Effect And Mechanism Of Aliskiren On Atherosclerotic Plaque Angiogenesis

BackgroudsAtherosclerosis(AS) is the main pathological basis of ischemic cardiovascular and cerebrovascular disease. Clinical studies have found that the unstable AS plaque hemorrhage or plaque rupture is the main cause of the acute ischemic vascular events.There was pathological neovascularization in AS plaque. The characteristics of neovascularization are high permeability and brittleness,and prone to rupture, which leads to bleeding in the plaque, causing acute cardiovascular events.Studies have confirmed that inflammation is closely related to the formation of new blood vessels in the plaque.The inflammatory factors can stimulate angiogenesis.TLR4/NF-κB signaling pathway can induce inflammatory cells to release inflammatory mediators to mediate the inflammatory response. Studies have shown that the TLR4/NF-κB pathway may induce MMPs expression and participate in the pathological process of AS. Research showed that the content of MMPs was increased in AS.The level of MMP-2 and MMP-9 in the arterial wall of AS was significantly increased, and the degree of increased was positively correlated with the severity of the lesion.Aliskiren is the newly discovered renin inhibitors.Aliskiren can significantly reduce the size of the atherosclerotic lesions. Studies have confirmed that RAAS can induce vascular inflammation in AS plaques of Apo E-/-mice by TLRs dependent signaling pathway,but the mechanism and pathway is not clear. Objective: To study the effect of Aliskiren on the formation of neovascularization and the stability of atherosclerotic plaque.To investigate whether Aliskiren can affect the formation of neovascularization by TLR4/NF-κB signaling pathway mediated inflammatory reaction and the formation of MMP-9 and MMP-2.Methods: A total of 30 Apo E-/- mice were divided into three groups randomly: the model group 10, Aliskiren low-dose group 10, Aliskiren high-dose group 10, and take C57BL/6 mice 10 as control group. Aliskiren low-dose group, high-dose group were given 25mg/kg/d and 50mg/kg/d Aliskiren by gavage for 12 weeks; C57BL/6 mice and model group were given normal saline 25mg/kg/d for 12 weeks. Immunohistochemical staining was used to observe and analyze the density of intraplaque neovascularization. HE staining was used to analysis plaque morphology and calculation plaque area; Oil red O staining was used to analysis the content of lipids; Masson staining was used to analysis the content of collagen; The serum levels of TNF-α,IL-,IL-1β and MCP-1were measured using ELISA kit; The m RNA levels of MMP-2,MMP-9 and TLR4 in the aortic tissue were analyzed by RT-PCR technique; The protein expressions of MMP-2, MMP-9, TLR4 and NF-κB in the aortic tissue were analyzed by Western Blot technique.Results:1. Serum lipid determination: Compared with the control group, the serum TC, TG and LDL-C concentrations in the model group were significantly increased(P<0.05); There was no significant difference between the model group, Aliskiren low-dose group and high-dose group(P > 0.05).2. HE staining: In the model group, the aortic AS plaque formation was significant, the lumen stenosis was more serious, the intima was not not smooth and discontinuous, and the endothelial cells were partly absent. Compared with model group, the plaque area, plaque area/lumen area ratio in Aliskiren low-dose group and high-dose group were decreased significantly(P<0.05); Compared with Aliskiren low-dose group, the Aliskiren high-dose group decreased more significantly(P< 0.05).3. Oil red O stain: There was a large number of lipid deposition in the AS plaque of the model mice.Compared with model group, the content of lipid in Aliskiren low-dose group and high-dose group were significantly decreased(P<0.05).Compared with the Aliskiren low-dose group, Aliskiren high-dose group decreased more significantly(P < 0.05).4. Masson trichrome staining: The content of collagen in AS plaque of the model group was decreased significantly. Compared with model group, the content of collagen in Aliskiren low-dose group and high-dose group were increased significantly(P<0.05). Compared with the Aliskiren low-dose group, Aliskiren high-dose group increased more significantly(P < 0.05).5. The density of neovascularization in the AS plaque: Immunohistochemical CD34 staining was used to identify the neovascularization in the plaque. Model group have a large amount of brown particles, positive expression of CD34.Compared with the model group, the neovascularization in Aliskiren low-dose group and high-dose group significantly were reduced(P<0.05), Compared with the Aliskiren low-dose group, Aliskiren high-dose group decreased more significantly(P < 0.05).6. TNF-α, IL-6, IL-1β, MCP-1 levels: Compared with the control group, the levels of TNF-α, IL-6,IL-1βand MCP-1 in model group were significantly increased(P < 0.05).The levels of TNF-α, IL-6, IL-1β and MCP-1 in Aliskiren low-dose group and high-dose group were significantly lower than the model group(P < 0.05), and Aliskiren high-dose group decreased more significantly(P <0.05).7. Expressions of MMP-2, MMP-9 and TLR4 m RNA in aortic tissue: Compared with the control group, the expressions of MMP-2, MMP-9 and TLR4 m RNA in model group was increased significantly(P<0.05); Compared with model group, the expression of MMP-2, MMP-9 and TLR4 m RNA in Aliskiren in low-dose group and high-dose group were decreased significantly(P<0.05), and Aliskiren high-dose group had the lower m RNA expressions(P<0.05).8. Expression of MMP-2, MMP-9, TLR4 and NF-κB protein in aortic tissue: Compared with the control group, the expression of MMP-2, MMP-9, TLR4 and NF- κB in model group was significantly increased(P<0.05); Compared with model group, the expression of MMP-2, MMP-9, TLR4 and NF- κB protein in Aliskiren in low-dose group and high-dose group were decreased significantly(P<0.05), and Aliskiren high-dose group had the lower protein expressions(P<0.05).Conclusion:1. Aliskiren can reduce the formation of neovascularization in the AS plaque and improve the stability of the plaque. 2.Aliskiren inhibits the formation of neovascularization in the AS plaque, which may be related to the inhibition of TLR4/ NF-κB signaling pathway mediated inflammatory reaction and the formation of MMP-9 and MMP-2.