Miguel Angel Hernández-Presa

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.

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation

The mechanism by which 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) induce apoptosis in vascular smooth muscle cells (VSMCs) is unknown. In this work, we demonstrate that treatment of VSMCs with simvastatin and atorvastatin inhibited Bcl-2 expression in a time and dose-dependent manner, while Bax expression was not modified. This effect was reversed by mevalonate (100 micromol/l), farnesylpyrophosphate (5 micromol/l) or geranylgeranylpyrophosphate (5 micromol/l), suggesting the involvement of protein prenylation. The treatment of VSMCs with lipophilic statins was associated with decreased prenylation of p-21 Rho A and mevalonate, farnesyl pyrophosphate (F-PP) and geranylgeranyl pyrophosphate (G-PP) reversed prenylation to basal levels. In addition, overexpression of constitutively active Q63L Rho A prevented, at least in part, apoptosis induced by statins and downregulation of Bcl-2. We also investigated the participation of caspases (proteases) in the apoptosis induced by statins. The treatment of VSMCs with lipophilic statins induced activation of the caspase 9, the first caspase of the mitochondrial pathway. Coincubation of VSMCs with the caspase inhibitor ZVAD-fmk (100 micromol/l) significantly inhibited lipophilic statin-induced apoptosis. These findings indicate that the downregulation of Bcl-2 by Rho GTPases mediates statin-induced apoptosis and suggest a new potential mechanism of action for these drugs on the regulation of cell number in the atherosclerotic lesions.

Red Wine Intake Prevents Nuclear Factor-κB Activation in Peripheral Blood Mononuclear Cells of Healthy Volunteers During Postprandial Lipemia

BackgroundSeveral epidemiological studies have demonstrated the beneficial effect of red wine intake in reducing total and cardiovascular mortality. This effect has been attributed in part to its antioxidant properties. Because the monocytes/macrophages and the nuclear transcription factor &kgr;B (NF-&kgr;B) are implicated in the pathogenesis of atherosclerotic lesions, we examined the effect of red wine intake on the activation of NF-&kgr;B in peripheral blood mononuclear cells. Methods and ResultsSixteen healthy volunteers were studied 3 times each: after a moderate dose, a low dose, and no wine with a fat-enriched breakfast. Lipid profile and NF-&kgr;B activation (electrophoretic mobility shift assay) were examined in blood samples taken before and 3, 6, and 9 hours after wine intake. In addition, mononuclear cells were incubated with VLDL in the presence of some antioxidants (quercetin and &agr;-tocopherol succinate) contained in red wine to study their effects on NF-&kgr;B activation. Subjects receiving a fat-enriched breakfast had increased NF-&kgr;B activation in peripheral blood mononuclear cells coinciding with the augmentation in total triglycerides and chylomicrons. Red wine intake prevented NF-&kgr;B activity even though it induced a certain increase in serum lipids, particularly VLDL, that did not increase after the fat ingestion alone. However, another form of alcohol intake (vodka) did not modify the NF-&kgr;B activation provided by postprandial lipemia. In cultured mononuclear cells, isolated human VLDL caused NF-&kgr;B activation in a time-dependent manner that did not occur in the presence of the red wine antioxidants quercetin and &agr;-tocopherol. ConclusionsOur results provide a new potential mechanism to explain the beneficial effects of red wine intake in the reduction of cardiovascular mortality.

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.

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.

Possible Role of Parathyroid Hormone–Related Protein as a Proinflammatory Cytokine in Atherosclerosis

Background and Purpose— Parathyroid hormone–related protein (PTHrP) is a vasodilator peptide. In addition, PTHrP appears to affect vascular growth and to be a mediator of inflammation in rheumatic and brain disorders. We examined the possible role of PTHrP in the inflammatory process in atherosclerosis Methods— We immunohistochemically analyzed the cellular localization of PTHrP, the type 1 PTH/PTHrP receptor (PTH1R), and monocyte chemoattractant protein-1 (MCP-1) in 26 human carotid atherosclerotic plaques. Results— The inflammatory region of plaques was characterized by high PTHrP, PTH1R, and MCP-1 immunostaining in relation to the cap (0.75±0.1 versus 0.29±0.04, 0.5±0.1 versus 0.25±0.05, 0.72±0.2 versus 0.29±0.05, respectively; P <0.05). PTHrP and MCP-1 were colocalized in both resident and inflammatory cells in the plaque. Moreover, in cultured vascular smooth muscle cells (VSMC), PTHrP(1–36) increased MCP-1 mRNA (3-fold at 6 hours) and MCP-1 protein (2.5-fold at 24 hours). This effect was inhibited by either PTHrP(7–34) or various protein kinase A inhibitors and by the nuclear factor-&kgr;B (NF-&kgr;B) inhibitor parthenolide. Furthermore, PTHrP(1–36) elicited an increase in NF-&kgr;B activation in VSMC. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin inhibited the PTHrP(1–36) induction of both NF-&kgr;B activity and MCP-1 overexpression, and this was reversed by mevalonate. Conclusions— PTHrP appears to be a novel proinflammatory mediator in the atheroma lesion and may contribute to the instability of carotid atherosclerotic plaques. Our data provide a new rationale to understand the mechanisms involved in the beneficial effects of statins in atherosclerosis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase the binding activity and nuclear level of Oct-1 in mononuclear cells

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are drugs very effective to decrease low-density lipoprotein (LDL) cholesterol. In addition, a number of studies suggest that statins have other beneficial clinical effects beyond cholesterol lowering. We recently reported that statins decrease nuclear factor kappa B (NF-kappaB) binding activity in monocytes and vascular smooth muscle cells. We now explored the effect of two different statins, simvastatin and atorvastatin, in the activation of the octamer transcription factor Oct-1 on the monocytic cell line THP-1. Oct-1 is a nuclear factor that represses the transcription of proinflammatory genes such as interleukin-8, CD11c/CD18, vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). Low concentrations of both statins increased Oct-1 DNA binding activity (electrophoretic mobility shift assay) that was resolved into two specific bands. The upper one was supershifted by preincubation of nuclear extracts with anti-Oct-1 antibody. The lower one was supershifted by preincubation of nuclear extracts with an anti-Oct-2 antibody, also partially competed with 100 mol/l excess of cold activator protein-1 (AP-1) and attenuated by anti-c-Jun antibody. Both statins increased Oct-1 and Oct-2 nuclear protein levels (Western blot). In contrast, neither had any effect on PMA-differentiated cells, suggesting a distinct sensitivity between circulating monocytes and resident tissular macrophages. In addition, statins did not increase Oct-lipoprotein lipase binding activity that contains an Oct-1 binding element. The mRNA expression of interleukin-8, a chemokine containing Oct sites in its promoter, was diminished by statin pretreatment. Our results indicate that simvastatin and atorvastatin increase the activity of the transcriptional repressor Oct-1 in mononuclear cells, and could thus contribute to decrease the activation of these cells. These data suggest a possible novel mechanism supporting a certain anti-inflammatory effect of these two 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.