Author + information
- Xhang Li,
- Zhenxing Sun and
- Mingxing Xie
We hypothesize that the inhibition of HIF-1α degradation via short hairpin RNA (shRNA) knockdown of PHD2 using UTMD in rats would promote transgene expression, facilitate angiogenesis, improve myocardial function and provide a potential therapy for ischemic and reperfusion myocardial injury in rats.
In vitro, after cationic microbubbles (CMBs) were prepared and identified, we assessed the gene-binding capacity of CMBs and examined the feasibility of UTMD for naked plasmid DNA transfection into H9C2 cardiac cells. The CMBs bound more plasmid DNA and exhibited a saturated loading capacity. UTMD-mediated gene delivery with CMBs enhanced transfection efficiency; however, this method requires optimization to maximize gene transfection. To better evaluate the therapeutic effect after shPHD2-EGFP gene transfer by UTMD, a flow cytometric analysis was used.
Silencing of PHD2 reduced the H9C2 cell apoptosis induced by oxygen glucose deprivation (OGD), and molecular studies revealed a significant inhibition of PHD2 and activation of HIF-1α and its downstream genes in the shPHD2-EGFP group (P < 0.05). In vivo, we also studied the transfection efficiency of naked plasmid DNA using ultrasound with CMBs transfected into the myocardium. Three days after transfection, EGFP in myocardium transfected with CMBs using ultrasound was significantly increased compared to that in the other treatment groups (P < 0.05). The impact of UTMD-mediated shPHD2 transfection on the protective effects was evaluated with a TUNEL assay. The number of TUNEL+ cardiomyocyte nuclei per mm2 was significantly reduced in shPHD2-EGFP-treated hearts at 48 hours post-MI compared with EGFP-treated hearts. Finally, we examined the feasibility of therapeutic angiogenesis using naked shPHD2-EGFP plasmid in a rat ischemia model using the UTMD method. Four weeks after transfection, compared with the MI-EGFP group, left ventricular function at the infarct area had improved and scar size was reduced in the MI-shPHD2-EGFP group (P < 0.05). PHD2 expression was lower and HIF-1α and its downstream expression were higher in the MI-shPHD2-EGFP group than in the sham, MI-EGFP and MI-shPHD2-EGFP groups (P < 0.05). The numbers of HIF-1α- and VEGF-positive cells/mm2 in the MI-shPHD2-EGFP group were greater than those in the MI-EGFP group (P < 0.05). Capillary densities in the MI-shPHD2-EGFP group were increased 2.29-fold (P < 0.05) over those in the MI-EGFP group.
In conclusion, ultrasound with CLMs successfully promoted the transfection efficiency of plasmid DNA in vitro as well as in vivo, thus improving the efficacy of therapeutic angiogenesis and myocardial function. The UTMD technique may serve as an ideal method for noninvasive, repeated gene delivery after an MI.