José Tuñón

Angiotensin-Converting Enzyme Inhibition Prevents Arterial Nuclear Factor-κB Activation, Monocyte Chemoattractant Protein-1 Expression, and Macrophage Infiltration in a Rabbit Model of Early Accelerated Atherosclerosis

BACKGROUND The migration of monocytes into the vessel wall is a critical event leading to the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is the main chemotactic factor involved in this phenomenon, and nuclear factor-kappa B (NF-kappa B) is one of the nuclear factors controlling its expression. ACE inhibitors have been useful in some experimental models of atherosclerosis. In this work, we addressed the hypothesis that angiotensin II (Ang II) may be implicated in the recruitment of monocytes into the vessel wall through the activation of NF-kappa B and the induction of MCP-1 expression. METHODS AND RESULTS Accelerated atherosclerosis was induced in the femoral arteries of rabbits by endothelial desiccation and atherogenic diet for 7 days. Atherosclerotic vessels exhibited an increase in NF-kappa B-like activity, and p50 and p65 NF-kappa B subunits were identified as components of this activity. MCP-1 (mRNA and protein) was also expressed in the injured vessels coincidently with the neointimal macrophage infiltration. ACE inhibition with quinapril reduced these three parameters. In cultured monocytic and vascular smooth muscle cells. Ang II elicited an increase in NF-kappa B activation and MCP-1 expression that was prevented by preincubation of cells with pyrrolidinedithiocarbamate, an inhibitor of NF-kappa B activation. CONCLUSIONS The present data support a role for Ang II in neointimal monocyte infiltration through NF-kappa B activation and MCP-1 expression in a model of accelerated atherosclerosis in rabbits. Our results suggest that ACE inhibitors may have a beneficial effect in early atherosclerosis.

HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis

OBJECTIVES To study the effect of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)-reductase inhibitor atorvastatin on the potential mechanisms involved in the recruitment of monocytic cells into the vessel wall. BACKGROUND Inhibitors of HMG-CoA-reductase reduce cardiovascular mortality though the mechanisms yet elucidated. Most ischemic events are secondary to disruption of atherosclerotic plaques highly infiltrated by macrophages. METHODS Atherosclerosis was induced in the femoral arteries of rabbits by endothelial damage and atherogenic diet for 4 weeks. Then, animals were switched to standard chow and randomized to receive either no treatment or atorvastatin (5 mg/kg/d) and killed after 4 weeks. RESULTS Atorvastatin induced a significant reduction in serum lipids and in lesion size. Arterial macrophage infiltration was abolished by the treatment, and monocyte chemoattractant protein-1 (MCP-1) was significantly diminished in the neointima and in the media. Nuclear factor kappa-B (NF-kappaB) was activated in the 60% of the lesions, both in macrophages and vascular smooth muscle cells (VSMC), of the untreated group while only in 30% of the atorvastatin group. NF-kappaB activity was also lower in the uninjured aorta and liver of treated compared with untreated rabbits. In cultured VSMC, MCP-1 expression and NF-kappaB activity induced by tumor necrosis factor alpha were downregulated by atorvastatin. CONCLUSIONS In a rabbit atherosclerosis model, atorvastatin diminishes the neointimal inflammation, and this could contribute to the stabilization of the atherosclerotic plaque. This may be an additional explanation for the reduction of acute ischemic events in patients treated with statins.

Atorvastatin reduces NF-κB activation and chemokine expression in vascular smooth muscle cells and mononuclear cells

Cardiovascular mortality, mainly due to the rupture of unstable atherosclerotic plaques, is reduced by 3-hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Inflammatory cells, attracted to the vascular lesion by chemokines, have been implicated in the process of the plaque rupture. In cultured vascular smooth muscle cells (VSMC) and U937 mononuclear cells we have studied the effect of Atorvastatin (Atv) on nuclear factor kappaB (NF-kappaB) activity, an inducer of the mRNA expression of chemokines such as interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein 1 (MCP-1). Angiotensin II (Ang II) and tumor necrosis factor alpha (TNF-alpha) increased NF-kappaB activity in VSMC (2 and 5-fold, respectively). Preincubation of cells with 10(-7) mol/l Atv diminished this activation (44 and 53%). The inhibition was reversed by mevalonate, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), but not by other isoprenoids. Coinciding with the NF-kappaB activation in VSMC, there was a diminution of cytoplasmic IkappaB levels that was recovered by pretreatment with Atv. Ang II and TNF-alpha induced the expression of IP-10 (1.5 and 3.4-fold) and MCP-1 (2.4 and 4-fold) in VSMC. Atv reduced this overexpression around 38 and 35% (IP-10), and 54 and 39% (MCP-1), respectively. Our results strongly suggest that Atv, through the inhibition of NF-kappaB activity and chemokine gene expression, could reduce the inflammation within the atherosclerotic lesion and play a role in the stabilization of the lesion.

Identification by a Differential Proteomic Approach of Heat Shock Protein 27 as a Potential Marker of Atherosclerosis

Background—We hypothesized that normal and pathological vessel walls display a differential pattern of secreted proteins. We have recently set up the conditions for comparing secretomes from carotid atherosclerotic plaques and control arteries using a proteomic approach to assess whether differentially secreted proteins could represent markers for atherosclerosis. Methods and Results—Normal endartery segments and different regions of endarterectomy pieces (noncomplicated/complicated plaques) were incubated in protein-free medium, and the released proteins were analyzed by 2D electrophoresis (2-DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27). Surprisingly, compared with control arteries, HSP27 release was drastically decreased in atherosclerotic plaques and barely detectable in complicated plaque supernatants. HSP27 was expressed primarily by intact vascular cells of normal arteries and carotid plaques (immunohistochemistry). Plasma detection of soluble HSP27 showed that circulating HSP27 levels are significantly decreased in the blood of patients with carotid stenosis relative to healthy subjects (0.19 [0.1 to 1.95] versus 83 [71.8 to 87.8]) ng/mL, P<0.0001). Conclusions—HSP27 secretion is decreased in complicated atherosclerotic plaques, and sHSP27 plasma levels are decreased in atherosclerotic patients compared with healthy subjects. Plasma sHSP27 levels could be a potential index of atherosclerosis, although further validation is needed in large patient cohorts.

ACE Inhibitor Quinapril Reduces the Arterial Expression of NF-κB-Dependent Proinflammatory Factors but not of Collagen I in a Rabbit Model of Atherosclerosis

Increasing evidence supports an association between inflammation and plaque rupture. Macrophages and vascular smooth muscle cells are a source of cytokines and growth factors, which contribute to ongoing inflammation during atherogenesis. In a rabbit model of atherosclerosis, we evaluated the effect of the ACE inhibitor quinapril on different parameters implicated in the pathogenesis of the plaque, such as the presence of chemokines (interleukin-8, monocyte chemoattractant protein-1), collagen I, and vascular smooth muscle cell proliferation (PDGF-B). Since nuclear factor kappaB (NF-kappaB) has been implicated in the control of chemokine transcription and cell proliferation, we also investigated its activation and localization in the lesion. Quinapril administration for 28 days caused a down-regulation in arterial expression of interleukin-8 and monocyte chemoattractant protein-1 (mRNA and protein). However, collagen I expression (mRNA and protein) was not modified. PDGF-B expression was reduced in both the intima and the media. Active NF-kappaB, found in both macrophages and vascular smooth muscle cells, was also reduced by quinapril. Nevertheless, no significant changes were noted in the mild neointima formation, although a certain trend toward normalization was found in the quinapril-treated group. In conclusion, our results show that quinapril treatment attenuates several parameters associated with inflammation within the atherosclerotic lesions that are controlled by NF-kappaB, although it has no effect on collagen I expression. Both effects could contribute to the stabilization of the atherosclerotic plaque.

Atorvastatin reduces the expression of cyclooxygenase-2 in a rabbit model of atherosclerosis and in cultured vascular smooth muscle cells

Inflammation is involved in the genesis and rupture of atherosclerotic plaques. We assessed the effect of atorvastatin (ATV) on the expression of cyclooxygenase-2 (COX-2) and other proinflammatory molecules in a rabbit model of atherosclerosis. Fourteen animals underwent injury of femoral arteries and 2 weeks of atherogenic diet. Afterwards, they were randomized to receive either 5 mg/kg per day of ATV (n=8) or no treatment (NT, n=6) during 4 weeks, and were finally killed. ATV reduced lipid levels, neointimal size (0.13 (0.03-0.29) mm(2) vs 0.65 (0.14-1.81) mm(2), P=0.005) and the percentage of neointimal area positive for macrophages (1% (0-3) vs 19% (5-32), P=0.001), COX-2 (32% (23-39) vs 60% (37-81) P=0.019), interleukin-8 (IL-8) (23% (3-63) vs 63% (25-88) P=0.015), and metalloproteinase-3 (19% (12-34) vs 42% (27-93), P=0.010), without significant differences in COX-1 expression (immunohistochemistry). In situ hybridization confirmed a decreased expression of COX-2 mRNA (22% (5-40) vs 43% (34-59) P=0.038). The activity of nuclear factor-kappaB, which controls many proinflammatory genes including COX-2, was reduced in atherosclerotic lesions (3538 (2663-5094) vs 8696 (5429-11312)) positive nuclei per mm(2), P=0.001) and circulating mononuclear cells (2966 vs 17130 arbitrary units). In cultured vascular smooth muscle cells, ATV reduced the expression of COX-2 mRNA induced by IL-1beta and TNF-alpha without affecting COX-1 expression. In conclusion, ATV, besides decreasing a number of inflammatory mediators in the atherosclerotic lesion, significantly downregulates COX-2 both in vivo and in vitro. These anti-inflammatory actions could partially account for the reduction of acute coronary events achieved by statins.

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.

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.

Simvastatin reduces NF-κB activity in peripheral mononuclear and in plaque cells of rabbit atheroma more markedly than lipid lowering diet

OBJECTIVE To study whether simvastatin reduces inflammation in atherosclerosis beyond its hypolipidemic effects. METHODS Twenty-four rabbits with induced femoral injury and on an atherogenic diet were randomized to normolipidemic diet (n=9), or to continue the atherogenic diet while receiving simvastatin 5 mg/kg/day (n=9) or no treatment (n=6) for 4 weeks. RESULTS As compared with no treatment, the normolipidemic diet significantly reduced lipid levels, while simvastatin produced nonsignificant reductions. In spite of this, NF-kappaB binding activity in peripheral mononuclear cells was reduced in the simvastatin group [2,958+/-5,123 arbitrary units (a.u.)] as compared with no treatment (49,267+/-20,084 a.u.; P<0.05) and normolipidemic groups (41,492+/-15,876 a.u.; P<0.05) (electrophoretic mobility shift assay). NF-kappaB activity in the atherosclerotic lesions was also reduced by simvastatin as compared to nontreated animals (4,108+/-3,264 vs. 8,696+/-2,305 nuclei/mm(2); P<0.05), while the normolipidemic diet induced only a nonsignificant diminution (P>0.05) (Southwestern histochemistry). Similarly, simvastatin decreased macrophage infiltration (4.6+/-12 vs. 19+/-12% of area staining positive; P<0.05) and the expression of interleukin-8 (24+/-12 vs. 63+/-21%; P<0.05) and metalloproteinase-3 (16+/-3 vs. 42+/-28%; P<0.05) (immunohistochemistry), while the reduction achieved by normolipidemic diet in all these parameters was again nonsignificant (P>0.05). CONCLUSIONS These findings suggest that simvastatin reduces inflammation in atherosclerotic plaques and in blood mononuclear cells more than expected for the lipid reduction achieved.

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.