Suppression Of Matrix Metalloproteinase Via RNA Interference Inhibits Atherosclerosis In Mouse Model

With the improvement of living standards and changes of lifestyle, the cardiovascular diseases such as acute coronary syndrome (ACS) and stroke which affect the people's health and quality of life seriously have been the main causes of death and disability both in domestic and worldwide and the incidence are sharply raised. Atherosclerosis is well known as the main pathological basis of cardiovascular diseases and the vulnerable plaque rupture and thrombosis are closely related to it.Atherosclerosis is a complex pathophysiological process referred to multiple factors, in which Systemic inflammation play roles in the initial and the development, including plaques' instability and the final patches' rupture.The unstable plaque, also called vulnerable plaque, is found to be related to Matrix metalloproteinases (MMPs), which are proteolytic enzymes which are strongly related to the degradation extracellular matrix proteins. They are secreted by the macrophages and blood vessels smooth muscle cells stimulated by inflammatory cells, e.g. IL-1, TNF-α, CD154 and the activated T lymphocytes. At present,23 kinds of MMPs have been identified and qualitative, in which MMP-9 is proved to be closed to the rupture of atherosclerosis plaque most. A lot of studies have demonstrated that the levels of MMP-9 in the plasma in ACS patients are significantly higher than those with stable angina. Further, it is showed in autopsy, the expression of MMP-9 in vulnerable plaque from coronary is much higher than that in stable plaque. Also, the atherosclerosis animal model indicated the rich expression of MMP-9 in the shoulders of atherosclerotic plaque. Besides, some atherosclerosis mouse models showed that when the macrophages in the plaque were induced to over-express activated MMP-9, the degradation of elastin was significantly increased, leading to plaque rupture. Hence, it is possible to benefit from suppressing the expression MMP-9 to prevent the rupture of vulnerable plaque. However, currently, a difficult problem people have to confront in clinical practice is how to detect and treat the vulnerable plaque in the asymptomatic individuals.At present,23 kinds of MMPs have been identified and qualitative, in which MMP-9 is proved to be closed to the rupture of atherosclerosis plaque most. A lot of studies have demonstrated that the levels of MMP-9 in the plasma in ACS patients are significantly higher than those with stable angina. Further, it is showed in autopsy, the expression of MMP-9 in vulnerable plaque from coronary is much higher than that in stable plaque. Also, the atherosclerosis animal model indicated the rich expression of MMP-9 in the shoulders of atherosclerotic plaque. Besides, some atherosclerosis mouse models showed that when the macrophages in the plaque were induced to over-express activated MMP-9, the degradation of elastin was significantly increased, leading to plaque rupture. Hence, it is possible to benefit from suppressing the expression MMP-9 to prevent the rupture of vulnerable plaque. However, currently, a difficult problem people have to confront in clinical practice is how to detect the vulnerable plaque in the asymptomatic individuals and treat them.In recent years, with the deeper awareness of the vulnerable plaque and the development of technologies, the diagnosis of vulnerable plaque becomes possible. For example, to detect vulnerable plaque in population with high risk factors via the measurement of MMPs, C-reactive protein (CRP) and high-sensitivity C-reactive protein (hsCRP), oxidized LDL (ox-LDL); to determine the vulnerability plaque via coronary angiography, intravascular ultrasound and intravascular optical coherence tomography, though which are limited as routine examinations.At present, the systemic drug intervention including statins application for prevention and treatment of atherosclerotic diseases has achieved remarkable results, but the coronary attacking still repeats. Most recently, the studies proposed a promising targeted therapy treatment for the prevention of repeated coronary attacking, which based on the mechanisms of vulnerable plaques and make use of molecular biology techniques. Considering the higher expression of MMP-9 is greatly contributed to the rupture of vulnerable plaques, so the present study hypnotized MMP-9 may be the ideal target of genetic intervention, and, the suppression of MMP-9 expression in atherosclerosis plaque may be prevent the rupture of plaque and treat ACS. RNA interference is a novel skill to inhibit the expression of gene in recent years, which can selectively inhibit specific gene expression and is described as double-stranded RNA molecules induce sequence-specific gene silencing in mRNA level after transcriptional. Now the RNAi is widely used in the study of gene function and gene therapy in some disorders. RNAi needs vectors to work in human's body, so the Lentviral vector derived from human immunodeficiency virus-1 that is characterized by infecting non-dividing cells, accommodating large fragments of exogenous gene, vulnerable immune response, and minute virus vector-mediated RNAi effect processes for long-lasting and difficult to induce host immune reaction, therefore, the present study aims to detect the effect of suppressed MMP-9 expression on atherosclerosis plaque via RNAi technology and the lentviral as vector, and try to explore a novel way for the prevention of vulnerable plaque.In this study, the lentiviral vector targeting interference MMP-9 in vitro is constructed first, and the interference effects are verified in vitro cell experiments; next, we transfect ApoE-/ - mice by lentiviral vector that RNA interferenced and establish mouse model of atherosclerosis, finally, we observe the influence of MMP-9 expression on atherosclerotic plaques in order to search for targeted therapy of vulnerable plaque in the new way.First:construction of the lentiviral vector of MMP-9 RNA interferenceMethods:Based on MMP-9 gene sequences of the mouse, using the public Web site in accordance with the principle of RNA interference sequences, design multiple RNA interference target sequence, using kinetic parameters to select the best target for interference sequence containing double-stranded synthetic DNA oligo, connecting to the pGCL-GFP digested vector to construct recombinant plasmid, then PCR analysis and sequencing were performed.Results:We successfully constructed pGCL-GFP/MMP-9 plasmid, agarose gel electrophoresis and gene sequencing showed that the obtained sequences were accordant with Gene Bank in the MMP-9 gene sequence.Second:Construction of eukaryotic expression vector for MMP-9 Methods:To obtain the MMP-9 gene by PCR from the cDNA library, and with pGCL-GFP vector were double-digested to recombinant, which were transformed into competent cells, PCR identificated; recombinant plasmid transfected 293T cells. Observe MMP-9 expression under fluorescence microscope after 48 hours.Results:Clones were identified positively by PCR, indicating that MMP-9 gene has been directed into pGCL-GFP vector; we can observe green fluorescent protein under a fluorescence microscope which expressed by 293T cells, indicating that MMP-9 expression in 293T cells.Third:MMP-9 RNA interference lentiviral vector silencing of the mouse in vitroExperimental Methods:MMP-9 RNA interference lentiviral vector and MMP-9 eukaryotic expression vector transfected into 293T cells, to detest MMP-9's silence effect in protein levels by Western blot; followed by co-transfected NIH3T3cells and Raw264.7 cells, then to detect MMP-9 expression in mRNA level by Real-time PCR, and then determine the effect of the disturbance; observed expression of green fluorescent protein under a fluorescence microscope, which determine the silencing effect.Results:Western blot showed that compared with the negative control group, group with MMP-9 interference was significantly knocked down in the role; Real-time PCR showed that interference group had interferential expression of MMP-9 mRNA gene significantly in NIN3T3 cells and Raw264.7 cells , interfering efficiency above 75%, and to the normal control group and positive control group, there was significant difference (P<0.001); under the fluorescence microscope we can observe, interference cells giving green fluorescent protein expression was significantly less than control group.Fourth:lentivirus-mediated MMP-9 RNA interference on atherosclerosis of ApoE-/ - miceMethods:10-week-old ApoE-/- mice was divided into 3 groups (each group n= 10), the PBS group, Lentiviruses-GFP group and Lentiviruses-MMP-9 intervention group, respectively. Injected PBS, Lentivirusesvector and Lentiviruses- MMP-9 by intravenous, given high fat feeding for 20 weeks then tested patch area; activity of antigens for MMP-9 in the plaque and vascular smooth muscle cells (SM-actin); simultaneous determinate MMP-9 and hs-CRP levels in serum of three groups.Results:HE staining showed that the average plaque area of the three groups had no significant difference in the average area. The three groups were:PBS group 0.99±0.14 mm2, GFP group 0.96±0.13 mm2, MMP-9 treated group 1.02±0.10 mm2, but compared PBS group and Lentiviruses-GFP group, Lentiviruses-MMP-9 in the intervention group macrophages reduced significantly in shoulder patch, and fibrous cap became thick. SM-actin antigen activity was significantly enhanced in MMP-9 intervention group compared with the other two groups, but the activity of MMP-9 antigen was significantly decreased. In PBS group and the GFP group, MMP-9 and serum hs-CRP levels were not different (respectively:798.7±115.1μg/L vs 801.3±129.4μg/L and 3.48±0.34mg/L vs 3.39±0.38 mg/L), and in MMP-9 interference group, MMP-9 and hs-CRP levels (728.5±208.1μg/L and 2.43±0.22 mg/L) in serum were significantly lower than that in the PBS group and the GFP group (P<0.01).The study found that the atherosclerosis and the expression of MMP-9 in plaque is inhibited in RNA interference lentiviral vector-transfected ApoE-/- mice, followed by the surface of the plaque cap became thick, and promotion the stability of the plaque, then protect the plaque from rupture. The possible mechanisms is:on the one hand, lentviral can infect non-dividing cells and act long-lasting, therefore, which may continue to inhibit the expression of MMP-9 within the plaque, thereby, the degradation of extracellular matrix is reduced, and the fibrous cap become thicker; on the other hand, vascular smooth muscle cells may promote migration to the plaque surface, resulting in the thicken plaque surface, besides, the reduction of inflammatory mediators and MMP-9's level in plasma may be as a mechanism for plaque stabilization also.Molecular cloning, immunofluorescence, Real-time-PCR and Western-blot and other experimental techniques have been used to study the MMP-9 RNA interference lentiviral vectors for high-fat feeding in ApoE-/- mice with atherosclerosis. We illustrate the expression of MMP-9. interference in vivo and vulnerability atherosclerosis plaque and provide information for further treatment of atherosclerosis vulnerable plaque from cellular and molecular prospectives.