Pilot Study On The Androgen Receptor Gene CAG Repeat Polymorphism And Relationship Between Androgen And Atherosclerosis

Background:Incidence and mortality rate of coronary artery disease(CAD)are both higher in men than age-matched premenopause women.Recently,more attention has been drawn to the relationship between androgen and atherosclerosis-associated disease in men,including CAD.Men with CAD have significantly lower levels of androgens than normal controls; Long-term supra-physiological oral or low-dose transdermal testosterone supplementation relieves symptoms of angina and signs of myocardial ischemia;Short-term intracoronary administration of testosterone at physiological concentrations induces coronary artery dilatation and increases coronary blood flow in men with established CAD;Testosterone level decreases dramatically in elderly men,who are also predisposed to atherosclerosis,indicating a relationship between testosterone and CAD. Meta-analysis of previous clinical epidemiological studies shows no positive relationship between testosterone and prevalence of CAD.Oxidization injure of endothelium by OX-LDL has been proved to be a critical initial event in the formation of atherosclerotic lesions.Moreover,androgen receptor(AR)has been identified to exist in atrial and ventricular muscle, endothelium and vascular smooth muscle cells.Androgen exerts its biological effects on target organs merely via AR,and the intensity of effects depends largely on the density of AR.Genetic studies in AR function have reported regulatory effects of CAG polymorphism in exon 1 of AR gene on AR protein expression and function.To elucidate the mechanism of how androgen influences atherosclerosis,AR is the pivotal mediator,whereas the role of AR gene CAG polymorphism in modulating androgen effects on cardiovascular risk factors remains unknown.So this study focuses mainly on the interrelationship among androgen,AR and atherosclerosis,and verifying the effects of AR gene CAG polymorphism on AR mRNA and protein expression,so as to illuminate the role AR plays in the development of atherosclerosis from genetic and cellular point of view.Objectives:(1)To evaluate the effects of AR gene CAG polymorphism on AR mRNA and protein expression;(2)To measure the levels of NO,PAI-1,ICAM-1,PDGF-B and TGF-β1 produced in ECV304 cell groups oxidative-injured by OX-LDL after pretreatment with different concentrations of testosterone,and to investigate the role testosterone plays in the formation and development of atherosclerosis;(3)To observe the effects difference of testosterone intervention on oxidative-injured ECV304 cell groups containing AR gene with different CAG repeats in exon 1.Methods:(1)Based on wild type plasmid pwtAR and genomic DNA extracted from leucocytes in which AR gene CAG repeat number had been identified,AR expression plasmids with 0,8,21 and 34 CAG repeats were constructed by recombinant PCR,restriction enzyme digestion,linkage reaction,etc.;(2)Transfecting HEK293 cells with the reconstructed plasmids with AR gene CAG repeats of 0,8,21 and 34,then extracting total RNA with TRIZOL for RT-PCR to detect AR mRNA;extracting total protein with cell lysis buffer for Western Blot to detect AR protein.Quantification and comparison of AR mRNA and protein band intensities were performed to evaluate effects of CAG repeat length on AR mRNA transcription and protein expression level;(3)Assaying effect of testosterone concentration on the proliferation of ECV304 by MTT method for 12,24,36,48,72 and 96h;(4)RT-PCR was carried out to detect and compare the level of AR mRNA in ECV304 cells treated with different concentrations of testosterone;(5)ECV304 cells were divided into groups,where OX-LDL(50mg/L),T 3nM to 30μM pretreatment for 4h then OX-LDL(50mg/L),Flutamide 10μM pretreatment for 4h then OX-LDL(50mg/L)and Flutamide 10μM pretreatment for 4h then T 30nM for 4h then OX-LDL(50mg/L)were added and incubated with the cells for 30h.Supernatants of culture media were collected and used for assay of NO by nitrate reductase method and of PAI-1 by ELISA method;cell pellets were solubilized in cell lysis buffer,centrifuged and supernatants were reserved for detection of ICAM-1,PDGF-B and TGF-β1 by Western Blot;(6)Transfecting ECV304 cells with the reconstructed plasmids with AR gene CAG repeats of 0,8,21 and 34,then injuring the cells with OX-LDL (50mg/L)for 12h,and then treating with testosterone of different concentrations for 24h.Assaying the factors mentioned above with corresponding methods.Results:(1)AR expression plasmids with 0,8,21 and 34 CAG repeats were constructed successfully.Restriction enzyme digestion,DNA sequence analysis and multiple sequence alignment of the 4 AR expression plasmids verified the size,orientation,reading frame,and CAG repeat length of the cloned inserts;(2)Relationship between CAG repeat length and AR mRNA or AR protein:as for AR mRNA,CAG₀ was at minimum,CAG₃₄was mediate,whereas CAG₈ and CAG₂₁at maximum;as for AR protein,CAG₈ was at minimum,CAG₃₄ was mediate,whereas CAG₀ and CAG₂₁at maximum.The differences of the relationship were not paralleled;(3)Low physiological and physiological testosterone concentrations (3-30nM)showed no deleterious effects on the growth of ECV304, whereas 36h later supra-physiological concentrations(3μM-30μM)and 48h later high physiological concentrations(300nM)slowed the cell growth.The growth difference became distinct as action duration of testosterone prolongated and the increase amplitudes of growth curves relaxed;(4)Treatment of synchronized ECV304 cells with testosterone for 24h increased intracellular AR mRNA expression in a concentration dependent fashion.During the low to high physiological testosterone concentration range of 3-300nM,the AR mRNA level keeped at the same higher level;whereas at supra-physiological concentration of 3-30μM, testosterone further induced increase in ARmRNA level;(5)Effects of testosterone pretreatment with different concentrations on the expression of various factors associated with OX-LDL injured ECV304 cells are as follows:in OX-LDL group,the level of NO and TGF-β1 decreased while that of PAI-1,ICAM-1 and PDGF-B decreased; compared with OX-LDL group,T 3nM and 30nM pretreatment had no reversal effects on the expression of PAI-1 and ICAM-1,however,PDGF-B levels further decreased.As for NO and TGF-β1,T 3nM further inhibited expression of both,while T 30nM up-regulated that of both.T 300nM to T 30μM exerted similar effects:inhibitory effects on ICAM-1 and PDGF-B, and stimulating effects on TGF-β1.However,T 3-30μM had no obvious reversal effects on the expression of NO and PAI-1.Flutamide pretreatment could oppose the changes caused by OX-LDL on ECV304 except for PAI-1;(6)Transfection results of the reconstructed AR expression plasmids with 0, 8,21 and 34 CAG repeats into ECV304 were not satisfying,and no further research into the effects of AR gene CAG repeat length on OX-LDL injured ECV304 cells(atherosclerosis)was done.Conclusions:(1)Recombinant PCR is a feasible and effective way for gene recombination and gene deletion mutation;(2)Base on the results of CAG repeat selected here,no linear relationship is found between AR gene CAG repeat length(0,8,21 and 34)and AR mRNA/AR protein level.Notably,the level of AR mRNA and AR protein is highest at CAG₂₁,which is close to the median of CAG repeat numbers investigated from general population;(3)Supraphysiological testosterone concentration has an obvious inhibitory effect on the growth of ECV304;(4)Treatment of ECV304 cells with testosterone increases intracellular AR mRNA expression in a concentration dependent manner;(5)Low physiological testosterone concentration has no protective effects against OX-LDL injure on ECV304;whereas mechanism underlying the protective effects of testosterone of physiological,high- or supraphysiological concentration against oxidative injure is not always the same;Flutamide(AR antagonist)also shows a protective effect against oxidation,mechanism of which remains unknown.