Type A Behavior Pattern And TLR4 Signaling Pathway Changes In Arteriosclerosis And Coronary Heart Disease

Objective : Although type A behavior pattern has been recognized as an independent predictor of cardiovascular disease, especially for coronary heart disease, the inflammatory mechanisms of thrombosis and arteriosclerosis in these patients are still not totally induced by acute stress events. This research comprises of three layers.First layer: To discuss the behavior and type pattern （A, B, intermediate） of different healthy individual on different activated pathway （TLR4, NF-κB, IL-6, IL-18） and to prove that in Type A personality the inflammatory activated pathway is continuously in an active state.Second layer: Using in vitro experiment on whole blood to observe the changes in TLR4 inflammatory activated pathway after addingβ₁ adrenergic receptor agonist, antagonist and TLR4 RNA interference in different behavior type healthy people.Third layer: Furthermore to validate that activated TLR4 inflammatory pathway in Type A behavior may influence the severity and prognosis of arteriosclerosis, especially on coronary heart disease.Subjects:From April 2009 to May 2010, 476 male university students from first year of Navy College and 424 female students from Dalian Medical University were randomly selected as first part research （health people） subjects. 8 subjects （4 male, 4 female） were randomly selected from Type A, Type B and intermediate type group respectively from the first part research as second part research subjects. We continuously collected subjects from Dalian Health Examination Center, PLA 210 Hospital, and The Central Hospital, they all accepted carotid ultrasonic check, and met with arteriosclerosis diagnosis criterion with 275 people as carotid arteriosclerosis group. And at the same time, 301 healthy persons were collected as control group for part three study; 182 patients with acute coronary syndrome （ACS） from The Second Affiliated Hospital of Dalian Medical University were consecutively collected, and also 152 patients with stable angina pectoris （Group SAP）, and 301 healthy persons as control group were also collected for part four study.Methods: （1） Part I study: Using Minnesota Multiphase Personality Investigation （MMPI-II） questionnaire, We used MMPI-2 Type A behavioural scale and self-made general situation investigation table for epidemic investigation; Flow type cell detection instrument were used to measure peripheral circulatory blood CD14+ single nuclear cell surface TLR4 of expression; ELISA method was used to detect the nor-epinephrine, epinephrine, IL-6 and IL-18 level in serum; （2） Part II study: The same methods was applied as above, RT-PCR technique for the determination of TLR4mRNA; immunohistochemical determination of NF-κBp65. For drug-inducedβ₁ receptor agonist 20, 30, 40umol/L dobutamine was used; Atenolol, PKA, PKC, P38 blocker 15umol/ L was add toβ₁ receptor agonist; TLR4siRNA transfect addβ₁ receptor agonist. （3） Part III and Part IV: Similar methods were used as Part I. For detection of cardiovascular risk factors, B-mode ultrasound imaging on carotid arteries, echocardiography, coronary angiography were performed. （4） Statistical analysis: For comparison between the groups used One-way ANOVA was used. Also Pearson correlation analysis and Logistic regression analysis methods were used. Spss 11.5 software was used for statistical analysis.Results:Part I: In healthy Type A behaviour young people, There were higher blood catecholamine level and higher expression of TLR4, and higher concentrations of IL-6, IL-18 in serum in a no stress state, they were significantly higher than Type B behavioural and intermediate type; Type A behaviour scores are significantly positive correlated to nor-epinephrine and epinephrine levels in serum and peripheral blood mononuclear cell TLR4 signaling pathway activation level.Part II:β₁ receptor agonist Dobutamine was gradually increased 20, 30, 40 umol/L in the TLR4 protein of expression, and NF-kBP65 protein expression, and IL-6, IL-18 serum levels in a dose-dependent manner , 40μmol/L Dobutamine was the optimal dose; andβ₁ antagonist 15umol/L atenolol totally suppressed TLR4 protein expression and 15umol/L PKA , P38 blocker partly blocked expression of TLR4 protein, but PKC could not block TLR4 expression and also up-regulate TLR4 expression; Transfect TLR4siRNA has suppressed up-regulation of NF-kBP65 protein expression, and IL-6, IL-18 serum levels after addingβ₁ receptor agonist induction, especially in Type A group down-regulation of NF-kBP65 protein expression, and IL-6, IL-18 serum levels is more obvious than in the other groups, and after transfect TLR4siRNA,the above index has no significant difference among the three behavioural groups. Before and after Dobutamine induction, both NF-κBp65 expression, TLR4 protein expression and serum IL-6, IL-18 concentrations in type A group were significantly higher than in the type B behavioural group and intermediate type.Part III and Part IV: （1）Type A behavioural carotid arteriosclerosis patients who suggested significantly higher CA level（catecholamine） in serum and higher TLR4 signal pathway activating in the single nuclear cell than type B and intermediate type behavior; TLR4 signal pathway activated degree positively correlated to type A behavioural scores; Regression risk factors analysis found that effect TLR4 signal pathway activating of factors include of type A behavioural total scores, scores of personality dimension, and scores of hostility dimension, and serum epinephrine and nor-epinephrine level; effect carotid artery average of IMT level and suffer from ACS events of logistic analysis display: TLR4, IL-6 and IL-18; （2）Under ACS stress statement, type A suggested persistant increase CA, and higher level activation state in mononuclear cell TLR4 signaling pathway; GRACE and Gensini scores were significantly higher in the type A behavioural group than the type B and intermediate type groups; Type A behavior scores of patients with ACS and TLR4 signaling pathway activation levels, serum catecholamine levels are correlated with GRACE marks and Gensini scores; Total score of type A behavior, personality dimension scores, hostile dimension scores entered to ACS stress protein expression of mononuclear cell TLR4 as dependent variable regression equation; TLR4 protein expression, serum levels of IL-6, IL-18, are also entered into GRACE marks and Gensini scores as dependent variable of the regression equation; （3） Under non-stress conditions, stable arteriosclerosis coronary heart disease and healthy control groups compared the three behavioural types depending on serum catecholamine levels and TLR4 signaling pathway activation, showed no significant differences among them. （4） Analysis of factors affecting the prognosis of ACS 1-year survival at statement: Logistic regression results displayed: type A behavior scores , personality dimensions scores, hostile dimension scores. and catecholamine levels in serum, mononuclear cell TLR4 protein expression , serum levels of IL-6, IL-18. Conclusions:（1）β₁ receptor agonist induced monotype TLR4/NF-κB/ proinflammatory factors signaling pathway activation in dose-dependent manner , Dobutamine 40 umol/L is the best, among three behavior pattern persons inducing effect of type A behavior is the most obvious;（2）β₁ receptor agonist induced TLR4/NF-κB /IL-6, IL-18 signaling pathway activation viaβ₁ receptor, TLR4, PKA, P38MAPK factors;（3） Discovered and confirmed that always TLR4/NF-κB signaling of peripheral blood mononuclear cells in the persons with typical TABP is highly activated under nonstress state and stress state. Activation of the signaling is positively related to TABP scores;（4） Carotid arteriosclerosis clinical trials reveal the similar results to above, and found that average thickness of the carotid artery wall is different between the higher and lower TLR4/NF-κB/proinflammatory factors signaling pathway activation; positively significant correlation have been found between the average thickness of the carotid artery wall and monocyte TLR4/NF-κB/proinflammatory factor signaling pathway activated level and TABP scores;（5） ACS severity and possibility of occurring of acute coronary events within one year’s prognosis are positively associated with TABP scores and correlated with TLR4/NF-κB/IL-6, IL-18 pathway activation.