Increased Expression Of Chitinase3-like1in Atherosclerosis And Suppression Of Atherosclerosis In Apolipoprotein E-Knockout Mice By Gene Silencing

Background and ObjectivesAs a systematic disease, atherosclerosis is the underlying pathology of the coronary artery disease (CAD) and is a chronic inflammatory disease. According to the "response-to-injury" hypothesis, the endothelial denudation and (or) endothelial dysfunction caused by the risk factors are the first step in the development of atherosclerosis. These activated endothelial cells facilitate monocytes infiltration into the vessel wall and the monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells mentioned above 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).Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1can be secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.Although the relationship between CHI3L1and CAD is important, there is a controversy in the association between blood CHI3L1levels and the severity of atherosclerosis. One study showed that severity of atherosclerosis is associated with higher blood CHI3L1levels, and another paper concluded that circulating CHI3L1was not specifically related to the size of atherosclerotic stenosis. In order to elucidate the relationship between CHI3L1and CAD, we investigated the correlation between CHI3L1expression levels and pathogenesis of atherosclerosis by measuring the changes of CHI3L1in the aortic tissues of patients undergoing coronary artery bypass graft (CABG) surgery.Materials and Methods1. Study populationFrom2010to2012,39patients with CAD scheduled for CABG surgery were investigated and defined as a research group. Eleven normal subjects who donated kidneys were investigated as the control group. Aortic specimens were obtained from the aorta that was routinely removed during the CABG surgery and the discarded renal arterial tissues were collected from the11subjects.2. Biochemical analysisVenous blood samples were obtained for the measurement of serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), lipoprotein (a)(Lp (a)), apolipoprotein A (ApoA), apolipoprotein B (ApoB), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL) as well.3. Coronary angiographyAll CAD patients were confirmed by the coronary angiography (CAG). Gensini score system was used to assess the CAD severity.4. Tissue preparation and histological analysisThe arterial tissues obtained from the CABG surgery and kidney donators were fixed in4%buffered formalin, embedded in paraffin and sectioned. After deparaffinage, hydration and antigen retrieve, the sections were incubated with the rabbit antihuman CHI3L1antibody.Results 1. Baseline characteristicsThe serum TG, TC, LDL-C, and Lp (a) levels in the research group were elevated, whereas the serum HDL-C, ApoA, TBIL, and IBIL levels were decreased, compared with the normal reference values (p<0.05).2. Coronary atherosclerotic lesions in the research groupCAG in the research group showed that19patients were with left main lesions,6patients were with two coronary arteries lesions, and14patients were with three coronary arteries lesions. The average Gensini score was62.25±21.77.3. Presence of CHI3L1in the human arterial tissuesIn the arterial tissues obtained from the healthy donors little CHI3L1expression could be demonstrated according to the immunohistochemical staining. CHI3L1was overexpressed in the arterial specimens of CAD patients.4. Correlation between the arterial CHI3L1expression and the clinical risk factors of,, atherosclerosisThe expression levels of CHI3L1did not differ between males and females, differ between smokers and non-smokers, hypertensives and non-hypertensives, diabetics and non-diabetics. The relative expression levels of CHI3L1was elevated in smokers, patients with hypertension, or diabetes mellitus, and gender had no significant effect. The linear correlation analysis revealed that arterial CHI3L1expression levels were correlated with the Gensini scores (r=0.61l,p<0.05).Conclusions1. In the present study, we found that CHI3L1was overexpressed in aorta of patients with coronary atherosclerosis and it might provide a new approach to the research of atherosclerosis.2. The relative expression levels were correlated with the atherosclerotic risk factors and the severity of CAD and CHI3L1could be a predictor of the coronary arterial stenosis. Background and ObjectivesAtherosclerosis is a chronic inflammatory disease and is the underlying pathology of the coronary artery disease (CAD). The endothelial denudation and (or) endothelial dysfunction are the first step in the development of atherosclerosis. The activated endothelial cells facilitate monocytes infiltration into the vessel wall. These monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells synthesize and release proinflammatory molecules, which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs).Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1is secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.The relationship between CHI3L1and atherosclerosis is important, in order to verify the therapeutic value of CHI3L1, we constructed lentiviral vectors, which could efficiently deliver small interfering RNAs (siRNAs), and aimed at knocking down CHI3L1to explore the mechanisms of CHI3L1in atherosclerosis in apolipoprotein E-knockout (ApoE-/-) mice.Materials and Methods1. Cell cultureThe293T human embryonic kidney cell line and the RAW264.7mouse macrophage cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with10%fetal bovine serum (FBS),100U/ml penicillin,100μg/ml streptomycin, in a humidified incubator with5%CO2.2. Lentivirus construction and target screening for RNAiFour different sequences (site A, site B, site C, and site D) of mice CHI3L1gene were designed as the target for RNA interference (RNAi).The sequence of site A was:5’-GCGACAACATGCTTAGCACATTTCAAGAGAATGTGCTAAGCATGTT GTCGCTT-3’;the sequence of site B was:5’-GGCCATTGACACTGGCTATGATTCAAGAGATCATAGCCAGTGTCAAT GGCCTT-3’;the sequence of site C was:5’-GCACTGGATTTGGATGATTTCTTCAAGAGAGAAATCATCCAAATCCA GTGCTT-3’;the sequence of site D was:5’-GCCAGAAGGACACTAGGTTTGTTCAAGAGACAAACCTAGTGTCCTT CTGGCTT-3’.As a negative control, the scrambled sequence (mock siRNA) was:5’-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAG AACTT-3’.The pShuttle vectors containing the different mouse CHI3L1RNAi sequences were constructed. A lentivirus was produced by cotransfection of the pShuttle vector, pGag/Pol, pRev, and pVSV-G into the293T cells. The lentiviruses were used to transfect the RAW264.7cells. The RAW264.7cells were collected for RT-PCR and western blot experiment at72h and96h after transfection. 3. Animal experimentWe obtained50male ApoE-/-mice,8weeks old. All the mice were fed a high-fat diet. The mice were divided into2groups:empty lentivirus control group and CHI3L1lentivirus silenced group. A constrictive silica collar was placed on the right common carotid artery of mice. Eight weeks after surgery, the lentiviral suspension at1×109TU/ml was instilled into the right common carotid artery.4. Tissue preparation and histological analysisThe right common carotid artery obtained from the mice was immersed in4%formaldehyde. Six crosssections in each mouse were used for a particular type of staining. One section was stained with hematoxylin and eosin. Another section was immunostained with rabbit antimouse CHI3L1antibody. Collagen and lipids were identified by Sirius red staining and oil red O staining. VSMCs and macrophages were immunostained with a-actin antibody and CD68antibody.5. Transmission electron microscopyThe fresh mice arterial tissues were used to undergo electron microscope examination. The arterial tissues were placed in2.5%glutaraldehyde, then the tissues were postfixed in1%osmium tetroxide followed by staining with2%uranyl acetate. Then the tissues were dehydrated through ethanol and were embedded in Spon812. Finally, the sections were stained with uranyl acetate followed by lead citrate and examined in a JEM-1010electron telescope.6. RT-PCR analysisThe mRNA expression levels of CHI3L1in RAW264.7cells, CHI3L1, TNF-a, MCP-1, IL-8, and MMP-9in mice were analysed using the RT-PCR.7. Western blot analysisThe protein expression levels of CHI3L1, p-ERK1/2, ERK1/2, p-AKT, and AKT in RAW264.7cells and in mice were assayed by western blot.Results1. Effects of lentiviral transfection in vitroThe RAW264.7cell line was transfected with four different CHI3L1siRNAs. Site A, site B, site C, and site D exhibited32%,17%,65%, and30%reduction in mRNA expression. Site A, site B, site C, and site D exhibited38%,18%,64%, and14%reduction in protein expression. Then the site C was the most effective vector in blocking CHI3L1expression. The site C lentivirus and mock lentivirus were produced at a viral titer of1×109TU/ml for further in vivo studies.2. Effects of lentiviral transfection on CHI3L1expression in plaquesIn the control group, CHI3L1expression could be demonstrated according to the immunohistochemical staining. However, little CHI3L1was expressed in the silenced group. Compared with the control group, the silenced group showed reduced CHI3L1protein expression by50%. In addition, there were significant differences of CHI3L1mRNA expression levels between control group and silenced group.3. Electron microscopy analysisFor electron microscopy, in control group most of the endothelial cells denudated and there were a large number of lipid granules under the basement membrane in the vessel wall. The atherosclerotic plaques were occupied with necrotic particles, calcification crystals and cellular debrises. However, in silenced group the number of lipid granules was relatively decreased. Collagen bundles and elastic fibers were seen on the vessel side of the endothelium.4. Effects of lentiviral transfection on plaque compositionThe relative content of lipids in plaque tissues of the control group and the silenced group was48.8%and35.2%, the relative reduction of lipids content in the silenced group was27.5%. The relative content of collagen was19.5%and29.8%, the relative increase of collagen content was53.2%. The relative VSMCs content was14.8%and22.5%, the relative increase of VSMCs content was51.2%. The relative macrophages content was11.9%and7.5%, the relative reduction of macrophages content was36%.5. Effects of CHI3L1gene silencing on inflammatory mediators and signal transduction protein in the lesionsThe CHI3L1silenced group showed lower mRNA expression levels of TNF-a, MCP-1, IL-8, MMP-9, compared with the control group (p<0.05). The silenced group showed lower protein expression levels of p-ERK1/2and p-AKT, compared with the control group (p<0.05).Conclusions1. In the present study, we applied lentivirus mediated shRNA to knock down the mice CHI3L1gene.2. The silence of CHI3L1diminished the atherosclerotic burden and increased plaque stability in ApoE-/-mice, which might provide a new therapeutic approach to the treatment of atherosclerosis.