Delivery Of TFPI-2 Using Microbubbles Ultrasound Contrast Agent Results In The Suppression Of Thrombosis And Arterial Stenosis

Atherosclerosis is characterized by thrombosis and arterial stenosis within the intima of the arterial wall, and is a multi-factorial chronic inflammatory disease. There is currently no "ideal" treatment for atherosclerosis. Gene therapy for atherosclerosis has been studied extensively but clinical application has not transpired. It is well known that tissue factor (TF), a transmembrane protein that triggers blood coagulation in vivo, contributes to atherosclerosis development. TFPI-2 inhibits TF activity by forming a TF-FVIIa-FXa-TFPI complex, which causes sustained repression of the TF pathway and regulates digestion and remodeling of the extracellular matrix. Therefore, TFPI-2 is a potential mechanism for preventing atherosclerosis. TFPI-2 may therefore be a candidate gene for suppressing thrombosis and arterial stenosis. Transporting the candidate gene into the targeted diseased tissue for reversal of the features of atherosclerosis is the main barrier. The interaction of microbubble contrast agents with ultrasonic waves would alter the permeability of biological membranes and facilitate gene transport into cells. This may therefore be a potential method of gene delivery into arterial walls. In the present study, we delivered the TFPI-2 gene by SonoVue (a kind of microbubble ultrasound contrast agent, Bracco Imaging B.V., Geneva, Switzerland) and observed the effects of this atherosclerosis gene therapy strategy in vitro and in vivo.PART ONE Study of TFPI-2 Delivered into Artery Endothelial Cells by Microbubbles Ultrasound Contrast AgentTo investigate the transfection efficiency of ultrasound microbubbles (SonoVue), which transfected the tissue factor pathway inhibitor-2 (TFPI-2) into human umbilical vein endothelial cells (HUVECs), and compared with that of other transfection methods. Firstly, we successfully constructed two recombination plasmids (pEGFP-TFPI-2 and pcDNA3.1-TFPI-2) and the adenovirus with TFPI-2 gene, and a great deal of amount preparation and purification. Optimizing the experimental conditions of the assay is required before experiments could be done and analyzed. We found that SonoVue and ultrasound did not cause significant harm to HUVECs and the high adherence of SonoVue was 90.1%. Next to compare the trasfection efficiency of different experimental groups:(1) control group:plasmids; (2) liposome group: plasmids+liposome; (3) SonoVue group:plasmids+SonoVue+ultrasound; (4) plasmids and adenovirus.24h, to detect the transfection efficiency and TFPI-2 expression in different groups using fluorescence microscope, RT-PCR and Western blot, respectively. The transfection efficiency of SonoVue and adenovirus groups was higher than that of the liposome group, but it was not obviously different among SonoVue and adenovirus groups. Similarly, the mRNA and protein expression of TFPI-2 gene in SonoVue and adenovirus groups were higher than other experimental groups. These results suggested that SonoVue may deliver TFPI-2 gene into arterial endothelium.Part TWO Delivery of TFPI-2 gene by SonoVue results in the Suppression of Thrombosis and Arterial StenosisAtherosclerosis was a disease which was characterized a chronic inflammatory response within the intima of the arterial wall to lead artery thrombosis and stenosis. It was ideal gene therapy method that delivered the treatment gene into the walls of arteries in free-flowing blood. In this study, we investigated that delivery of TFPI-2 gene using SonoVue into arterial wall results in the suppression of thrombosis and arterial stenosis. Before the experiments, a rabbit model of a damaged carotid artery was established using the 2 F balloon catheter to mimic the positive group of atherosclerosis. And we found that SonoVue may deliver TFPI-2 gene into arterial endothelium and the transfection efficiency of SonoVue was higher than that of adenovirus. On the base of these results, we researched the suppression of thrombosis and arterial stenosis of TFPI-2 in different experimental groups:(1) the first negative group:no any treatment; (2) the second negative group:PBS+plasmids+ultrasound; (3) positive group:arterial damaged model; (4) SonoVue group:plasmids+SonoVue +ultrasound; (5) adenovirus group:adenovirus with TFPI-2 gene. On the tenth day, five group rabbits of arterial tissue were checked by ultrasound and assessed for arterial tension, pathology, and western blot. The two negative groups have not found any plaques and stenosis in arteries. Compared with the positive group, the SonoVue and adenovirus groups have a fewer plagues and stenosis in arteries, a bigger arterial diameter and tension, and a smaller arterial blood flow velocity. With consulting theβ-actin, the TFPI-2 expression of SonoVue and adenovirus groups in arterial endothelium was higher than that of other experimental groups. These results supported that delivery of TFPI-2 gene using SonoVue into arterial endothelium results in the suppression of thrombosis and arterial stenosis.