Angiotensin Ⅱ Induces Atrial Fibrosis Through Activating Platelet-derived Growth Factor/Rac1/Nuclear Factor-kappa B Axis In Atrial Fibroblasts

BackgroundAtrial fibrillation (AF) is the most encountered arrthythmia in clinicalpractice and its incidence increases with age. Atrial fibrillation is associatedwith many forms of cardiac pathology, including coronary artery disease,arterial hypertension, valvular diseases, heart failure, and cardiac surgery,but a substantial proportion of patients with AF have no detectable heartdisease. Hemodynamic impairment and thromboembolic events related toAF result in significant morbidity, mortality, and cost. At present, theunderlying mechanisms responsible for its induction and perpetuationremain incompletely understood and its Clinical effect is not satisfied.Pharmacological approach to AF therapy is target to the underlyingsubstrates. Atrial fibrosis is one of the most important substrates in AF andrefers to the progressive architectural deterioration of the atria afterrepeated episodes of AF. The molecular mechanism of atrial fibrosis islargely unknown. The present study found that, transforming growth factor-β（TGF-β）、mitogen-activated protein kinase (MAPKs)、Januskinase(JAK)/signal transducers and activators of transcription (STAT)pathway、 connective tissue growth factor(CTGF)、 OxidizedCa(2+)/calmodulin-dependent protein kinase II and so on，which wereparticipated in formation of atrial fibrosis. The renin-angiotensin system(RAS) is involved in the pathogenesis of various cardiovascular diseases.Activation of local RAS in the heart as an initiate factor play a vital role inthe pro-fibrosis of the atria. Ang Ⅱ, the main active factor of RAS,stimulates a lot of signal pathway activation, and facilitates the process ofthe atrial fibrosis. It has been demonstrated exact signaling pathwaysinducing cardiac structural remodeling have AngⅡ/Rac1/STAT3、AngⅡ/Rac1/CTGF/Cx43（N-Cadherin）、 Ang/Cn/NFAT/MMPs、AngⅡ/MAPKs/TGF-beta1/TRAF6、the mast cell/PDGF-A axis and soforth.Our previous studies have found that the degree of atrial fibrosis elevatedin the atrial tissue of transverse aortic constriction (TAC) rats, andup-regulation of AngⅡ concentration in the atiral was coupled withelevated expression level of PDGF, activated-Rac1and NF-ΚB. Followingtreatment with irbesartan or/and simvastatin, the degree of atrial fibrosisdecreased, and the expression level of PDGF、activated Rac1and NF-κBattenuated significantly concomitant with decreased AngⅡ concentrationin the atiral. These results indicated that the interaction among Ang II, PDGF, Rac1, NF-κB is profoundly implicated in the pathogenesis of atrialfibrosis, and that irbesartan and simvastatin could effectively inhibit theformation of atial fibrosis.ObjectivesIn order to verify the results from patients with AF and TAC rats models,The purpose of this study further explored whether they have interactionsamong AngII,PDGF,Rac1,NF-κB and their upstream and downstreamrelationship.MethodsBy isolating and culturing primary atrial fibroblasts of Sprague-Dawley,Transient transfection of atrial fibroblasts with lentiviral vector encodingthe wild-type PDGF(WT-PDGF), constitutively active PDGF (PDGF-C),dominant negative PDGF (PDGF-D), constitutively active Rac1(RacV12)and dominant negative Rac1(RacN17). Treatment of atrial fibroblasts withAng Ⅱ, simvastatin, irbesartan and the specific Rac1small moleculeinhibitor NSC23766, NF-κB protein expression was measured by westernblot, the activated Rac1was detected by PAK-PBD pull down assay,Intracellular PDGF-AA quantity was determined by ELISA, fibrosismarkers of COL1A1mRNA expression and transfection efficiency oflentiviral vector encoding PDGF and Rac1were detected by real timequantitative PCR assay. Results1. Intracellular PDGF-AA quantity increased5miniutes later (p＜0.05),activated Rac1markedly increased30miniutes later（p＜0.01） andNF-κB protein expression significantly up-regulated2hours later (p＜0.01) in atrial fibroblasts after treatment by AngⅡ（10-6mol/L). Whenthe atrial fibroblasts was incubated with AngⅡ in differentconcentration24hours later, the concentration of PDGF intracellularwas markedly increased when the concentration of Ang Ⅱ＞10-8mol/L（p＜0.01）,and the expression of Rac1activity and NF-κB protein wereboth significantly increased in the condition of Ang Ⅱ concentration＞10-7mol/L （p＜0.01）.2. An increase and decrease in Rac1activity and NF-κB protein expressionby over-expressing PDGF-C and PDGF-D were found, respectively (allP <0.01).3. Transfection of RacV12increased the activation of Rac1（p＜0.01）inatrial fibroblasts, whereas Rac1activity was down-regulated（p＜0.01）by RacN17.4. Reduction of Rac1activity and NF-κB protein expression by stimulatingwith simvastatin or irbesatan was observed (all P <0.01). Moreover, theattenuation effect was even greater when simvastatin and losartan werecombined.5. NF-κB protein expression was attenuated by the specific Rac1smallmolecule inhibitor NSC23766in atrial fibroblasts.ConclusionIn summary, the study identity a critical signal transduction PDGF/Rac1/NF-κB initiated by AngⅡ contributes to the pathogenesis ofatrial fibrosis from the level of cultured cells. Treatment with RASinhibitors and statins could improve atrial fibrosis by regulating theexpression of AngⅡ/PDGF/Rac1/NF-κB axis, and it indicated interventionof PDGF may be one of the new targets for prevention and treatment of AF.