An Experimental Study Of The Effect Of Renin Overexpression And Combined Drugs On Atherosclerosis And Plaque Inflammation

Atherosclerosis (AS) is a common disease which severely threatens human health. It is also a main reason of coronary artery disease and acute myocardial infarction. A variety of studies indicated that AS is a chronic inflammatory process based on the damage of vascular endothelial cells (VECs).Acute coronary syndromes, including unstable angina, acute myocardial infarction and sudden cardiac death, result from fissure, erosion, or rupture of a vulnerable atherosclerotic plaque. The intrinsic features that characterize a plaque vulnerable are an increased macrophage, T-lymphocyte, foam content, an increased lipid content, and reduced collagen and smooth muscle cell content. The characteristics of stable plaque are composed of solid fibrous or fibrocellular tissue and only small amount of extracellular lipid or no lipid and less inflammatory cell. The plaque nature depends on intrinsic features. Accumulation of macrophage, and T-lymphocyte in plaque may decrease its stability, because these inflammatory cells secrete cytokines and matrix metalloproteinases (MMP), which results in destruction of extracellular matrix and apoptosis of smooth muscle cell.Renin angiotensin system (RAS) has been demonstrated to play a critical role in the initiation and progression of atherosclerosis. AngiotensinⅡ(AngⅡ), a major substrate in RAS, stimulates atherosclerosis through various deleterious effects such as endothelial dysfunction, cellular proliferation and inflammation. AngⅡplays a main role in the RAS by interacting with its specific receptor, AngⅡtype 1 receptor (AT1R). AngiotensinⅠ(AI) and angiotensinⅡ(All) are generated from hepatic angiotensinogen (AGT), after a cascade of proteolytic cleavages involving a rate-limiting enzyme, renin. Renin is produced by the cleavage of prorenin, originating from the juxtaglomerular cells of the kidney. Renin and prorenin binding to the prorenin receptor not only target and facilitate angiotensin generation but also lead to activation of prorenin receptor signal transduction pathways, which is distinct from classical RAS signaling. In the study, we constructed the overexpression vector of human renin and detected the effect on atherosclerotic cytokines.A growing body of evidence has suggested that blood lipid metabolism disorders and angiotensinⅡ(AngⅡ) have synergistic effects on the occurrence of atherosclerosis, and the combination of dyslipidemia and the activation of the renin-angiotensin system (RAS) plays an important role in the pathogenesis of atherogenesis. Oxidized low-density lipoprotein (ox-LDL) can upregulate the expression of the angiotensinⅡtype 1 receptor (AT1R) on the surface of endothelial cells and macrophages. AngⅡfacilitates LDL oxidization and uptake by endothelial cells, smooth muscle cells and macrophages. The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of AS. Recent studies showed that Lox-1 was a key molecule in the generation of endothedial dysfunction and played an important role in endothelial injury, monocyte adhesion to VECs and foam cells form in response to ox-LDL and angiotensinⅡ(AngⅡ). Monocyte chemoattractant protein-1 (MCP-1) is the main chemotactic factor and plays a crucial role in monocyte adhesion to endothelial cells. Monocyte and T-lymphocyte migrate into the subendothelial space by MCP-1, which plays a key role in the subendothelial recruitment of monocyte. The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of AS. In the study, we investigated the effects and mechanisms of the combinational use of fluvastatin and losartan on the development of atherosclerosis and inflammation within the atherosclerotic plaques. Part One Construction and Identification of the Overexpression Vector of Human Renin GeneObjectiveTo construct and identification of the overexpression vector of human renin gene, purified and concentrated the recombinant overexpression virus.Methods1. The sequence encoding renin was amplified by RT-PCR and was cloned to SalⅠand BglⅡsites of pDC316, the positive plasmid constructs named pDC316-Ren. Then the pDC316-Ren plasmid was confirmed by electrophoresis and sequencing.2. The sequence mcmv-ren-polyA was amplified by PCR and was cloned to BamHI sites of pSNAV2.0-tdRFP, the positive plasmid constructs named pSNAV2.0-tdRFP-mCMV-Ren.Then the pSNAV2.0-tdRFP-mCMV-Ren plasmid was confirmed by electrophoresis and sequencing.3. pSNAV2.0-tdRFP-mCMV-Ren was transfected into BHK-21 by Lipofectamine 2000, then along with HSV1-rc/AUL2 into BHK/HIF-2 to package virus particles rAAV2-Ren-RFP. Then the rAAV2-Ren-RFP plasmid was confirmed by electrophoresis. The functional titer was determined.Results1. A fragment about 1.2 kb was amplified, the recombinant plasmid pDC316-Ren was constructed successfully and confirmed by SalⅠand BglⅡdigestion and DNA sequencing.2. A fragment about 1.9 kb was amplified, the recombinant plasmid pSNAV2.0-tdRFP-mCMV-Ren was constructed successfully and confirmed by BamH digestion and DNA sequencing.3. The recombinant virus rAAV2-Ren-RFP was packaged successfully and confirmed by DNA sequencing.4. The functional titer of the recombinant virus rAAV2-Ren-RFP is 8.3×1011vg/ml.Conclusions 1. The recombinant plasmids pDC316-Ren and pSNAV2.0-tdRFP-mCMV-Ren were constructed successfully2. The recombinant overexpression vector rAAV2-Ren-RFP was packaged successfully and concentrated effectively. It was necessary for subsequent research. Part Two The Effect of Human Renin Gene Overexpression on Atherosclerosis and Plaque Inflammation in vitro and in vivoObjectiveAssess the effect of human renin gene overexpression on atherosclerosis and plaque inflammation in vitro and in vivo, and provide a potential therapeutic target in cardiovascular disease.Methods1. Atherosclerosis (AS) was induced in the aortic arteries of New Zealand White Rabbits by endothelial injury after anesthesia and atherogenic diet for 12 weeks.2. Rabbits were randomly divided to 3 groups of high-cholesterol group (CH) and rAAV2-Ren-RFP group and rAAV2-RFP group (n=10 in each group) at the end of 12 weeks, the rAAV2-Ren-RFP group and rAAV2-RFP group were received a suspension of the recombinant renin overexpression virus (5×1010vg per rabbit) through a catheter into the abdominal aortic segments rich in atheromatic plaques and the rAAV2-RFP group were injected with empty vector.3. At the end of 16 weeks, the rabbits were anesthetized and their aortas were collected for histological and biochemical analysis. The protein and mRNA level of Renin, Macrophages, MCP-1 and VCAM-1 were detected by immunohistochemistry and RT-PCR.4. Human umbilical vein endothelial cells (HUVECs) were cultured and identified. HUVECs in passages 3 to 8 were utilized for in vitro experiments.5. HUVECs were incubated with 100MOI rAAV2-Ren-RFP, the rAAV2-RFP viral vector served as control group. Then HUVECs were harvested at 48 hours after gene transfection for Western blot and RT-PCR analysis. HUVECs were treated with rAAV2-Ren-RFP to evaluated the effect of renin expression on the mRNA and protein level of cytokines and signal transduction molecular.Results1. The protein and mRNA expression of renin gene were markedly increased at 48 hours, and the expression of VCAM-1 and MCP-1 were also increased at 48 2. Overexpression of renin leads to more macrophages and increased unstable lesion.ConclusionsRenin overexpression could increase the expression of VCAM-1 and MCP-1 and macrophage accumulation, leading to plaque unstability in the rabbit madel. It suggested that renin could play an important role in atherogenesis and maybe provide a potential therapeutic target in cardiovascular disease.Part Three The Effect of Combined Drugs on Atherosclerosis and Plaque InflammationObjectiveTo determine if the combination of fluvastatin and losartan has synergistic effects on relieving atherosclerosis and plaque inflammation induced by a high-fat diet in rabbits.MethodsThe rabbits (n=36) were provided with regular food for 1 week and were subsequently provided with high-cholesterol food (1% cholesterol and 5% pig oil particles) for 16 weeks to generate the atherosclerosis model. They were randomly divided into 4 groups. The high-cholesterol group (CH) contained rabbits (n=9) that were provided with high-cholesterol food without other treatments. The fluvastatin group (F) contained rabbits (n=9) that were provided with high-cholesterol food and fluvastatin (10mg/kg/d). The losartan group (L) contained rabbits (n=9) that were provided with high-cholesterol food and losartan (25mg/kg/d). The fluvastatin and losartan combination group (F+L) contained rabbits (n=9) that were provided with high-cholesterol food and treated with a combination of fluvastatin (10mg/kg/d) and losartan (25mg/kg/d). Blood samples were drawn from the marginal ear vein at the end of 16 weeks. Total cholesterol (TC) and LDL-cholesterol concentrations were evaluated. Macrophage infiltration, smooth muscle cells (SMCs) and MCP-1 protein expression were measured by immunohistochemistry. The protein and mRNA level of MCP-1 and p38MAPKs were analysis by western blot and RT-PCR. ResultsAlthough the combination of losartan and fluvastatin did not demonstrate a higher reduction in blood cholesterol levels than the individual losartan and fluvastatin groups, the combination of losartan and fluvastatin had synergistic effects on decreasing the intimal and media thickness of the thoracic aortas, which reduced the extent of macrophage infiltration and monocyte chemotactic protein-1(MCP-1) expression in the plaques.ConclusionsThe combinational use of losartan and fluvastatin significantly inhibited atherosclerotic evolution and reduced inflammation associated with atherosclerotic plaques.