Transfection of Ang-1 gene via ultrasound-mediated SonoVue microbubble destruction in vitro and in vivo

Abstract

Objective To investigate the transfection efficacy and expression of Ang-1 gene and proangiogenesis in vitro and vivo by ultrasound-mediated microbubble destruction.Methods 293T cells were divided into three groups:group A was given hAng-1 plasmid and microbubbles plus ultrasonic irradiation,group B was given hAng-1 plasmid and ultrasound,group C was given hAng-1 plasmid only (without ultrasound).Forty-eight hours after transfection,the transient expression rate was observed under fluorescence microscopy and flow cytometry.RT-PCR and Western blot analysis were taken to evaluate the mRNA and protein expression of Ang-1 respectively.Twenty-seven rabbit models of ligated left circumflex branch coronary artery were divided into 3 groups randomly as follow:group Ⅰ (accepted intravenous injection of SonoVue microbubble and Ang-1 plus ultrasonic irradiation),group Ⅱ (accepted intravenous injection of Ang-1 with ultrasound),group Ⅲ (control group).Myocardial contrast echocardiography (MCE) was executed on all animals before and after the treatment.Two weeks after gene delivery,RT-PCR and Western blot analysis were taken to evaluate mRNA and protein expression of Ang-1 respectively.Microvessel density (MVD) counting of infracted myocardium,observed by Factor Ⅷ immunochemical staining,was performed to value the proangiogenesis effect of Ang-1 delivered by ultrasound mediated cavitation of microbubble.Results Green fluorescence was observed in group A and B by fluorescence microscopy,which was negative in group C.The transfection expression rate was significantly improved in group A ( P ＜ 0.01).In vivo,Microbubbles could be observed in former ischemic myocardium in MCEexamination and the Ang-1 mRNA and protein could be detected in group Ⅰ.On the other hand,the contrast agent was defected obviously and none of the animals showed Ang-1 mRNA and protein expression in other two groups.The MVD counting showed significant improvement in group Ⅰ whereas other two groups didn't.Conclusions Microbubble-enhanced ultrasound exposure can improve the Ang-1 gene transfection expression rate observably both in vitro and in vivo.This strategy for delivering has great proangiogenesis effect in vivo. Key words: Ultrasonography; Microbubbles; Transfection; Angiopoietin-1