerotic lesions. Nuclear factor-&kgr;B (NF-&kgr;B) is involved in the transcriptional regulation of the proapoptotic protein Fas ligand (FasL). We have analyzed NF-&kgr;B activation and FasL expression in atherosclerotic plaques and peripheral blood mononuclear cells (PBMCs) of patients with carotid stenosis. Methods— NF-&kgr;B activation and FasL and active caspase-3 expression were analyzed in 32 human carotid plaques. NF-&kgr;B activation and FasL mRNA were tested in PBMCs of patients and healthy volunteers. We analyzed whether the NF-&kgr;B inhibitor parthenolide regulates FasL expression and cytotoxicity in human T cells. Results— The inflammatory region of plaques showed an increase in NF-&kgr;B activation (3393±281 versus 1029±100 positive nuclei per mm2, P <0.001) and FasL (16±1.4% versus 13±1.8%, P <0.05) and active caspase-3 (3.3±0.6 versus 1.5±0.3%, P <0.05) expression compared with the fibrous area. Activated NF-&kgr;B and FasL protein were colocalized in plaque cells. In PBMCs obtained from those patients the day of endarterectomy, NF-&kgr;B activation and FasL expression were significantly increased compared with healthy controls (1.5±0.1 versus 0.5±0.1 and 2.1±0.1 versus 1.2±0.1 arbitrary units, respectively; P <0.001). There was a significant correlation between NF-&kgr;B activation and FasL expression. In activated T cells, parthenolide decreased NF-&kgr;B activation, FasL promoter activity, and mRNA expression. Parthenolide also decreased cytotoxicity of activated Jurkat cells on FasL-sensitive cells. Conclusions— NF-&kgr;B activation and FasL overexpression occur in PBMCs and atherosclerotic lesions of patients with carotid stenosis. The NF-&kgr;B-FasL pathway could be involved in the mechanisms underlying plaque instability in humans.

Leukotriene B4 enhances the activity of nuclear factor-κB pathway through BLT1 and BLT2 receptors in atherosclerosis

AIMS Leukotriene B4 (LTB4) is a powerful chemoattractant and pro-inflammatory mediator in several inflammatory diseases, including atherosclerosis. It acts through its two membrane receptors, BLT1 and BLT2. The aim of this study was to determine the molecular mechanism involved in the proatherogenic effect of LTB4, BLT1 and BLT2 in atherosclerosis. Moreover, we characterized the expression of 5-lipoxygenase (5-LO) pathway and LTB4 receptors in blood and plaques from patients with carotid atherosclerosis. METHODS AND RESULTS In cultured monocytic cells, LTB4 induced a rapid phosphorylation of mitogen-activated protein kinases (MAPKs ERK1/2 and JNK1/2) and PI3K/Akt via BLT1 and BLT2 in a pertussis toxin (PTX)-dependent mechanism (assessed via western blotting) and also increased nuclear factor-kappaB (NF-kappaB) DNA binding activity (assessed via EMSA) in a MAPK- and reactive oxygen species-dependent mechanism. Furthermore, LTB4 elicited interleukin-6, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha mRNA overexpression also via BLT1 and BLT2 by a PTX- and NF-kB-dependent mechanism (assessed by real-time PCR), promoting an inflammatory environment. When compared with healthy subjects, patients with carotid atherosclerosis showed a significant increase in the expression of all the components of the 5-LO pathway and BLT1 and BLT2 mRNA (real-time PCR) in peripheral blood mononuclear cells and LTB4 plasma levels (ELISA). In these patients, an overexpression of 5-LO, leukotriene A-4 hydroxylase (LTA4-H) and BLT1 was noted in the inflammatory region of carotid plaques when compared with the fibrous cap (assessed by immunohistochemistry). CONCLUSION The 5-LO pathway is enhanced in patients with carotid atherosclerosis. Furthermore, its product LTB4 phosphorylates MAPKs and stimulates NF-kappaB-dependent inflammation via BLT1 and BLT2 receptors in cultured monocytic cells. The blockade of this pathway could be a novel and potential therapeutic target in atherothrombosis.

Tumor Necrosis Factor–Like Weak Inducer of Apoptosis (TWEAK) Enhances Vascular and Renal Damage Induced by Hyperlipidemic Diet in ApoE-Knockout Mice

Objective—Tumor necrosis factor–like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of cytokines. TWEAK binds and activates the Fn14 receptor, and may regulate apoptosis, inflammation, and angiogenesis, in different pathological conditions. We have evaluated the effect of exogenous TWEAK administration as well as the role of endogenous TWEAK on proinflammatory cytokine expression and vascular and renal injury severity in hyperlipidemic ApoE-knockout mice. Methods and Results—ApoE−/− mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 &mgr;g/kg/d), anti-TWEAK neutralizing mAb (1000 &mgr;g/kg/d), TWEAK plus anti-TWEAK antibody (10 &mgr;g TWEAK +1000 &mgr;g anti-TWEAK/kg/d), or nonspecific IgG (1000 &mgr;g/kg/d) daily for 9 days. In ApoE−/− mice, exogenous TWEAK administration in ApoE−/− mice induced activation of NF-&kgr;B, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE−/− mice with an anti-TWEAK blocking mAb decreased NF-&kgr;B activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion. Conclusion—Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid–rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with hyperlipidemia.

Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis

AIMS Heat shock protein 90 (HSP90) is a ubiquitous chaperone involved in the folding, activation, and assembly of many proteins. HSP90 inhibitors [17-allylamino-17-demethoxygeldamycin (17-AAG)/17-dimethyl aminothylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG)] bind to and inactivate HSP90, increasing the heat shock response and suppressing different signalling pathways. We aim to investigate the effect of HSP90 inhibitors in the modulation of inflammatory responses during atherogenesis. METHODS AND RESULTS In human atherosclerotic plaques, HSP90 immunostaining was increased in inflammatory regions and in plaques characterized by lower cap thickness. In cultured human macrophages and vascular smooth muscle cells, treatment with either 17-AAG or 17-DMAG increased HSP70 expression and reduced transcription factor [signal transducers and activators of transcription (STAT) and nuclear factor-kappaB (NF-kappaB)] activation and chemokine expression induced by proinflammatory cytokines. In vivo, hyperlipidaemic ApoE(-/-) mice were randomized to 17-DMAG (2 mg/kg every 2 days, n = 11) or vehicle injected (n = 9) during 10 weeks. Atherosclerotic plaques of mice treated with 17-DMAG displayed increased HSP70 expression and diminished NF-kappaB and STAT activation, along with decreased lesion, lipid, and macrophage content, compared with vehicle-injected mice. In addition, treatment with 17-DMAG significantly reduced monocyte chemoattractant protein-1 levels, both in plaques and in plasma. CONCLUSION HSP90 expression is associated with features of plaque instability in advanced human lesions. HSP90 inhibitors reduce inflammatory responses in atherosclerosis, suggesting that HSP90 could be a novel therapeutic target in atherosclerosis.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors decrease Fas ligand expression and cytotoxicity in activated human T lymphocytes.

Background—HMG-CoA reductase inhibitors reduce cardiovascular mortality, although the mechanisms of action have not been completely elucidated. The presence of T cells and apoptotic cells in atherosclerotic plaques is well established, the reduction of cellular content being a marker of their vulnerability. One of the main mechanisms of cell death activation is the Fas-Fas ligand (FasL) system. Methods and Results—We studied whether HMG-CoA reductase inhibitors can regulate FasL expression and cytotoxicity in human T cells (Jurkat cells). Activation of Jurkat cells with phorbol esters and ionomycin increased FasL expression, an effect prevented by atorvastatin or simvastatin. Mevalonate and geranylgeranylpyrophosphate but not farnesylpyrophosphate prevented the effect of atorvastatin, indicating that protein geranylation was involved in FasL expression. The C3 exotoxin, which selectively inactivates Rho proteins, also decreased FasL expression on T cells. Overexpression of constitutively active RhoA increased FasL expression in Jurkat cells, and dominant-negative RhoA decreased FasL expression in activated cells, indicating that RhoA is implicated in FasL expression. Atorvastatin also decreased cytotoxic activity of activated Jurkat cells on FasL-sensitive cells. Finally, atorvastatin treatment reduced FasL expression in peripheral blood mononuclear cells and human carotid atherosclerotic plaques. Conclusions—Atorvastatin regulates FasL expression in T cells, probably because of the inhibition of RhoA prenylation. These results provide novel information by which atorvastatin may regulate the cytotoxic activity of T cells and the number of cells in the atherosclerotic plaque.