| BackgroundAtherosclerosis (AS) is a systematic disease, and an underlying pathology of the coronary artery disease (CAD). Up to date, the developing process of AS is still not clear. According to the widely-accepted "response-to-injury" hypothesis, the first step in the development of atherosclerosis is the endothelial denudation and (or) endothelial dysfunction caused by the risk factors. Then these activated endothelial cells facilitate monocytes infiltration into the vessel wall and the monocytes differentiate into macrophages. The accumulated lipid remains in the vessel wall, leading to foam cells. The cells release proinflammatory molecules such as tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), and interleukin (IL), which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs). Studies have shown that, with the stimulation of inflammatory cytokines, VCAM-1 expression levels in the endothelial cells could be significantly elevated.VCAM-1 belongs to the immunoglobulin superfamily, with a molecular weight of 100-110 kD. Its ligand is VLA-4, distributed on the surface of leukocytes. VCAM-1 is expressed in vascular endothelial cells, and promotes the adhesion of leukocytes to endothelial cells. VCAM-1 can also accelerate the migration of adherent leukocytes along the endothelial surface, and promote the proliferation of SMCs. Therefore, it is speculated that VCAM-1 might play an important role in the pathogenesis of AS, and accelerate the development and progression of the disease.VCAM-1 molecules on the surface of endothelial cells are usually shed into the blood to form soluble proteins (sVCAM-1). Due to the relative difficulty in evaluating VCAM-1 in vascular endothelial tissues, the detection of sVCAM-1 in blood has always used as an indirect indicator for VCAM-1 expression levels. Hackmen et al. found that the level of sVCAM-1 in the blood was elevated in patients with higher TG.To further understand the relationship between VCAM-1 and CAD, we first select the whole wall samples acquired from CABG (coronary artery bypass graft) operation, measure the expression of VCAM-1 of these samples, and investigate the effect of VCAM-1 expression on AS development.Materials and methods1. Patients and sample collection--- PatientsA total of 36 patients undergoing CABG,27 males and 9 females, aged 46 to 78 years (mean63.6±9.2years), were included in the study. These patients had been admitted to the Qianfoshan Hospital, Shandong University, from December 2008 to February 2012. In the present study, indications for CABG surgery included left main lesions or bifurcation lesions insensitive to medical treatments, severe proximal left anterior descending artery stenosis, three-vessel disease (especially accompanied with cardiac dysfunction or diabetes), and intervention failure. Exclusion criteria were as follows:acute and/or chronic infectious diseases, autoimmune diseases, cancers, liver and/or kidney dysfunction, acute cerebrovascular disease, and peripheral vascular disease. After hospitalization, patients were subjected to physical examination, and the general information was recorded, including gender, age, and histories of smoking, hypertension, and diabetes. Out of these 36 patients,6 cases were smokers,15 cases were with hypertension, and 12 cases were with diabetes.The control group consists of normal arterial tissues, which were collected from 12 cases of kidney transplantation. As indicated by comprehensive physical examinations, these kidney transplant donors were free from organic diseases, and did not have histories of coronary heart disease, hypertension, or diabetes mellitus.---Sample collectionIn patients undergoing CABG, full-thickness aortic wall tissue samples were collected from the surgery, and in the control group, renal artery tissues were obtained from the kidney transplant donors.2. Biochemical determinationIn the next morning after admitted to the hospital, 6 ml venous blood was collected from these subjects in fasting states. Serum triglycerides (TG) and total cholesterol (TC) levels were determined by colorimetric endpoint method. Serum levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C)、total bilirubin (TBIL)、direct bilirubin (DBIL)、indirect bilirubin (IBIL) were detected by chemical modification-enzymatic method, and the levels of lipoprotein (a) [Lp (a)], apolipoprotein (Apo) A, and ApoB were measured by immunoturbidimetry. A MODULAR biochemical analysis system (Roche, Basel, Switzerland) was used for all the above analyses.---Coronary angiography and coronary Gensini scoringCoronary angiography (CAG) was performed via the right femoral artery approach by the Judkins technique. The lesions were directly exposed to do left and right coronary angiography. Angiography images were analyzed by two interventional cardiologists, and the extent of coronary artery stenosis was determined with naked eyes. Gensini score was applied to assess the severity of coronary artery disease, mainly.---ImmunohistochemistryThe samples were fixed in 10% formalin, and then subjected to gradient alcohol dehydration and xylene treatment. After embedded in paraffin, the samples were cut into slices. The sections were dewaxed in xylene and then dipped in gradient alcohol. Then the samples were treated with citrate antigen retrieval buffer under high temperature and pressure, followed by 3% hydrogen peroxide treatment. Rabbit anti-human VCAM-1 polyclonal antibody (dilution 1:300; Bioss, Beijing, China) was used to incubate the sections at 4 overnight. HRP-labeled goat anti-mouse/rabbit IgG polymers was added for a further incubation at room temperature for 30 min, and then DAB reaction was performed, followed by hematoxylin staining. The sections were sealed with neutral resin, and then visualized by an auto image analysis system. Three high-power fields were randomly selected from each slice, and the averaged OD was applied to quantify the expression levels of VCAM-1.---tatistical analysisData were expressed as mean ± SD. SPSS 13.0 software was applied to perform statistical analysis. The t test was used for comparison, and linear correlation analysis was performed to assess the correlation between two variables. p< 0.05 was considered as statistically significant.Results---Measurement of blood lipid levels in AS patients.The blood lipid levels were first measured in these AS patients before surgery. Our results showed that, in AS patients, the serum levels of TG, TC, LDL-C, Lp (a) and ApoB were significantly elevated compared with the normal reference values (p< 0.05). On the other hand, the levels of HDL-C,ApoA, TBILandIBIL in serum were significantly lower than the normal controls (p< 0.05). These results indicate that the significant differences exist in the blood lipid levels between AS patients and normal control subjects, which is in line with occurrence and development of the disease.---CAG analysisAccording to the results from CAG and Gensini scores, coronary heart disease patients could be divided into the following three categories:left main (LM) coronary disease (14 cases), three-vessel disease (14 cases), and two-vessel disease with proximal LAD-lesions (8 cases). The Gensini score ranges from 24 to 128(66.64 ±29.11).---Expression levels of VCAM-1 in arterial tissues in AS patients.In order to investigate the role of VCAM-1 in the pathogenesis and development in AS, the expression levels of VCAM-1 in arterial tissues were measured by immunohistochemistry in these patients. We observed that brown staining of VCAM-1 was mainly located in the cytoplasm in endothelial cells and smooth muscle cells, as granular, flake, and strip clusters. Our results indicated that the expression level of VCAM-1 in arterial tissues in AS patients was 0.21 ± 0.05 OD units, ranging from 0.14 - 0.40 OD units, which was significantly higher than that in the control group (0.08 ± 0.03 OD units, ranging from 0.06 - 0.10 OD units). These results suggest that the expression levels of VCAM-1 in arterial tissues in AS patients are significantly elevated, compared with the normal control subjects.---Relationship between the expression levels of VCAM-1 in aortic tissues and coronary lesion severity in AS patients.In order to investigate whether aortic VCAM-1 expression levels were associated with the lesion severity of AS, these patients were subjected to coronary angiography (CAG) and the Gensini scores were evaluated. According to the results from CAG and Gensini scores, coronary heart disease patients could be divided into the following three categories:left main (LM) coronary disease (14 cases), three-vessel disease (14 cases), and two-vessel disease with proximal LAD-lesions (8 cases). We also observed that higher Gensini score in these categories was accompanied with higher VCAM-1 expression levels. The correlation analysis showed that the Gensini scores (i.e., the lesion severity) were positively correlated with the expression levels of VCAM-1 in AS (r = 0.431, p< 0.05). These results suggest that the expression level of VCAM-1 is linked with the extent of coronary artery disease, support the role of VCAM-1 in the pathogenesis of the disease.---Relationship between VCAM-1 expression in aortic tissues and the main risk factors for AS in AS patients.We next investigate whether the expression of VCAM-1 in AS patients would be affected by AS risk factors. Subgroup analyses were performed:gender, smoking, hypertension, and diabetes. Our results showed that, there were no significant differences in VCAM-1 expression levels in aortic tissues between male and female AS patients (0.21 ± 0.04 vs 0.22 ± 0.07 OD units, p> 0.05). On the other hand, the VCAM-1 expression levels were significantly higher in smokers (0.27 ± 0.05 OD units) than non-smokers (0.15 ± 0.04 OD units) (p< 0.05). The expression levels of VCAM-1 in aortic tissues were significantly higher in AS patients with either hypertension (0.23 ± 0.06 OD units) or diabetes (0.27 ± 0.08 OD units), compared with non-hypertensive (0.19 ± 0.04 OD units) and non-diabetic (0.19 ± 0.02 OD units) patients, respectively (p< 0.05). These results indicate that, as risk factors for AS, smoking, hypertension, and diabetes are also associated with elevated VCAM-1 expression in AS patients. Conclusions1. The study focuses on the whole wall samples acquired through CABG, and demonstrates that VCAM-1 shows a significant high expression relative to control group.2. The study utilizes the samples collected from living objects; thus, the experimental results are more objective and accurate relative to the existing approaches.3. The study shows a positive correlation between VCAM-1 expression and AS risking factors, implying that AS risking factors can promote VCAM-1 expression, and VCAM-1 in turn leads to the development of AS.Backgrounds:Atherosclerosis related diseases have been the main threats of human health, and cardiovascular diseases caused by atherosclerosis have been the leading cause of mortality in lots of countries. Atherosclerosis is a chronic progressing disease in which plaques are formed in large and mid-sized arteries. Plaques cause the stenosis and thrombosis in arteries, eventually leading to the severe clinical outcomes. Current studies have found that lots of immune cells and inflammatory mediators involve in the formation and the progression of atherosclerosis. Inflammatory hypothesis has been accepted by most scholars.Vascular cell adhesion molescule-1 (VCAM-1) plays a crucial role in the pathophysiology of atherosclerosis. The adhesion and aggregation of leukocytes are very important parts in the early stage of atherosclerosis. Normal endothelial cells are not able to be adhered by leukocytes. However, in the location of atherosclerosis, leukocytes adhere to the surface of intima, and penetrate the intima through the connections between endothelial cells. Adhesion molecules expressed in the membrane of endothelial cells involve in this progression. VCAM-1 expressed in endothelial cells could accelerate the adhesion of leukocytes and endothelial cells, and promote the proliferation of smooth muscle cells (SMC). As a result, VCAM-1 plays a vital role in atherosclerosis, accelerating the progression of atherosclerosis.RNA interference (RNAi) is a gene silencing progress mediated by double stranded RNA (dsRNA). The dsRNA can induce a homology dependent degradation of cognate mRNA, resulting in gene silencing. As a substitution of gene knockout, RNAi has been widely used.There are two kinds of dsRNA vectors used in RNAi technology. One is the nonviral vector, mainly the plasmid carrying dsRNA, the main disadvantage of which is the low transfection efficiency. The viral vector is the other kind of dsRNA vector. Among which, the lentiviral vector is most widely used, for its stable gene expression, efficient gene transfer, low immunogenicity, and high biological safety. The interference mediated by lentiviral vectors causes the most stable and persistent target gene changes.Objectives:The aim of this study was to determine whether inhibition of VCAM-1 signaling by lentivirus-mediated RNAi could reduce atherosclerosis in apolipoprotein E (ApoE) knockout mice.Methods:1. Animal protocolA total of 30 male ApoE deficient mice were fed a high-fat diet. After 4 weeks, mice were divided into control group and gene interfering group, which were injected via femoral vein with mock viral suspension (1x108 TU/ml,20ul) and VCAM-1 interfering viral suspension (1x108 TU/ml,20ul). Four weeks after the lentivirus injection, mice were anesthetized and perfused with normal saline with needle inserted into the left ventricle. The aortic root vessels were removed and embedded in optimal cutting temperature (OCT) compound. The rest of aortas were collected for protein extraction.2. Serum lipid level measurementTotal triglycerides (TG), total cholesterol (TC), High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were detected with an automated enzymatic technique.3. Western BlotThe VCAM-1 protein expression in aortas of mice in the control and gene interfering group was determined by western blot.4. Histological analysisOCT compound-embedded aortic root vessels were cross-sectioned into pieces. Sections were stained with hematoxylin and eosin (H&E), oil red O staining for lipids and monocyte/macrophage-specific antibody (MOMA)-2 for macrophages.Results:1. All mice were apparently healthy, and no mice were lost before the day of sacrifice. Gene silencing resulted in no significant differences in comparison with controls in body weight.2. Compared with the control group, the protein levels of NLRP3 were significantly downregulated (p<0.05).3. Serum TC, TG, LDL and HDL in the VCAM-1 interfering group did not differ significantly from those in the control group.4. H&E staining showed that compared with control group, the atherosclerotic plaque areas were significantly smaller in gene silencing group (p<0.05). In the atherosclerotic lesion area, lipid content was lower with VCAM-1 lentivirus than control transfection (p<0.05). Number of macrophages determined by MOMA-2 was significantly lower with VCAM-1 lentivirus than control transfection (p<0.05).Conclusion:1. Lentiviral vectors expressing siRNA provide an efficient, specific and safe tool to knock down VCAM-1 genes in mice.2. Plaques of gene silencing group showed smaller plaque areas and lower content of lipid than did the control group.3. The lower content of macrophages in plaques in VCAM-1 silencing group might be the mechanism by which VCAM-1 silencing reduces atherosclerosis. |
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