Chun Xi Liu

Overexpression of ACE2 Enhances Plaque Stability in a Rabbit Model of Atherosclerosis

Objective—The purpose of this study was to test the hypothesis that ACE2 overexpression may enhance atherosclerotic plaque stability by antagonizing ACE activity and converting angiotensin II to angiotensin 1–7. Methods and Results—Atherosclerotic plaques were induced in the abdominal aorta of 114 rabbits by endothelial injury and atherogenic diet. Gene therapy was performed in group A at week 4 and in group B at week 12, respectively. Each group of rabbits were randomly divided into 3 subgroups which received, respectively, a recombinant ACE2 expressing vector (AdACE2), a control vector AdEGFP and AdACE2+A779, an antagonist of angiotensin 1–7 receptor. Local ACE2 overexpression attenuated the progression of lesions from week 4 to week 8, but not progression of plaque size from week 12 to week 16. In group B rabbits, local ACE2 overexpression resulted in stable plaque compositions, ie, fewer macrophages, less lipid deposition and more collagen contents, higher plaque stability scores, decreased angiotensin II levels, and increased angiotensin 1–7 levels in plaque tissues in the AdACE2 subgroup compared with those in the AdEGFP subgroup. Conclusions—Overexpression of ACE2 results in stabilized atherosclerotic plaques and the mechanism is probably the conversion of vasoconstrictive angiotensin II to vessel protective angiotensin 1–7.

Angiotensin-converting enzyme 2 attenuates atherosclerotic lesions by targeting vascular cells

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered homolog of ACE whose actions oppose those of angiotensin II (AngII). However, the underlying mechanisms by which ACE2 effectively suppresses early atherosclerotic lesions remain poorly understood. Here, we show, both in vitro and in vivo, that ACE2 inhibited the development of early atherosclerotic lesions by suppressing the growth of vascular smooth muscle cells (VSMCs) and improving endothelial function. In a relatively large cohort animal study (66 rabbits), aortic segments transfected by Ad-ACE2 showed significantly attenuated fatty streak formation, neointimal macrophage infiltration, and alleviation of impaired endothelial function. Segments also showed decreased expression of monocyte chemoattractant protein 1, lectin-like oxidized low-density lipoprotein receptor 1, and proliferating cell nuclear antigen, which led to the delayed onset of atherosclerotic lesions. At the cellular level, ACE2 significantly modulated AngII-induced growth and migration in human umbilical vein endothelial cells and VSMCs. The antiatherosclerotic effect of ACE2 involved down-regulation of the ERK-p38, JAK-STAT, and AngII-ROS-NF-κB signaling pathways and up-regulation of the PI3K-Akt pathway. These findings revealed the molecular mechanisms of the antiatherosclerotic activity of ACE2 and suggested that modulation of ACE2 could offer a therapeutic option for treating atherosclerosis.

Angiotensin-Converting Enzyme-2 Overexpression Improves Left Ventricular Remodeling and Function in a Rat Model of Diabetic Cardiomyopathy

OBJECTIVES The aim of this study was to test the hypothesis that angiotensin (Ang)-converting enzyme-2 (ACE2) overexpression may inhibit myocardial collagen accumulation and improve left ventricular (LV) remodeling and function in diabetic cardiomyopathy. BACKGROUND Hyperglycemia activates the renin-Ang system, which promotes the accumulation of extracellular matrix and progression of cardiac remodeling and dysfunction. METHODS Ninety male Wistar rats were divided randomly into treatment (n = 80) and control (n = 10) groups. Diabetes was induced in the treatment group by a single intraperitoneal injection of streptozotocin. Twelve weeks after streptozotocin injection, rats in the treatment group were further divided into adenovirus-ACE2, adenovirus-enhanced green fluorescent protein, losartan, and mock groups (n = 20 each). LV volume; LV systolic and diastolic function; extent of myocardial fibrosis; protein expression levels of ACE2, Ang-converting enzyme, and Ang-(1-7); and matrix metalloproteinase-2 activity were evaluated. Cardiac myocyte and fibroblast culture was performed to assess Ang-II and collagen protein expression before and after ACE2 gene transfection. RESULTS Four weeks after ACE2 gene transfer, the adenovirus-ACE2 group showed increased ACE2 expression, matrix metalloproteinase-2 activity, and LV ejection fractions and decreased LV volumes, myocardial fibrosis, and ACE, Ang-II, and collagen expression in comparison with the adenovirus-enhanced green fluorescent protein and control groups. ACE2 was superior to losartan in improving LV remodeling and function and reducing collagen expression. The putative mechanisms may involve a shift in balance toward an inhibited fibroblast-myocyte cross-talk for collagen and transforming growth factor-beta production and enhanced collagen degradation by matrix metalloproteinase-2. CONCLUSIONS ACE2 inhibits myocardial collagen accumulation and improves LV remodeling and function in a rat model of diabetic cardiomyopathy. Thus, ACE2 provides a promising approach to the treatment of patients with diabetic cardiomyopathy.

Traditional Chinese medication Tongxinluo dose-dependently enhances stability of vulnerable plaques: a comparison with a high-dose simvastatin therapy

This study was carried out to test the hypothesis that Tongxinluo (TXL) as a Chinese herbal medicine enhances stability of vulnerable plaque dose dependently via lipid-lowering and anti-inflammation effects, similar to a high-dose simvastatin therapy. After abdominal aortic balloon injury, 75 rabbits were fed a 1% cholesterol diet for 10 wk and were then divided into five groups for 8-wk treatment: control group, low-dose TXL group, moderate-dose TXL group, high-dose TXL group, and high-dose simvastatin group. At the end of week 16, an adenovirus containing p53 was injected into the abdominal aortic plaques. Two weeks later, plaque rupture was induced by pharmacological triggering. The incidence of plaque rupture in all treatment groups (14.3%, 7.1%, 7.7%, and 7.1%) was significantly lower than that in control group (73.3%; P>0.01). TXL dose-dependently lowered serum lipid levels and inhibited systemic inflammation. Corrected acoustic intensity and fibrous cap thickness of the aortic plaques were significantly increased, whereas plaque area, plaque burden, vulnerable index, and expression of oxidized low-density lipoprotein (ox-LDL) receptor 1, matrix metalloproteinase 1 (MMP-1), MMP-3, tissue inhibitor of MMP 1, and NF-kappaB in plaques were markedly reduced in all treatment groups when compared with the control group. Similar to high-dose simvastatin group, high-dose TXL group exhibited a low serum level of low-density lipoprotein cholesterol and ox-LDL, a low expression level of systemic and local inflammatory factors and a low plaque vulnerability index, with no differences in the incidence of plaque rupture among all treatment groups. TXL dose-dependently enhances the stability of vulnerable plaques and prevents plaques from rupture. Simvastatin and TXL offer similar protection in terms of lipid-lowering, anti-inflammation, and antioxidation effects.

ACE2 Overexpression Ameliorates Left Ventricular Remodeling and Dysfunction in a Rat Model of Myocardial Infarction

The purpose of this study was to test the hypothesis that overexpression of angiotensin-converting enzyme 2 (ACE2) may favorably affect left ventricular (LV) remodeling and function after myocardial infarction (MI). The left anterior descending coronary artery was ligated to produce anterior MI in 100 Wistar-Kyoto rats that were randomly divided into Ad-ACE2, Ad-ACE2+A779, Ad-EGFP, model, and sham groups. Two weeks later, rats in the Ad-ACE2 and Ad-EGFP groups received direct intramyocardial injection of Ad-ACE2 and Ad-EGFP, respectively. Rats in the Ad-ACE2+A779 group received both intramyocardial injection of Ad-ACE2 and a continuous intravenous infusion of A779 for 15 days. LV volume and systolic function, the extent of myocardial fibrosis, and levels of ACE2, angiotensin II (Ang II), and collagen I protein expression were evaluated. Four weeks after ACE2 gene transfer, the Ad-ACE2 group showed reduced LV volume, extent of myocardial fibrosis, and expression levels of ACE, Ang II, and collagen I in the myocardium, and increased LV ejection fraction and levels of ACE2 activity and expression in comparison with the Ad-EGFP and model groups. These results suggest that ACE2 overexpression attenuated LV fibrosis and improved LV remodeling and systolic function. In conclusion, overexpression of ACE2 favorably affected the pathological process of LV remodeling after MI by inhibiting ACE activity, reducing AngII levels, and up-regulating Ang-(1-7) expression, thus providing a potential therapeutic target in the treatment of heart failure.

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.

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.

Peak radial and circumferential strain measured by velocity vector imaging is a novel index for detecting vulnerable plaques in a rabbit model of atherosclerosis

AIMS To evaluate the reliability of velocity vector imaging (VVI) for detecting vulnerable plaques. METHODS AND RESULTS After aortic balloon injury, 60 rabbits were fed a 1% cholesterol diet for 10 weeks and normal chow for another 6 weeks. Adenovirus containing p53 or lac Z was then injected into the aortic plaques and rabbits were divided into p53-treated group (n=20), lac Z-treated group (n=20) and blank control group (n=20). Peak longitudinal (LSp), radial (RSp) and circumferential (CSp) strain of plaques was measured using VVI at the end of week 18 before pharmacological triggering. Higher RSp and CSp and lower LSp were found in ruptured than those in non-ruptured plaques, and RSp, CSp and LSp correlated well with the fibrous cap thickness and plaque content of macrophages, smooth muscle cells and collagen (all p<0.01). A logistic regression model showed that both RSp (RR: 8.96, 95% CI: 5.3575-10.4857, p<0.001) and CSp (RR: 8.45, 95% CI: 5.9043-9.1043, p<0.001) were significant predictors of plaque rupture. RSp and CSp had a sensitivity of 88.0% and 88.6% and a specificity of 88.6% and 92.0% to predict plaque disruption, respectively. CONCLUSION VVI offers a new and noninvasive technique for measuring the peak strain of atherosclerotic plaques and RSp and CSp are a novel index with a high sensitivity and specificity for detecting plaques vulnerable to rupture.

Intraplaque injection of Ad5‐CMV.p53 aggravates local inflammation and leads to plaque instability in rabbits

This study aims to develop a new animal model of vulnerable plaques and investigate the potential mechanisms of exogenous p53‐induced plaque instability. Forty rabbits underwent aortic balloon injury, were fed a 1% cholesterol diet for 10 weeks and then normal chow for 6 weeks. Rabbits were divided into Ad5‐CMV.p53‐treated group (n= 16), Ad5‐CMV.lac Z‐treated group (n= 16) and blank control group (n= 8). Under the guidance of intravascular ultrasound, a 50‐μl suspension of adenovirus containing p53 or lac Z was injected into the largest plaque of the first two groups, respectively, and these rabbits received pharmacological triggering 2 weeks later. In 76.9% of rabbits with p53 transfection, plaque rupture was found, which was significantly (P < 0.05) higher than that in the Ad5‐CMV.lac Z‐treated plaques (23.1%), or blank controls plaques (0%). Increased apoptotic cells, and subsequently, decreased vascular smooth muscle cells and collagen content, enhanced intima macrophage accumulation, increased C‐reactive protein (CRP) and matrix metalloproteinases staining and high serum levels of high sensitive CRP (hs‐CRP) and monocyte chemoattractant protein‐1 (MCP‐1) were observed in Ad5‐CMV.p53‐treated rabbits. However, a binary logistic regression model revealed that hs‐CRP concentration rather than apoptosis rate played an independent role in plaque rupture with an odds ratio as 1.314 (95% CI: 1.041–1.657, P= 0.021), and there were high positive correlations between inflammatory biomarkers (hs‐CRP or MCP‐1) and apoptosis (R2= 0.761, and R2= 0.557, respectively, both P < 0.01). Intraplaque injection of p53 gene provides a safe and effective method for inducing plaque vulnerability in rabbits. The destabilizing effect of p53 overexpression is mediated mainly through apoptosis‐enhanced inflammation rather than cell apoptosis itself.

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.