Abstract
Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE−/− mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE−/−) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P<0.001). Compared with the Ad-EGFP group, carotid plaques in the Ad-MCP-1i group showed increased levels of collagen and smooth muscle cells, and decreased levels of lipid and macrophages. The expression of inflammatory cytokines and activities of matrix metalloproteinases (MMPs) were lower in the Ad-MCP-1i group than in the Ad-EGFP group. In conclusion, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption. A marked suppression of the local inflammatory cytokine expression may be the central mechanism involved.
Highlights
Atherosclerotic plaque disruption has been identified as the major cause of acute coronary syndrome [1]
RT-PCR and western blot analyses further demonstrated that the mRNA and protein expression levels of Monocyte chemoattractant protein-1 (MCP-1) in the carotid plaque were significantly lower in the Ad-MCP-1i group than in the Ad-enhanced green fluorescent protein (EGFP) group (Figure 2B and 2C)
Colocalization of adenovirus vector and MCP-1 expression by immunostaining further confirmed that MCP-1 expression was especially reduced in the GFP-positive lesion in the Ad-MCP-1i group, compared with the Ad-EGFP group (Figure 2D)
Summary
Atherosclerotic plaque disruption has been identified as the major cause of acute coronary syndrome [1]. A plaque with a large necrotic core and a thin fibrous cap infiltrated by inflammatory cells is vulnerable to disruption. MCP-1 has become an important therapeutic target for atherosclerosis and three treatment approaches have been developed in experimental studies. The first approach involved systemic blocking MCP-1 by injection of an N terminal-deletion mutant of the MCP-1 gene (7ND) and Inoue S et al first showed that 7ND treatment attenuated progression of the atherosclerotic lesions in ApoE2/2 mice [11]. It is difficult to translate this approach into clinical application because long-term injection of 7ND may cause hypersensitivity reaction and systemic immunosuppression. The third approach entails local gene silencing of MCP-1 using RNA interference (RNAi) and if this technique proves effective and safe, it would be feasible to translate RNAi into clinical application by combining small interference
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