The Study Of The Effect Of Lipidemia On The Vascular Adventitial Inflammation Of Atherosclerosis

Introduction: For a long period, atherosclerosis (As) has been regarded as endothelial inflammatory disease, and the function of vascular adventitia is just nourishing and supporting vessels. However, recent researches have found that vascular adventitia plays an important role in the regulation of vascular function. The sympathetics and parasympathetics nerve terminal in vascular adventitia release neurotransmitters such as norepinephrine, neuropeptide Y, substance P, acetylcholine and nitric monoxide. These substances act on medial smooth muscle cells and adjust vasomotor function. The main components of vascular adventitia, fibroblast cell, can be activated in pathological conditions. Then the fibroblast cells may have phenotype conversion, proliferation, migration and synthesis of a variety of cytokines and take part in the development of a variety of diseases. Further researches on vascular adventitia can help to understand the pathophysiological mechanisms of vascular lesions fully and to develop methods of prevention and cure of vascular lesions.It has been confirmed that the adventitial inflammation exists in blood vessels of As. The infiltration of inflammatory cells in vascular adventitia has a positive correlation with vascular membrane lesions of As. Some reports show that adventitial inflammation has some relations with intimal hyperplasia and the occurrence of acute coronary syndrome. The vasa vasorum of blood vessel may be the gateway of the inflammatory cells invasion. However, the mechanism of adventitial inflammation of As is not yet clear. The researches are rarely in the world that about the role of the adventitial fibroblasts in the adventitial inflammation and whether the statins drugs can inhibit adventitial inflammation in addition to the lipid-lowering effects. Thus, through a series studies of cell culture and animal experiments, we aim to observe the relationship between hyperlipidemia and adventitial inflammation of atherosclerosis, fibroblast phenotype conversion, proliferation and inflammatory factor synthesis as well as the interventional function of fluvastatin.Part OneTHE STUDY OF THE RELATIONSHIP BETWEEN LIPIDEMIA AND VASCULAR ADVENTITIAL CHANGE, AND THE INTERVENTION EFFECT OF FLUVASTAIN IN THE ATHEROSCLEROSIS RABBITSBackground: More evidence shows inflammation of vascular adventitia may be one of the reasons of the development of atherosclerosis (As). Inflammatory cells infiltrated in vascular adventitia secrets cytokines and adhesion molecules; It not only can affects the intima and induces the occurrence of As, but also affects the media directly through nerve endings or by acting on receptors on smooth muscles so that cause development of As. Pathogenesis of As correlate with hyperlipidemia is definite. Past studies mainly focus on hypercholesterolemia (HC), especially the effects of oxidized low-density lipoprotein (ox-LDL) on vascular endothelium and the protecting role of statins. However, the reports on the influences of hyperlipidemia on atherosclerotic vascular adventitia and the effects of statins on adventitial inflammation of As are rarely. Therefore, we observe the relationships between hyperlipidemia and changing of vascular adventitia and to explore the role of intervention of fluvastatin by useing As model established by a high-fat diet.Objective: Use As model establishing by a high-fat diet to observe the follow questions:â‘ The relationship between lipidemia and infiltration of inflammatory cells in vascular adventitia, the relationsip between lipidemia and phenotype transformation of adventitial fibroblasts and the relationship between lipidemia and the activity of nuclear factor p65 (Nuclear factor kappa B, NF-Kappa B).â‘¡The relationship between infiltration of inflammatory cells in vascular adventitia and the seriousness of As.â‘¢The relationship between activity of NF-κBp65 and phenotypic transformation of fibroblasts.â‘£The effects of fluvastatin on the infiltration of inflammatory cells in adventitia of As and the phenotype transformation, the activity of NF-κBp65.in adventitial fibroblasts.Methods: (1)Establish As model: choose 34 healthy adult male New Zealand rabbits and divide randomly into the following groups.â‘ Control group: ten rabbis are given ordinary particles feeding;â‘¡High fat diet group: twelve rabbits are given high fat diet for 12 weeks (cholesterol + lard + egg yolks + ordinary particles feed) to establish As rabbit model;â‘¢Fluvastatin group: twelve rabbits are given high-fat diet and fluvastatin 10mg/kg/d for 12 weeks; (2) The rabbits aorta were taken and were stained with HE method to observe formation of As plaque and inflammatory cell infiltration of vascular adventitia by light microscopy; (3) Analysize blood lipid using automatic biochemical analyzer; (4) Use immunohistochemistry to detect the expression ofα-smooth muscle actin and the activation of NF-κBp65 in adventitial fibroblast.(5) Analysize the relationship between serum lipids and the infiltration of inflammatory cells in adventitia; the relationship between serum lipids and the activity of NF-κBp65 in adventitial fibroblasts and expression ofα-SMactin; the relationship between infiltration of inflammatory cells in vascular adventitia and area of As plaque; the relationship between activity of NF-κBp65 and expression ofα-SMactin. in fibroblasts. Results: (1) Total cholesterol (TC) and low-density lipoprotein (LDL) in the rabbits in high fat diet group and fluvastatin group were significantly higher than the in normal control group (P <0.01), but they decreased significantly in fluvastatin group than in high-fat diet group (P <0.01); (2) The arteries intima in high fat diet group and fluvastatin group are thicker than those of the control group (P<0.01); obvious plaque formation can be observed. However, the arteries intimal thickness and plaque area in fluvastatin group decreased significantly than in the high-fat diet group (P <0.01). (3) In the normal control group nearly have no infiltration of inflammatory cells in vascular adventitia. Adventitial fibroblasts almost has no expression ofα-SMactin and no activity of .NF-κBp65. In the high-fat diet group and fluvastatin group there are significantly infiltration of inflammatory cell in adventitia. The NF-κBp65 in fibroblasts has significantly activity in both groups. The expression of theα-SMactin also significantly increases. The infiltration of inflammatory cell in vascular adventitia decrease significantly in fluvastatin group than in high-fat diet group. The activity of NF-κB in fibroblasts and the expression ofα-SMactin also decrease obviously than in high-fat diet group (P <0.05). (4) In high-fat diet group and fluvastatin group the LDL and TC have positive relationship with the number of aortic adventitial inflammation cells, the activity NF-κB and the expression ofα-SMactin (P <0.01). The HDL has negative relationship with them. The TG has no significantly relationship with them.(P> 0.05). (5) In the high-fat diet group and fluvastatin group, the activity of NF-κB in fibroblasts has positive relationship with the expression ofα-SMactin [(r =0.633, 0.649, respectively ), (P <0.05)]. The infiltration of inflammatory cells in adventitia has positive relationship with plaque area [(r =0.648,0.663, respectively) (P <0.05)].Conclusion: (1)The experimental atherosclerosis model has been established successfully The hyperlipemiaxan be cause by high-fat diet.(2) HC, particularly hyperlipemia of LDL is related to adventitial inflammation, phenotype transformation of adventitial fibroblasts and the activity of NF-κB in atherosclerosis. (3)The infiltration of inflammatory cells in vascular adventitia has positive relationship with severity of atherosclerosis. (4) The activity of NF-κB in adventitial fibroblasts has positively relationship with its phenotype transformation. (5) The infiltration of inflammatory cells in adventitia of atherosclerosis can be inhibited by fluvastatin. The phenotype transformation and activity of NF-κB of vascular adventitial fibroblasts also can be inhibited by fluvastin.Part TwoTHE EFFECTS OF THE OXIDATIVE LOW-DENSITY LIPOPROTEIN ON THE PHENOTYPE TRANSFORMATION, PROLIFERATION AND THE CYTOKINE SYNTHESIS OFTHE FIBROBLASTSBackground: The adventitial fibroblasts which are at "inactive" in normal condition are the principal cell in vascular adventitia. However, the inactive AF could transform to myofibroblasts(MF) in some pathology condition. The MF is fibroblasts in fact which own the characteristic of the smooth muscle cells. The MF could secrete lots of cytokine and mediators of inflammation which participate immunization and inflammatory reaction. Our study confirmed that the AF could be activated and transform to MF in atherosclerosis, and the transformation of AF was positive relationship with HC, especially with LDL. Now there are rarely study about the relationship between the hyperlipidema with the phenotypic modulation, proliferation and inflammatory cytokine synthesis of the fibroblast. We applied the ox-LDL directly to fibroblasts in culture, and observed the effects of the oxidative low-density lipoproteins on phenotypic modulation, proliferation and inflammatory cytokine synthesis of the fibroblast.Objective: To observe the effects of the oxidative low-density lipoproteins on phenotypic modulation, proliferation and inflammatory cytokine synthesis of the fibroblastsMethods: The human embryon fibroblast cell line (HEFs) was traditional cultured. Use vimentin andα-SMactin immunocytochemistry for characterized tests. Take generations of 3～8 for experiment. Divide the samples into the following groups.â‘ Normal control group: the HEF without irritant.â‘¡ox-LDLl group: Add 25μg/ml ox-LDL into the medium.â‘¢ox-LDL2 group: Add 50μg/ml ox-LDL into the medium.â‘£ox-LDL3 group: Add 100μg/ml ox-LDL into the medium.⑤BAY11-7082 group: Add 10μg/ml BAY11-7082 into the medium and incubate the HEFs for one hour and then added 100ug/ml ox-LDL into the medium. The HEFs of each group were cultured 24h. (1)Use the test of MTT to detect the proliferation of HEFs in each group. (2)Use the flow cytometry to detected the cell generation cycle. (3)Use the immunocytochemical method to detect the activity of NF-KBp65 and the express of the a-Smactin, Transforming growth factorβ₁ and Monocyte chemoattractant protein. (4)Use hybridization in situ to detect the express of NF-κBp65mRNA,TGF-β₁mRNA and MCP-1mRNA. (5)Use ELISA to measure the IL-1αand IL-6 level in the cell culture medium of different groups. (6)To analysis the effects of the different density ox-LDL on proliferation and cell cycle of the HEFs, the activity of the NK-KBp65, the express of theα-Smactin,TGF-β₁ and MCP-1, and the synthesis of the IL-1αand IL-6.Result: 1. The identification of the cell characteristics showed that the dyeing of the specificity vimentin antigen were positive andα-SMactin were negative. 2. Compare with the control group, the ratio of the cell proliferation which detected by the MTT were higher in ox-LDL2 and ox-LDL3 group (P<0.05) , and in the BAY11-7082 group there were no significant difference with control group (P>0.05). 3. Compare with the control group, the fibroblast cells in G₁ or S period were increased significantly (P<0.05) in each group of the ox-LDL, especially in the ox-LDL3 group(100ug/ml), and there were no significantly difference in BAY11-7082 group than in control group (P>0.05). 4. There were almost no activity of the NF-KBp65 and the express ofα-SMactin in the HEFs in normal control group and BAY11-7082 group. The detection by hybridization in situ showed that there were almost no express of NF-ΚBp65mRNA in normal control group, otherwise, it is obviously in BAY11-7082 group, the express of TGF-BimRNA in two groups are weak. The activity of NF-KBp65 and express of theα-SMactin and TGF-β₁ in each ox-LDL group are coincidence with the express of NF-KBp65mRNA and TGF-β₁mRNA detected by hybridization in situ. Each group had significant difference (P<0.05). 5. There were no express of the MCP-1 and MCP-1mRNA, and also no secretion of the IL-1αand IL-6 in normal control group and BAY11-7082 group. The HEFs which were motivated by ox-LDL could express MCP-1 and MCP-1mRNA, and could secrete IL-1αand IL-6 in concentration dependent, and the difference were significance in each group. The contents of IL-1αand IL-6 were positive correlation with the express of the MCP-1, and the coefficient correlation were (r=0.932, P<0.01) and (r=0.834, P<0.01) respectively.Conclusion: (1) the ox-LDL could induce phenotype transformation of the fibroblast, and displayed with concentration dependent. (2) The ox-LDL with difference density all could affect the cell cycle of the fibroblast and increase the cell proliferation. (3) the ox-LDL could induce the fibroblast to express inflammatory factor(MCP-1 , IL-1αand IL-6) with concentration dependent. (4) The ox-LDL could induce the fibroblast to synthesis IL-1αand IL-6, which may be related with increased express of the MCP-1. (5) The ox-LDL induces phenotype transformation, proliferation and inflammatory cytokine synthesis of the fibroblast maybe by the NF-KB way.Part Three THE INTERVENTION EFFECT OF FLUVASTATIN ON THE MIGRATION AND CYTOKINE SYNTHESIS OF THE FIBROBLASTS INDUCTION BY OX-LDLBackground: Many clinical experiments have confirmed that 3-hydroxy-3-methyl-coenzyme A reductase inhibitors (HMCI) can significantly reduce coronary heart disease events; its lipid-lowering effect is known. In recent years, its non-lipid-lowering effects, expecially the anti-inflammatory effect, have been a hot topic; however, most researches focus on the effect of HMCI on inflammation of the vascular endothelium. HMCI's effect on AS vascular adventitia is less reported. In animal experiments, we observed that HMCI drug, fluvastatin, could significantly inhibit As' inflammation of the vascular adventitia inflammation cell infiltration, phenotypic transformation of adventitial fibroblasts and the activity of NF-κBp65. This demonstrated that fluvastatin has the function of anti adventitial inflammation in As, inhibit of fibroblast cell phenotype, and help to remodel vascular. However, whether this function is independent of its role in lowering lipid level is not yet clear. Therefore, we use fluvastatin and ox-LDL with HEFs directly in cell culture to observe its impact on the migration of HEFs and cytokine synthesis.Objective: Cultivate fibroblast cells in the medium that it contains fluvastatin and ox-LDL.To observe the fibroblast migration and changes in cytokine synthesis and to observe the inhibiting effects of fluvastatin on ox-LDL-induced changes in the fibroblasts.Methods: (1) The human embryon fibroblast cell line (HEFs) was traditional cultured. Use vimentin andα-SMactin immunocytochemistry for characterized tests. Take generations of 3～8 for experiment. Divide the samples into the following groups.â‘ Normal control group: the HEFs without irritant.â‘¡ox-LDL group: Add 100μg/ml ox-LDL into the medium.â‘¢Statins Group I: add 1×10^-7mol/L fluvastatin in the medium and incubate the HEFs for four hours, and then add 100μg/ml ox-LDL.â‘£Statins Group II : add 1×l0^-6mol/L fiuvastat in the medium and incubate th HEFs for four hours, and then add 100μg/ml ox-LDL.⑤Statins Group III: add 1×10^-5mol/L fiuvastat in the medium and incubate the HEFs for four hours, and then add 100μg/ml ox-LDL. Cultivate these HEFs for 24 hours. (2) Use Sarkar's method to measure the distance of cell migration. (3) Use method of immuocytochemistry to detect of MCP-1 expression. (4)Use ELISA to measure the IL-1αand IL-6 level in the cell culture medium of different groups. (5) Analysing the relevance between the expression of MCP-1 and the synthesis of IL-1αand IL-6.Results: (1) Identification of cells is the same as that of the second part. (2)Fluvastatin in each concentration can inhibit the migration of fibroblast. The effect of concentration-dependent was significant differences between the groups (F=23.947. .P<0.01). (3) In the fluvastatin group, Fluvastatin can dose-dependently inhibit the induction of ox-LDL on the expression of monocyte chemoattractant protein-1 and the synthesis of IL-αand IL-6 in HEFs. but it is still more significant than the normal control group (P <0.05) (4)The synthesis of IL-1αand IL-6 is positive correlation with the expression of MCP-1 [ (r=0.947, P<0.01) and (r=0.757, P<0.05) respectively.]Conclusion: 1. In addition to the lipid-lowering effect, fluvastatin can dose-dependently inhibit the migration of fibroblast that induced by ox-LDL. 2. In addition to fluvastatin's lipid-lowering effect, fluvastatin can dose-dependently inhibit the expression of MCP-1 and the synthesis of IL-1αand IL-6 of fibroblasts induced by ox-LDL. 3.The inhibiting effect of fluvastatin on the synthesis of IL-1αand IL-6 has relationship with the inhibiting effect on the MCP-1.of fibroblasts induced by ox-LDL...