| Background: Atherosclerosis(AS) is the pathophysiologic basis of ischemic cardiovascular and cerebrovascular diseases, its pathogenesis has not yet been clarified. In recent years, AS has been considered as an inflammatory disease and innate immunity mediated by macrophages play an important role in the initiation and progression of atherosclerosis. As an important biomarker of macrophage activation, siglec-1 (sialic acid-binding immunoglobulin-like lectin 1, also known as CD169 or sialoadhesin) is highly expressed in the peripheral blood and organ-specific lesions in SLE, systemic sclerosis, HIV infection and other autoimmune diseases and inflammatory diseases. And it can participate in the secretion of cytokines and activation of T lymphocytes and other inflammatory processes. But the exact role of siglec-1 in AS has not been elucidated so far.Objective: By the method of peripheral blood analysis, in vitro cell culture and in vivo animal experiments, we want to explore the expression of siglec-1 in coronary heart disease patients and the exact role of siglec-1 in the process of AS. And to evaluate the possibility of siglec-1 as an indicators for monitoring disease severity or as a potential therapeutic target.Methods:(1) Phenotypic analysis of peripheral blooda) Flow cytometric analysis of CD14CD169 double-positive cells in peripheral blood mononuclear cells (PBMCs) in patients with coronary heart disease and normal controls.b) To separate PBMCs, extracte total RNA, reverse transcript into cDNA and detect siglec-1 mRNA expression by QRT-PCR.c) To calculate Gensini Score, the score to reflect the degree of coronary stenosis, according to the results of coronary angiography, and to do correlation study of Gensini Score with siglec-1 mRNA level.d) To determine high sensitivity C-reactive protein (hs-CRP) and homocysteine (HCY), the risk factors for coronary heart disease patients, and to do correlation analysis of hs-CRP, HCY with siglec-1 mRNA level.(2) In vitro cell culturea) In vitro culture of RAW264.7 macrophage cell line and primary mouse bone marrow-derived macrophages (BMMs).b) A variety of stimulating agents such as ox-LDL, M-CSF, INF-α, etc. to up-regulate the expression of siglec-1 by macrophages; small interfering RNA or blocking antibodies to inhibit the expression of siglec-1 by macrophages.c) To determine chemokine MCP-1, MIP-1alpha, MIP-2 secretion and phagocytosis of ox-LDL by macrophages.d) Peripheral blood CD4+, CD8+ and CD14+ cells were seperated by magnetic activated cell sorting (MACS) and mixed lymphocyte reaction (MLR) of monocytes (CD14 +) and lymphocytes (CD4 +, CD8 +) was performed. Then siglec-1 on monocytes was inhibited or enhanced and CD4 + and CD8 + T cell proliferation and cytokines secretion in MLR were determined.(3) Animal experimentsa) To establish atherosclerotic animal model by feeding ApoE-/ - mice with high fat diet.b) Construction of lentiviral vector with siglec-1 si-RNA sequence and control lentiviral vector with scrambled sequence. Injection of mice by tail vein and to observe atherosclerotic plaque formation.c) Immunohistochemistrical analysis of CD4 + T cells, CD8 + T cells and macrophages in the plaque.d) To determine inflammatory cytokines (IL-1β, TNF-α) secretion of aorta by QRT-PCR.Results:(1) Phenotypic analysis of peripheral blooda) Siglec-1 protein was not found in lymphocytes and neutrophils in both CAD patients and healthy controls. The positive rate of monocytes expressing Siglec-1 in CAD group was significantly higher than that in healthy controls [(11.5±3.9)% versus (1.8±2.0)%, P<0.01]b) Siglec-1 mRNA level was 3.17 times higher in CAD group than that in control group. But no significant differences were observed in different CAD groups (AMI, SA and UA) and in CAD patients with normal and abnormal serum lipids.c) When correlation studies were performed, close correlations between Siglec-1 mRNA level and Gensini score, hs-CRP and HCY were observed (r=0.338, P=0.015; r=0.316, P=0.016; and r=0.224, P=0.042, respectively).(2) In vitro cell culture a) A variety of stimulating agents such as ox-LDL, M-CSF amd INF-αcan enhance the expression of siglec-1 by macrophages; small interfering RNA or blocking antibodies can specifically inhibit the expression of siglec-1 by macrophages.b) Enhanced expression of siglec-1 on macrophage can significantly increase MCP-1, MIP-1alpha, and MIP-2 secretion and strengthen phagocytosis of ox-LDL by macrophages. When siglec-1 was down-regulated, all these effect reversed.c) CD14+ monocytes from CAD patients has a more robust role in stimulating T cells proliferation and pro-inflammatory cytokine production compared with monocytes from healthy controls. Furthermore, up-regulation of siglec-1 on healthy monocytes by IFN-αtreatment could enhance CD4+ or CD8+ T cell proliferation and pro-inflammatory cytokine (IL-2, IL-12 and INF-γ) production, decrease anti-inflammatory cytokine (IL-10) production. All these effect could be reversed by siglec-1 knockdown.(3) Animal experimentsa) The percentage of atherosclerotic plaque area of the total aorta was smaller in si-RNA group than that in control group [(14.043±1.342)% vs. (33.487±2.715)%, p <0.001].b) CD4+ T cells, CD8+ T cells and macrophages infiltration in plaques of si-RNA group was slighter than that in control group. The mRNA level of IL-1βand TNF-αwere lower in si-RNA group than those in control group (0.2646±0.0387 vs. 1.0032±0.0969, p <0.001 and 0.1820±0.0193 vs. 1.0119±0.1964, p <0.01, respectively).Conclusions: Siglec-1 may be considered as a potential noninvasive indicator for monitoring disease severity and a biomarker for predicting the relative risk of cardiovascular events. By the stimulation of ox-LDL and other stimuluses, siglec-1 can be highly expressed on macrophage and involved in macrophage uptake of lipid and turn into foam cells. Furthermore, it can active macrophages and enhance the secretion of MIP-1 alpha, MCP-1 and IL-8, attracting more macrophages and lymphocytes to the site of inflammatory plaque. Highly experssed siglec-1 can also activate CD4+ and CD8+ T cells. Targeted inhibition of siglec-1 can reduce macrophage uptake of lipid, secretion of chemokines and activation of CD4+/CD8+ T cells. Therefore, inhibition of siglec-1 expression by molecular techniques and antibody engineering may possibly serve as a new approach to treat or prevent the initiation and progression of atherosclerosis.
|
PDF Full Text Request