Ultrasound Targeted Microbubble Destruction Mediate PHD2-shRNA Interference For The Therapy Of Ischemic Heart Disease

Ischemic heart disease(IHD) is the leading cause of morbidity and mortality in the world.Conventional treatment for IHD consists of medical therapy as the first-line strategy followed by percutaneous coronary intervention or coronary artery bypass graft. It can significantly reduce mortality and morbidity. However, a significant number of patients will still have refractory angina despite these treatments. For such patients, the alternative approach of delivering potent angiogenic factors to stimulate new vessel growth has undergone intense investigation over the past decade.The progress of gene therapy largely depends on the development of gene delivery technologies, including viral vector and non-viral vector. Among non-viral techniques, ultrasound-targeted microbubble destruction(UTMD) has evolved as a new promising tool for organ-specific gene delivery. Recent studies showed that mechanical and cavitation effects caused by UTMD were able to increase membrane permeability and enhance exogenous genetic materials into targeted cells.In this article, we assessed whether the novel combination of UTMD and cationic lipid microbubbles(CLM) was available and useful tool for gene delivery and transfection. Furthermore, we demonstrated that the inhibition of HIF-1αdegradation through shRNA knockdown of PHD2 in the ischemic heart represents a novel angiogenic therapy approach. Objective:①To prepare cationic lipid microbubble (CLM), and to evaluate its physical and chemical properties and toxicity;②To evaluate the gene transfection efficiency by UTMD accompanied with CLM. Methods①The CLM was prepared by the thin film hydration, and its morphology was observed with electron microscopy in 1h,24h,3d,7d, and 14d, respectively, the nano-particle size were measured and the stability was tested; acute toxicity were observed;②Different transfection methods of the green fluorescent protein (EGFP) gene transfection enhancement. Experimental groups:naked plasmid group (P group), Ultrasonic irradiation and plasmid (P-US group), plasmid and CLM(P-CLM Group), naked plasmid and ultrasound and CLM (UTMD Group). The expression of EGFP was observed.Results①CLMs were spherical, the similar size and good distribution degree under the light and electron microscopy. The nano-size of cationic lipid microbubbles was varied from (250.4±88.32)nm to(399±99.8)nm and the Potential was varied from (18.8±4.97) mV to (20.1±3.1) mV. Vivo and in vitro toxicity tests have shown no acute toxicity.②The EGFP expression was the strongest intensity in UTMD group, P-CLM group was secondary, and that in P group was the weakest; the results from flow cytometry analysis were coincidental to fluorescence microscopy results. The transfection efficiency rate of P-US was almost 7 times than that of P group; That in P-CLM group than in P group increased by about 10 times; UTMD group than in P group increased nearly 30 times.Conclusions:Nano-size CLM prepared by film hydration was characteristic as uniform diameter, non-toxic side effects; Ultrasound combined with CLM can significantly increase the transfection of EGFP to HUVEC. Objective:To construct the targeting PHD2 shRNA eukaryotic expression vector by combining UTMD with RNA interference technology. To investigate the feasibility of PHD2-shRNA transfection to human umbilical vein endothelial cells (HUVEC) induced by hypoxic condition and the PHD2 gene silencing effect, HIF-1αand its downstream angiogenesis factor expression under hypoxic condition. Methods:We constructed the targeting PHD2 shRNA eukaryotic expression plasmid (shPHD2) and the control plasmid (shScramble), inserted the hypoxia response element (HRE) oligonucleotide fragments into plasmid, and transfected shPHD2 and shScramble to HUVEC cells mediated by UTMD. Under In vitro microenvironment; we observed shPHD2 transfection efficiency and detected PHD2, HIF-1αand its downstream angiogenesis factor expression with RT-PCR, Western blots. Results:Fluorescence microscope and luciferase detection were confirmed that the luciferase activity of shPHD2 transfection group significantly increased in normal oxygen and hypoxic conditions comparing with control group(P<0.05). Under hypoxic conditions, there was more angiogenesis factor expression. Western blot confirmed the HIF-1a stable expression increased in hypoxia condition. In order to verify the HIF-1a expression is induced by the shRNA silence PHD2 gene, RT-PCR results further confirmed that after shPHD2 transfection treatment, the expression on the PHD2 mRNA reduced, however, the expression of HIF-1a and its downstream 3 angiogenesis-related expression gene significantly increased comparing with the control group (P<0.05). Conclusions:UTMD mediated PHD2 interference expression vectors can effectively promoted silence PHD2, HIF-1αand its downstream angiogenesis-related factor expression. Objective:To construct the recombinant expression vectors targeted PHD2 gene and to analyze the silencing effect in myocardial ischemic in rats; To investigate direct injection of shRNA targeting PHD2 can improve ventricular function and enhance neoangiogenesis in rat model of myocardical infarction by UTMD techniques associated with RNAi techniques. Methods:shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in rat. Animals were randomized into sham operation group(n=30) shPHD2 MI experimental group(n=60) and MI shScramble control group (n=60). Echocardiography was performed before and after(7d,14d,28d) the left anterior descending artery ligation. Explanted heart from study and control groups were embedded into Masson's trchome and HE for immunostaining. Results: echocardiography showsd the shPHD2 group had improved fractional shortening compared with the shSchamble group at 14 days. RT-PCR analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of HIF1RT-PCR analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of HIF1α,VEGF,bFGF and TGFβm RNA.Postmortern analysis show increased presence of small capillaries and venules in the infracted zones by CD34 staining. Conclusions:Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility in Myocardial ischemic heart disease in rats. With further validation, the combination of shRNA therapy and UTMD can be used to track novel cardiovascular gene therapy application in the future.