Shi Fang Ding

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.

Atherosclerotic plaque disruption induced by stress and lipopolysaccharide in apolipoprotein E knockout mice

To establish an animal model with disruptions of atherosclerotic plaques, 96 male apolipoprotein E knockout (apoE(-/-)) mice were randomly divided into stress, lipopolysaccharide (LPS), stress+LPS, and control groups (n = 24 each). All mice were fed a high-fat diet throughout the experiment, and carotid atherosclerotic lesions were induced by placement of a constrictive perivascular collar. Four weeks after surgery, mice in the LPS and stress+LPS groups were intraperitoneally injected with LPS (1 mg/kg twice per week for 8 wk). Eight weeks after surgery, mice in the stress and stress+LPS groups were treated with intermittent physical stress (electric foot shock and noise stimulation) for 4 wk. Morphological analysis revealed a plaque disruption rate of 16.7% in control, 34.8% in LPS, 54.2% in stress, and 60.9% in stress+LPS groups. The disruption rates in stress and stress+LPS groups were both significantly higher than those of controls (P = 0.007 and P = 0.002, respectively). Luminal thrombosis secondary to plaque disruption was observed only in the stress+LPS group. Both stress and LPS stimulation significantly decreased fibrous cap thickness and increased macrophage and lipid contents in plaques. Moreover, the combination of stress and LPS stimulation further lowered cap thickness and enhanced accumulation of macrophages and expression of inflammatory cytokines and matrix metalloproteinases. Stress activated the sympathetic nervous system, as manifested by increased blood pressure and flow velocity. Plasma fibrinogen levels were remarkably elevated in the stress and stress+LPS groups. In conclusion, stress- and LPS-costimulated apoE(-/-) mice provide a useful model for studies of plaque vulnerability and interventions.

Local gene silencing of monocyte chemoattractant protein-1 prevents vulnerable plaque disruption in apolipoprotein E-knockout mice.

Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE−/− mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE−/−) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P<0.001). Compared with the Ad-EGFP group, carotid plaques in the Ad-MCP-1i group showed increased levels of collagen and smooth muscle cells, and decreased levels of lipid and macrophages. The expression of inflammatory cytokines and activities of matrix metalloproteinases (MMPs) were lower in the Ad-MCP-1i group than in the Ad-EGFP group. In conclusion, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption. A marked suppression of the local inflammatory cytokine expression may be the central mechanism involved.

A causal relationship between shear stress and atherosclerotic lesions in apolipoprotein E knockout mice assessed by ultrasound biomicroscopy.

The present study was undertaken to examine the hemodynamic state using the latest ultrasound biomicroscopy (UBM) technique and to investigate the effect of local shear stress on the development of atherosclerosis in the constrictive collar-treated carotid arteries of apolipoprotein E-deficient (apoE(-/-)) mice. Fifty-six male apoE(-/-) mice fed a high-lipid diet were divided into an interventional group (n = 48) and the control group (n = 8). Constrictive and nonconstrictive collars were placed around the carotid artery of the mice in the interventional group and the control group, respectively. The carotid lumen diameters and flow velocities were measured by UBM, and shear stress in the lesion region was calculated. Histopathology and electron microscopy were performed to observe the morphological changes in the carotid artery. In the region proximal to the constrictive collar, shear stress was significantly reduced 2 days after collar placement and remained low over time compared with the baseline level. In contrast, within the constrictive collar region, shear stress was increased significantly. Although endothelial permeability was enhanced in both regions, monocyte chemotaxis protein-1 (MCP-1) expression, macrophage infiltration, and atherosclerotic lesions were more prominent in the region proximal to the constrictive collar. Moreover, increased MCP-1 expression was observed as early as 2 days after constrictive collar placement, which preceded the morphological changes of the vessel wall. In conclusion, UBM offers a noninvasive and reliable technique for measuring shear stress in apoE(-/-) mice. Persistent low shear stress promotes endothelial permeability and enhances MCP-1 expression and macrophage recruitment, which were essential in the pathogenesis of atherosclerosis in apoE(-/-) mice.

Enhanced Stabilization of Atherosclerotic Plaques in Apolipoprotein E-Knockout Mice by Combinatorial Toll-like Receptor-1 and -2 Gene Silencing

Because both Toll-like receptor-1 (TLR1) and TLR2 are expressed in atherosclerotic plaques, we hypothesized that TLR1 and TLR2 may play different roles in the formation of vulnerable plaques and that combinatorial knockdown of TLR1 and TLR2 genes may enhance the effects of isolated knockdown of the TLR1 or TLR2 gene on plaque stabilization. Lentiviruses carrying small interfering RNAs of TLR1 or TLR2 were constructed, which knocked down mRNA and protein expression of TLR1 or TLR2 significantly in vitro. One hundred and forty apolipoprotein E-deficient (apoE(-/-)) mice were randomly allocated to control, mock, TLR1 interference (TLR1i), TLR2 interference (TLR2i), and TLR1+2 interference (TLR1+2i) subgroups and a constrictive collar was placed around the carotid artery of these mice to induce plaque formation. TLR1i and TLR2i viral suspension was transfected into the carotid plaques separately in the TLR1i and TLR2i subgroups or together in the TLR1+2i subgroup. Four weeks after lentivirus transfection, expression of both TLR1 and TLR2 in the carotid plaques was remarkably attenuated. Plaques of the TLR1i subgroup showed lower macrophage content and interleukin (IL)-6 expression and a thicker fibrous cap compared with the control or mock subgroups. Plaques of the TLR2i subgroup showed a higher content of collagen and lower content of lipid and macrophages, a thicker fibrous cap, lower vulnerability index, and lower mRNA expression of IL-6 and monocyte chemoattractant protein-1 than the TLR1i subgroup. In the TLR1+2i subgroup, the macrophage and smooth muscle cell content, and the vulnerability index, were ameliorated as compared with those in the TLR2i subgroup. Lentivirus-mediated RNA interference can be used to efficiently knock down TLR1 and TLR2 genes in carotid plaques of apoE(-/-) mice. Although isolated knockdown of TLR1 or TLR2 is effective in attenuating plaque vulnerability, combinatorial interference with TLR1 and TLR2 exhibits enhanced improvement of plaque stability, and thus provides a useful approach to the stabilization of vulnerable plaques.

Combinatorial interference of toll-like receptor 2 and 4 synergistically stabilizes atherosclerotic plaque in apolipoprotein E-knockout mice.

To test the hypothesis that combinatorial interference of toll‐like receptor 2 (TLR2) and TLR4 is superior to isolated interference of TLR2 or TLR4 in stabilizing atherosclerotic plaques, lentiviruses carrying small interfering RNA of TLR2 or TLR4 were constructed and proved efficacious for knocking down mRNA and protein expression of TLR2 or TLR4 significantly in vitro. One hundred and fifty apolipoprotein E−/− mice fed a high‐fat diet were divided into the control, mock, TLR2i, TLR4i and TLR2 + 4i subgroups and a constrictive collar was placed around carotid artery of these mice to induce plaque formation. TLR2i and TLR4i viral suspension was transfected into carotid plaques, respectively, in TLR2i and TLR4i subgroups, or in combination in TLR2 + 4i subgroup. Four weeks after lentivirus transfection, mRNA and protein expression of TLR2 or TLR4 was attenuated markedly in carotid plaques, leading to reduced local inflammatory cytokine expression and plaque content of lipid and macrophages, increased plaque content of collagen and lowered plaque vulnerability index. Factorial ANOVA analysis revealed that there was a synergistic effect between TLR4i and TLR2i in stabilizing plaques. In conclusion, combinatorial interference of TLR2 and TLR4 reduces local inflammation and stabilizes plaques more effectively than interference of TLR2 or TLR4 alone.

Plaque volume compression ratio, a novel biomechanical index, is independently associated with ischemic cerebrovascular events.

Objective The purpose of this study was to develop a new biomechanical index for assessing the elastic characteristics of carotid plaques and to test the association between carotid plaque elasticity and ischemic cerebrovascular events (ICEs). Methods One hundred and eighteen carotid plaques were detected with real-time three-dimensional ultrasonography in 104 patients. All patients received MRI and were divided into two groups according to the history of ICEs: patients with ICEs (n = 58, including 20 patients with transient ischemic attack and 38 with ischemic stroke) and patients without ICEs (n = 46). The carotid plaque volume at end diastole (Vd) and end systole (Vs) was measured by use of a TomTec (Munich, Germany) workstation. Plaque volume compression ratio (VCR) was calculated as (Vd − Vs)/Vd × 100 and the reproducibility of VCR measurement was analyzed. The carotid intima–media thickness, plaque area and plaque acoustic density were also measured. Multivariate logistic regression was used to test the association between ICEs and plaque ultrasonic parameters or traditional risk factors including age, sex, smoking, blood pressure, history of coronary heart disease, levels of serum low-density lipoprotein, triglyceride and glucose. Results Satisfactory images of carotid plaques were obtained in all patients by real-time three-dimensional ultrasonography. Patients with ICEs and patients without ICEs differed significantly in VCR (22.19 ± 8.42 vs. 13.95 ± 7.86, P < 0.01). Regression analysis revealed that systolic blood pressure, [odds ratio (OR) = 1.054, 95% confidence interval (CI) = 1.028–1.081, P < 0.001] and VCR (OR = 1.074, 95% CI = 1.022–1.128, P < 0.001) were associated with ICEs independently. Plaque volume had only a marginal association with ICEs (OR = 1.007, 95% CI = 1.000–1.013, P = 0.05). Conclusion Measurement of VCR provides a noninvasive approach to the evaluation of the elasticity of carotid plaques, which is associated independently with ICEs. Thus, real-time three-dimensional ultrasonography-derived VCR holds a great potential in identifying patients with high risk of ICEs.