Ultrasonic microbubble-mediated gene delivery causes phenotypic changes of human aortic endothelial cells

Ultrasound, in combination with microbubbles, serves as a feasible nonviral method in vascular gene delivery. However, the effects of ultrasonic microbubble transfection (UMT) on vascular endothelial cells remained unclear. We therefore investigated whether UMT itself causes phenotypic changes of the human aortic endothelial cells (HAEC) in vitro. HAEC were cultured with solution containing luciferase reporter gene and microbubbles followed by exposure to ultrasound of selected parameters. Thereafter, the proliferation and migration activities of HAEC were investigated. Real-time RT-PCR and/or western blotting were performed to assess expression profile of HAEC, including growth-related factors (vascular endothelial growth factor, fins-like tyrosine kinase-1 [Flt-1] and kinase insert domain-containing receptor [KDR]), coagulatory factor (von Willebrand factor), vasodilatory enzyme (endothelial nitric oxide synthase), gap junctional protein connexin43 and adhesion molecules (P-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1). The results showed that in conditions where UMT lead to expression of luciferase, proliferation capacity is enhanced (p<0.001), partly attributable to the effect of ultrasound (p<0.05), after excluding the effect of contact inhibition. In addition, the expression of KDR and Flt-1 were found increased at either the mRNA level, protein level, or both (p<0.05). Other markers did not have significant changes (all p>0.2). Similarly, the migration capacity was minimally changed (p>0.3). In conclusion, UMT causes phenotypic changes of HAEC by enhancing proliferation and upregulating KDR and Flt-1, while possesses no obvious adverse effect on viable transfected cells. Further investigation is required to clarify the impact of these changes by UMT in vivo.