| Atherosclerosis is a complicated inflammatory progress characterized by lipid deposition, leukocyte infiltration, and vascular smooth muscle cell proliferation in the vascular walls. Because inflammation in the atherosclerotic process mostly occurs in the absence of microbial infection, it is considered sterile inflammation. Growing evidences suggests that some types of sterile inflammation are mediated by the inflammasome, a large multiprotein complex in the cytosol and a component of the innate immune system. The Nod-like receptor family pyrin domain containing3(NLRP3) inflammasome, the best-characterized inflammasome to date, contains the Nod-like receptor scaffold protein NLRP3associated with the adapter protein, apoptosis-associated speck-like protein containing a CARD (ASC), which recruits caspase-1and induces its activation. Caspase-1induces secretion of interleukin-1β (IL-1β) and interleukin-18(IL-18), which plays a vital role in promoting the development of lipid plaques and destabilizing the plaques. These findings suggest the involvement of the NLRP3inflammasome in the development of atherosclerosis.Recent studies have shown that cholesterol crystal activate the NLRP3inflammasome and release of IL-1β, which indicates that the NLRP3inflammasome may have a role in the development of atherosclerosis. However, another study of double-mutant mice did not find any differences in atherosclerosis progression with or without the NLRP3inflammasome. The reason for this discrepancy is unclear. The role of the NLRP3inflammasome in atherosclerosis remains controversial.In this study, we investigated the expression of NLRP3in aorta of patients with coronary atherosclerosis. Furthermore, we constructed lentiviral vectors to knock down NLRP3to evaluate the role of the NLRP3inflammasome in atherosclerosis in apolipoprotein (Apo) E-deficient mice..Part1. NLRP3Inflammasomes Show High Expression in Aorta of Patients with atherosclerosisObjectives:To investigate the expression of NLRP3in aorta of patients with coronary atherosclerosis and to explore the association between aortic expression levels of NLRP3and atherosclerotic risk factors.Methods:1. The research group consisted of36patients scheduled for CABG surgery at Qianfoshan Hospital. The36patients included25males and11females, aged62.1±10.1. We collected arterial tissues without atherosclerotic lesions from10subjects who donated kidneys to their blood relatives, which composed the control group. These10healthy donors consisted of6males and4females, aged56.2±4.89.2. Coronary angiographies were performed using the transfemoral Judkins approach.3. Specimens were obtained from the aorta that was routinely removed during CABG surgery as a button hole according to the location of atherosclerotic lesions.4. Aortic tissues from CABG surgeries and arterial tissues from kidney donors were fixed in formalin and embedded in paraffin for immunohistochemistry (IHC). A monoclonal mouse anti-NLRP3antibody (Abeam, Cambridge, MA) was used as the primary antibody. Sections after immunohistochemical staining were analyzed using a digital camera equipped with a microscope. We randomly selected3high-power fields per section and then tested the integral optical density (IOD) for positive signals from each field. The mean value of IOD was considered the expression level of NLRP3for each section.Results:1. Among the36patients in the research group who underwent CABG,25patients suffered from unstable angina and11had a history of old myocardial infarction. In addition,13patients had a history of diabetes,19had hypertension, and 8had cardiac dysfunction. Table1shows the serum lipid levels of these patients. Plasma TC, TG, LDL-C, and Lp(a) values increased, whereas the plasma HDL-C values decreased compared with the reference values.By contrast, the10kidney donors in the control group had no history of coronary artery disease, hypertension, or diabetes. Moreover, all were non-smokers and reported no long-term drug use.2. NLRP3proteins were dramatically expressed in all aorta tissues of36patients, and the mean expression level of NLRP3was calculated at879.98±337.39. The renal artery sections of the10kidney donors in the control group were normal as determined by macroscopic observation and routine pathological (histological) examination.3. Spearman’s rank-order correlation revealed that aortic NLRP3expression was highly correlated with Gensini coronary severity scores. The mean Gensini score of all patients in the research group was84.81±23.78.4. NLRP3expression increased in smokers, patients with diabetes, or hypertension (p<0.05). However, no significant differences in the NLRP3expression between male and female patients were indicated (p>0.05).5. Linear correlation analysis revealed that the aortic NLRP3expression was positively correlated with TC (r=0.487,p<0.01), TG (r=0.498,p<0.01),LDL-C (r=0.586,p<0.01), and Lp(a)(r=0.789,p<0.01). By contrast, the NLRP3expression was negatively correlated with serum HDL-C levels (r=-0.694, p<0.01). No direct correlation between the aortic NLRP3expression and TG (r=0.114,p=0.69) was observed.Conclusion:1. NLRP3was overexpressed in the aortas of patients with coronary artery disease.2. The NLRP3expression level was also correlated with the severity of coronary stenosis and clinical risk factors. Part2. Silence of NLRP3Suppresses Atherosclerosis and Stabilizes Plaques in Apolipoprotein E-Deficient MiceObjectives: The role of the NLRP3inflammasome in atherosclerosis remains controversial. The aim of this study was to determine whether inhibition of NLRP3signaling by lentivirus-mediated RNA interference could reduce atherosclerosis and stabilizes plaques. We also tried to explore the mechanisms of the impact of NLRP3inflammasome on atherosclerosis.Methods:Apolipoprotein E-deficient mice aged8weeks were fed a high-fat diet and were injected with NLRP3interfering or mock viral suspension after4weeks. Lentivirus transfer was repeated in2weeks. Four weeks after the first lentivirus injection, we evaluated the effects of NLRP3gene silencing on plaque composition and stability and on cholesterol efflux and collagen metabolism, by histopathologic analyses and real-time PCR.Results:1. All mice were apparently healthy, and no mice were lost before the day of sacrifice. Compared with the control group, the mRNA and protein levels of NLRP3were significantly downregulated, the caspase-1activity was strongly decreased and the content of IL-1(3and IL-18was also decreased after silencing NLRP3(p<0.05).2. In the atherosclerotic lesion area, SMCs content was higher by39%and collagen content was46%higher with NLRP3lentivirus than control transfection. Number of macrophages in the atherosclerotic lesion area, as determined by MOMA-2, was significantly lower by nearly22%and lipid content was33%lower with NLRP3lentivirus than control transfection. The vulnerability index was significantly lower with NLRP3lentivirus than control transfection.3. NLRP3silencing resulted in no significant differences in comparison with controls in body weight. Likewise, serum total cholesterol, triglycerides, LDL and HDL in the NLRP3i group did not differ significantly from those in the control group. 4. In comparison with control, mRNA of ABCA1and ABCG1increased significantly in aortas of ApoE-deficient mice treated with NLRP3silencing (p<0.05).5. Prolyl-4-hydroxylase al (P4Hα1) are rate limiting for collagen synthesis. Matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) contributes to the breakdown of the collagen. The mRNA expression of P4Hα1was markedly higher in the NRLP3i group than the control group (p<0.05), and the mRNA expression of MMP-2and MMP-9was significantly lower in the treatment group than the control group (p<0.05). These two effects led to the increasing of the collagen and thickness of the fibrous cap.Conclusions:NLRP3inflammasomes may play a vital role in atherosclerosis, and lentivirus-mediated NLRP3silencing would be a new strategy to inhibit plaques progression and to reduce local inflammation. |
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