Effects of Antiatherosclerosis in Carotid Artery by RNAi-Mediated Silencing of MCP-1 Expression

Background

Our objective was to identify the effects of MCP-1 siRNA in vivo transfection in an atherosclerosis model on local expression of MCP-1 and pathogenesis of atherosclerosis.

Methods

Carotid atherosclerosis was induced in 28 New Zealand white rabbits. Rabbits were divided into three groups randomly: RNAi group, model group, and blank plasmid group. siRNA-expressing vector was transfected to blood vessels by liposomes. The carotid arteries were processed for morphological evaluation. Local expression of MCP-1 was detected by immunohistochemistry, RT-PCR, and Western blot.

Results

On hematoxylin and eosin–stained sections, partial endothelial cells detached while intimae were less thickened in the RNAi group compared to the model and blank plasmid groups; the I:M ratio was significantly reduced to 1.46 in the RNAi group compared to the model and blank plasmid groups (5.55 and 5.27, respectively). The results of immunohistochemistry showed that MCP-1 expression was less colorized and less positive in the RNAi group. RT-PCR and Western blot showed reduced expression in the RNAi group than in the model and blank plasmid groups. There were highly positive correlations between semiquantitative RT-PCR and the I:M ratio (r = 0.968).

Conclusion

Expression of MCP-1 was successfully inhibited by transfecting MCP-1 siRNA expression plasmid to the carotid artery, and the progression of atherosclerosis was restricted by RNAi-mediated silencing of MCP-1 expression.

Introduction

Carotid artery stenosis is a major health threat that causes disability and death and is one of the leading causes of stroke.¹ The mechanisms behind the development of atherosclerosis (AS) have been studied for half a century. Three main theories—namely, lipid infiltration, injury and repair, as well as thrombosis—have been put forward to explain atherogenesis. As experimental pathological studies go ahead, researchers have discovered that AS plaques show not only substantial accumulation of lipids but also infiltration of large numbers of inflammatory cells and secretion of inflammatory cytokines. In 1976, Ross and Glomset² proposed the “injury-repair” theory to explain the formation of AS plaques. In the 1980s, he further proposed the “endothelial injury-repair” theory. In the 1990s, many scholars conducted numerous studies related to inflammatory factors, inflammatory markers, and AS plaques and thought that the inflammatory response was involved in the development and progression of AS plaques. In 1999, Ross explicitly proposed that AS was a kind of inflammatory proliferative disease. He believed that the formation of AS lesions resulted from the inflammatory fibroplasia of the artery after responding to arterial endothelial dysfunction caused by various major risk factors for this disease.3, 4, 5, 6, 7 The earliest pathomorphological changes in AS are characterized by the migration of monocytes/macrophages into the tunica intima and formation of subendothelial foam cells, followed by the migration (from tunica media to tunica intima) and hyperplasia of smooth muscle cells (SMCs). Currently, it is generally believed that monocyte chemotactic protein-1 (MCP-1)-mediated migration of monocytes into the arterial endothelium plays a key role in the early development of AS. MCP-1, as a member of the CC chemokine subfamily, is a glycoprotein with a molecular weight of 14 × 10³. Studies show that vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), mononuclear cells (MCs), macrophages, and fibroblasts express MCP-1. Through specifically acting upon peripheral blood MCs and attracting them to migrate into the subendothelium, MCP-1 plays an important role in the development and progression of AS.8, 9 Yla-Herttula et al.¹⁰ examined the expression of MCP-1 in rabbit and human AS lesions using Northern hybridization, in situ hybridization, and immunohistochemistry. They found that MCP-1 was positively expressed in rabbit AS arteries and Mφ-derived foam cells isolated from macrophage (Mφ)-rich AS lesions. In contrast, MCP-1 was negatively expressed in normal arteries and alveolar Mφ isolated from the same animal. In situ hybridization showed that MCP-1 mRNA was positively expressed in human and rabbit macrophage (Mφ)-rich AS lesions, while immunohistochemical analysis further proved that MCP-1 protein was expressed in MCP-1 mRNA-positive regions. In MCP-1-postive regions, ox-LDL was also positively expressed, which is consistent with the observation that ox-LDL could induce the in vitro expression of MCP-1 in VECs and VSMCs, suggesting that ox-LDL may be involved in the development of AS lesions by inducing the expression of MCP-1. Through studying the tunica intima of the carotid artery in patients undergoing endarterectomy and comparing that in subjects with normal arteries, Nelken et al.¹¹ found that MCP-1 was positively stained in 16% of cells in AS carotid arteries, while the percentage of MCP-1-positive cells in normal arteries was <0.1%, proving that MCP-1 plays a role in the migration of monocytes into the wall of AS arteries. Schwartz et al.¹² proved that MCP-1 was a strong monocyte chemotactic factor and could regulate the expression of relevant adhesion molecules in the vascular endothelium. After crossing CCR2 (a receptor of MCP-1)-deleted mice (CCR2-/-) with apolipoprotein E (apoE)-deleted mice (apoE-/-), Boring et al.¹³ discovered that CCR2 deletion could significantly reduce the incidence of AS. Similar studies also showed that in apoE-/- mice MCP-1 overexpression–induced acceleration of AS was associated with an increase in the number of macrophages in AS plaques.14, 15, 16 These discoveries indicate that MCP-1 plays an important role in the formation of AS, and it has been recognized as a new target for the prevention and treatment of AS.

Domestic and foreign scholars have made many attempts to explore the possibility using MCP-1 as a target for anti-inflammatory therapy.17, 18, 19, 20 Theoretically, many technologies, such as antisense technology, ribozyme technology, as well as peptide nucleic acid- and small-molecule peptide–based technologies, can be used to specifically inhibit the expression of target genes. However, these molecules or drugs are not easily designed. Moreover, it is possible to obtain the molecules that can specifically and efficiently suppress the expression of target genes only after many trial-and-error and screening tests are performed. The emergence of RNA interference (RNAi) technology provides a new clue for blocking the expression of MCP-1. As a new technology to inactivate target genes, RNAi has the advantages of easier design as well as higher inhibitory efficiency and specificity when compared with the above-mentioned technologies. Therefore, it has played important roles in studying gene function and screening drug targets in recent years.21, 22, 23, 24 At present, two strategies are mainly used in RNAi. One is to directly transfect small interfering (si) RNA (chemically synthesized or transcribed in vitro) into cells; the other is to introduce a vector system (plasmid or viral vectors) into cells, which can endogenously express siRNA.

In the present study, we constructed a eukaryotic expression vector of siRNA specific for MCP-1 and established a rabbit model of carotid AS. After liposome-mediated local transfection, we observed the impact of MCP-1 siRNA on the expression of MCP-1 in the artery and further analyzed the effect of MCP-1 siRNA on AS. The results will provide new clues for developing new interventional approaches for the prevention and treatment of this disease.