The Role Of NADPH Oxidase-2 In Calcific Aortic Valve Disease

Background: Calcific aortic valve disease(CAVD),currently lack of effective clinical pharmacotherapy,is the third-most prevalent cardiovascular disease after coronary artery disease and hypertension.Emerging evidence indicates reactive oxygen species(ROS)are involved in the process of CAVD,however the sources of ROS and their regulatory roles in CAVD are elusive.NADPH oxidase-2(Nox2)is an important ROS-generating enzyme in cardiovascular system,but its roles in the process of CAVD are not fully understood,even lack of basic knowledge about its distribution and expression in normal aortic valves.Objectives: To investigate: 1 the physiological expression patterns of NADPH oxidase main isoforms Nox2 and Nox4 in normal porcine aortic valves,and in cultured aortic interstitial cells(AVIC)and endothelial cells(EC);2 the change profiles of Nox2 levels during AVIC calcification and CAVD in rabbits;3 the effects of Nox2 inhibitor Celastrol on AVIC calcification in vitro and rabbit CAVD in vivo.Methods: 1 The m RNA and protein levels of Nox2 and Nox4 were evaluated by RT-PCR,Western blot and immunofluorescence in different parts of aortic valves and cultured AVIC and EC.2 The Nox2 protein levels and calcification of AVIC with or without Celastrol treatment(10 n M,14 days)were examined by Western blot or Alizarin red staining.3 The protective effects of Celastrol(20 mg/day,18 weeks)on an established rabbit CAVD model were evaluated by echocardiography.The changes of Nox2 levels and calcification marker Runx2 were also examined by immunohistology,and/or RT-PCR.Results: 1 The protein levels of Nox2 and Nox4 were both significantly higher in the root of porcine aortic valves than that of in the tip and middle parts.Compared to VIC,the endothelial Nox4 levels were markedly higher,but Nox2 proteins were significantly higher in VIC.2 The protein levels of Nox2 were increased during AVIC calcification.Nox2 inhibitor Celastrol significantly diminished AVIC calcification.3 There was an increase of Nox2 levels in aortic valves of a rabbit CAVD model.Oral treatment of Celastrol could effectively attenuate the severity of aortic valve calcification evaluated by echocardiography and molecular analyses.Conclusions: 1 The expressions of Nox2 and Nox4 in normal aortic valves are tissue and cell-specific.2 Nox2 levels increase during CAVD,Nox2 inhibitor Celastrol significantly attenuates AVIC calcification in vitro,and rabbit CAVD in vivo.