| Since Russel Ross developed his popular "response to injury" hypothesis of atherogenesis, the view that atherosclerosis is indeed a chronic inflammatory disease initiated by monocyte/lymphocyte adhesion to activated endothelial cells (EC) is now widely accepted and substantiated by experimental and clinical observations. Inflammatory is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines, including monocyte chemotactic protein MCP-1 and interleukin IL-8, and adhesion molecules, including intercellular adhesion molecule ICAM-1, vascular adhesion molecule VCAM-1, E- andP -selectin, leading to monocyte/ lymphocyte recruitment and infiltration into the subendothelium . FKN, a new discovered chemokine CX3CL1(FKN) which is over expressed in atherosclerosis contributes to platelet activation and increased thrombogenesis in vascular diseases. Combination with its receptor CX3CR1 mostly expressed on monocytes, lymphocytes, and natural killer (NK) cells the FKN can inhance the NK cell-mediated endothelium damage, which may result in vascular injury. At the same time, FKN can also mediate capture , firm adhesion and migration of monocytes and lymphocytes to the vascular endothelial cells under physiologic flow conditions. It is reported that FKN and its receptor CX3CR1 were overexpressed by the blood vessel endothelium and monocytes of atherosclerotic lesions .These indicate that FKN/ CX3CR1 play an important role in the initiation and development of atherosclerosis and now people are paying close attention to significance of them.Up to now ,studies merely demonstrated that FKN stimulated angiogenesis by activating the MAPK system and PI3K signal pathways via the G protein- coupled receptor CX3CR1, but the underlying mechanism has not been exclu- dated. NF- B is a redox-sensitive transcription factor, and the activation of which controls the expression of proinflammatory cytokines ,inclding kinds of chemokines and adhesion molecule , such as MCP-1,IL-8,VCAM-1,ICAM-1 at al . Activated NF- B has been identified in SMC, macrophages, and EC of human atherosclerotic lesions, and not been identified in healthy vascular. When the signal transduction pathway is concerned , PKC,PI3K and AKT may take part in the activation of NF- B. Tumor necrosis factor–α(TNF-α) is one of the aimed-genes of activated NF- B ,it may participate in the progre- ssion of atherosclerosis by impairing vessel endothelium and break- ing down the balance between blood coagulation and anticoagulation system . Captopril is known for its protecting cardiovascu- lar .Tranditional opinion about the mechanism of antiatherosclerosis is that Captopril may protect the vessel endothelium by cutting down blood pressure and adjust the blood fat . Nowadays people have posed that captopril may have other mechanisms which may inhibit the expression of inflammatory factors such as PKC,NF- B and TNF-α.This research connect the above four together and then investigate their relationships and the intervention of angiotensin converting enzymeinhibitor captopril.This research will isolate the peripheral blood monocyte from human , and use the technology of molecular biology and immunohistochemistry to investi- gate the the possible mechanism of FKN/CX3CR1 affecting the progression of athecrosclerosis and the invention of captoprilq. Meanwhile, we will investigate the gene polymorphism of CX3CR1 in atherosclerotic underwent cardiaccathe- terization to study whether the gene polymorphism of CX3CR1 will affect the development of atheroclerosis .The experiment is summaried as follow:1. Association between polymorphism in the chemokine receptor CX3CR1 and coronary atherosclerosisThe 249th and 280th nucleotides located in the sixth and seventh transmem- brane domains of the CX3CR1 protein have polymorphism , Since the 249th and 280th condon locate in the area of coding region ,the polymorphism will affect the biological function of CX3CR1.It is found that the affinity of 249I280M haploid homozygote to its ligand is significantly lower than the wild ones, which demonstrate that the mutation influence the integrity of the receptor. Binding sites which mediate the combination of ligand and receptor in cells of mutation homozygote are much less than that in wild ones. The experiment is the first research on the association between CX3CR1 I249 allele and cardiovascular desease .Case control and PCR-PELP were used to study the CX3CR1 polymorphism in a cohort of 94 yellow individuals who underwent cardiacctheterization .The result shows that the freguencies of the V249I and T280M polymorphisms showed no deviation from Hardy-Weinberg equilibrium. However ,a statistically significant difference in genotype frequencies was observed in cases compared with controls .The adjusted odds ratios (ORs) associated with the presence of the M280(TM 1 MM versus TT genotype) and I249 alleles (VI+Ⅱversus VV genotype) were 0.49(95%confidence interval [CI],0.27-0.89;P=0.002)and 0.43(95% CI,0.26-0.72;P=0.001),respectively .Thus ,these alleles were associated with a reduced risk of acute coronary events .To calculate the OR for each genotype carrying the I249 or M280 allele ,we also tested the genotype as a 3-class variable .The adjusted Ors were 0.47(95% CI,0.26-0.88;P<0.02) and 0.68 (95%CI,0.09-5.45; P<0.7)for the TM and MM genotypes ,respectively;they were 0.44 (95 % CI , 0.26-0.75 ;P<0.003)and 0.39(95 % CI,0.13-1.19;P<0.099)for the VI andⅡgenotypes , respectively .AS shown recently ,the T280M and V249I polymorphismsare in complete linkage disequilibrium and generate combined genotypes of the 9 theoretically possible and only 3 haplotypes (V249T280 ,I249T280,and I249 M280). Indeed ,all subjectscarrying allele M280 also carry allele I249 ,whereas in some subjects ,allele I249is associated with allele T280.However ,when the I280M polymorphism was used in the same logistic equation as the V249I polymorphism, only the effect of the I249 allele remained significant:Adjusted Ors were 0.84(95%CI,1.84-0.39; P<0.669) and 0.48(95% CI,0.92-0.25;P<0.028) for the M280 and I249 alleles ,respectively .Therefore, we considered that the protective effect was due to the I249 allele and performed .2.Research on signal transduction of FKN affecting atherosclerosis and the invention of captoprilMolecular biological technology were used to investigate the effect of G-protein ,PKC,PI3K,NF-κB and TNF-αon the progression of atherosclerosis induced by FKN/CX3CR1 ,and to study the possible cell signal transduction pathway ,and at last to explore the invention of captopril .1. To study the effect of signal transduction pathway of FKN/CX3CR1/PKC/ NF-κB in the atherosclerosis and the invention of Captopril ,we divided peripheral blood monocytes into six groups ,which are control group ,soluble FKN group , pertusis toxin PTX + FKN group , PKC inhibitor RO31-8220 + FKN group, NF-κB inhibitor PDTC + fractaline group,and captopril + FKN group , sepratively, then measure the nuclear factor–κappaB(NF-κB) expression of monocytes from each group by immune histochemsitry ,and then collect the supernatant of monocytes from each group ,determin the expression of TNF-αby euzyme-linked immunosorbent assay(ELISA). The result shows that (1)The expression of NF-κB and TNF-αfrom FKN group is increased compared with the control group .(2)The expression of NF-κB and TNF-αfrom G-protein inhibitor PTX group is decreased compared with the FKN group .(3)The expression of NF-κB and TNF-αfrom PKC inhibitor RO31-8220 group is decreased compared with the FKN group .(4)The expression of TNF-αfrom inhibitor NF-κB PDTC group is decreased compared with the FKN group .(5)The expression of NF-κB and TNF-αfrom captopril group is decreased compared with the FKN group .The differences above had statistical significan- ce (P<0.05).2. To study the effect of signal transduction pathway of FKN/CX3CR1/PI3K / NF-κB in the atherosclerosis and the invention of Captopril , we divided peripheral blood monocytes into five groups ,which are control group , soluble FKN group , PI3K inhibitor ly294002+FKN group, NF-KB inhibitor PDTC + fractaline group ,and captopril + FKN group, then measure the nuclear factor–κappaB(NF-κB) expression of monocytes from each group by western blotting ,and then collect the supernatant of monocytes from each group ,determin the expression of TNF-αby euzyme-linked immunosorbent assay(ELISA). The result shows that : (1) there are much more NF-κB and TNF-αexpression in the monocytes from FKN group compared with control group (2) there are less NF-κB and TNF-αexpression in the monocytes from LY294002 group compared with FKN group (3) there are less NF-κB and TNF-αexpression in the monocytes from PDTC group compared with FKN group ,but more than control group (4) there are less NF-κB and TNF-αexpression in the monocytes from CAP group compared with FKN group ,but more than control group3.To study the effect of signal transduction pathway of FKN/CX3CR1/AKT/ NF-κB in the atherosclerosis and the invention of Captopril , we divided peripheral blood monocyte into five groups ,which are control group , soluble FKN group ,PI3K inhibitor ly294002+FKN group, NF-κB inhibitor PDTC + fractaline group ,and captopril + FKN group, then measure the nuclear Akt expression of monocytes from each group by western blotting ,and then collect the supernatant of monocytes from each group ,determin the expression of TNF-αby euzyme-linked immunosorbent assay(ELISA).The result shows that : (1)Phospho-Akt and TNF-αlevels of FKN group increased significantly compared with those of the control group .(2) By pre-incubation with LY294002 Phospho-Akt levels increased compared with those of FKN group.(3) By pre- incubation with LY294002 , TNF-αlevels decreased compared with those of FKN group.(4) By pre-incubation with PDTC, Phospho-Akt levels decreased compared with those of FKN group.(5) By pre-incubation with PDTC, TNF-αlevels decreased compared with those of FKN group.(6)By intervention with captopril, Phospho-Akt and TNF-αlevels decreased compared with those of FKN group.We have conclusion from above information that (1)CX3CR1 I249 polymorphism is an independent risk factor of CAD .(2) The frequences of CX3CR1 I249 allele in subjects with CAD is significantly different from control subjects, and the I249 allele can protect human from suffering atherosclerosis (3) FKN/CX3CR1 may increase the expression of NF-κB and TNF-αin monocytes ,which takes part in the progression of atherosclerosis (4)CX3CR1 is a G-protein coupled receptor ,and it will initiate the cell signal transduction when combined with FKN. (5)PKC, PI3K play an important part in increasing the expression of NF-κB and TNF-αin monocytes induced by FKN. FKN-CX3CR1 may increase the expression of NF-κB and TNF-αin monocytes through the G-protein activating PKC, PI3K.(6) NF-κB play an important role during the process that Peripheral blood monocytes synthesize TNF-αinduced by FKN -CX3CR1. Mechanism of FKN -CX3CR1 initiating intracellular signal transduction perhaps is through the process that FKN-CX3CR1 activate NF-κB by PKC ,PI3K ,which activated by G-protein and then improve the expression of TNF-α. (7) PI3K is not the upstream of Akt in the signal process of TNF-αupregulation(8) Akt mediates the signal process of TNF-αupregulation depending on NF-κB (9) Inhibition of Akt-mediated TNF-αupregulation induced by FKN may be one of mechanism of ACEI repressing atherosclerosis progression. (10) FKN-CX3CR1 promote the progression of atherosclerosis through the cascade reaction possibly as follow:1) FKN-CX3CR1→→G-protein→→PKC→→NF-κB→→TNF-α;2)FKN-CX3CR1→→PI3K→→NF-κB→→TNF-α;3 ) FKN-CX3CR1→→AKT→→TNF-α(11) Captopril may be anti-inflammatory and prevent the progression of atherosclerosis by decreasing the synthesis of TNF-αinduced by FKN-CX3CR1 ,which may be partly linked to inhibiting the expression of NF-κB.We obtain further understand from the research about the relationship between FKN-CX3CR1 and the formation of atherosclerosis ,as well as the signal transduction mechanism involved, which provide us theoric and experi- mental foundation of the feasibility of finding new ways to treat atheroscle- rosis ;It also provide us new theory and evidence-based proof of the specific mechanism of antiatherosclaerosis ;Meanwhile ,it is the first research in our country which collect and analyze the genotype of the receptor of chemotatic factor FKN CX3CR1 from people in the northeast of China ,and it also provide theory evidence for early intervention to people with different genotypes for clinical work. |
PDF Full Text Request