Ultrasound-targeted cationic microbubbles combined with the NFκB binding motif increase SDF-1α gene transfection: A protective role in hearts after myocardial infarction

Abstract

Treatment of myocardial infarction (MI) remains a major challenge. The chemokine family plays an important role in cardiac injury, repair, and remodeling following MI, while stromal cell-derived factor-1 alpha (SDF-1α) is the most promising therapeutic target. This study aimed to increase SDF-1α expression using a novel gene delivery system and further explore its effect on MI treatment. In this study, two kinds of plasmids, human SDF-1α plasmid (phSDF-1α) and human SDF-1α- nuclear factor κB plasmid (phSDF-1α-NFκB), were constructed and loaded onto cationic microbubble carriers, and the plasmids were released into MI rabbits by ultrasound-targeted microbubble destruction. The transfection efficiency of SDF-1α and the degree of heart repair were further explored and compared. In the MI rabbit models, transfection with phSDF-1α-NFκB resulted in higher SDF-1α expression in peri-infarct area compared with transfection with phSDF-1α or no transfection. Upregulation of SDF-1α was shown beneficial to these MI rabbit models, as demonstrated with better recovery of cardiac function, greater perfusion of the myocardium, more neovascularization, smaller infarction size and thicker infarct wall 1 month after treatment. Ultrasound-targeted cationic microbubbles combined with the NFκB binding motif could increase SDF-1α gene transfection, which would play a protective role after MI.

Keywords: myocardial infarction; nuclear factor κB; stromal cell-derived factor 1 alpha; ultrasound targeted microbubble destruction.

FIGURE 1

Plasmid structure. (A) Structure of the pcDNA3.1(−). (B) Structure of the human SDF‐1α plasmid (phSDF‐1α). The hSDF‐1α DNA is inserted downstream of the CMV promoter. (C) Structure of the hSDF‐1α‐nuclear factor κB plasmid (phSDF‐1α‐NFκB). The 5× NFκB binding motif (five repeats of 5′‐GGGACTTTCC‐3′) is inserted downstream of the CMV promoter. The sequence encoding hSDF‐1α cDNA follows the 5× NFκB binding motif

FIGURE 2

Characteristics of cationic microbubbles (MBs) and plasmid conjugation. (A) Plasmid‐loaded cationic MBs under light and fluorescence microscopy (scale bar, 5 μm). The red fluorescence at the periphery of cationic microbubbles in the fluorescence field indicated that the Cy3‐labeled plasmids were successfully connected to the surface of cationic MBs. (B) Transmission electron microscopy image of the cationic MBs (scale bar, 1 μm). (C) The size distribution and zeta potential of cationic MBs dissolved in phosphate‐buffered saline over a prolonged period (0.5, 1, 1.5, and 2 h). Data are shown as mean ± SD

FIGURE 3

SDF‐1α expression in the peri‐infarct myocardium 3 days following transfection. (A) Real‐time quantitative polymerase chain reaction detects SDF‐1α mRNA expression. The relative expression of the mRNAs is normalized by glyceraldehyde 3‐phosphate dehydrogenase. (B) Western blot analysis of SDF‐1α protein expression. The relative expression of SDF‐1α protein is normalized by β‐actin. (C) Quantitative analysis of the SDF‐1α protein expression by using enzyme‐linked immunosorbent assay. Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. SDF‐1α, stromal cell‐derived factor‐1 alpha

FIGURE 4

Effects of neovascularization 1 month after the gene transfection. Neovascularization of the peri‐infarct areas was detected following immunostaining for CD31. (A) Representative histopathological staining of myocardial sections (scale bar, 20 μm). (B) Quantification of microvascular density (MVD). Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 5

Cardiac morphology 1 month after the gene transfection. (A) Representative histological Masson's trichrome staining of transverse sections of infarcted hearts (scale bar, 5 mm). Normal heart muscle appears red and fibrotic scar tissue appears blue. (B) Quantification of the infarct size and wall thickness. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001