doi: 10.1089/hgtb.2013.028.
Noninvasive imaging of hypoxia-inducible factor-1α gene therapy for myocardial ischemia
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- PMID:23937265
- PMCID: PMC3798229
- DOI: 10.1089/hgtb.2013.028
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Noninvasive imaging of hypoxia-inducible factor-1α gene therapy for myocardial ischemia
Ian Y Chen et al. Hum Gene Ther Methods.2013 Oct.
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Abstract
Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.
Figures
Schematics of the bidirectional TSTA plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc) for indirect imaging of HIF-1α-VP2 expression. The experimental vector, pcTnT-HIF-1α-VP2-TSTA-fluc, uses the cTnT promoter to drive the expression of the GAL4-VP2 fusion protein, which can bind to any of the eight tandem repeats of a GAL4-binding sequence (8xGAL4bs) on the same vector, leading to recruitment of RNA polymerase, followed by enhanced bidirectional transcription of a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2) gene and the firefly luciferase (fluc) gene. The translated HIF-1α-VP2 can subsequently stimulate angiogenesis by activating the expression of downstream angiogenic genes (e.g., VEGF) via binding to their HRE in the promoter region. Given a strong correlation between the expression of HIF-1α-VP2 and the firefly luciferase enzyme (FLuc), the HIF-1α-VP2 expression can be inferred from the FLuc enzyme activity, which can be imaged with BLI using a systemically delivered probe, D-Luciferin. Upon entering the cytosol of a transfected cell (yellow background), D-Luciferin interacts with FLuc to emit bioluminescence, which can be detected and quantified using an ultrasensitive cooled CCD camera. BLI, bioluminescence imaging; cTnT, cardiac troponin T; E4TATA, adenovirus E4 minimal promoter; HRE, hypoxia response element; PA, SV40 poly(A) tail; TSTA, two-step transcriptional amplification; VEGF, vascular endothelial growth factor; VP2, 2 tandem repeats of herpes simplex virus VP16 transcriptional activator. Color images available online atwww.liebertpub.com/hgtb
In vitro characterization of pcTnT-HIF-1α-VP2-TSTA-fluc. HL-1 cells co-transfected with increasing doses of pcTnT-HIF-1α-VP2-TSTA-fluc, fixed doses of both p5xHRE-hRluc and pCMV-β-gal, as well as appropriate doses of pcDNA3.1(+) (empty vector) were assayed for FLuc, RLuc, and β-GAL activities 36 hr later. Cytotoxicity, in terms of cell proliferation at 36 hr post-transfection, was additionally assessed in a repeat transfection experiment.(A) FLuc and(B) RLuc activities normalized by total protein and transfection efficiency (β-GAL activity) are plotted for the plasmid dose indicated. VEGF from cell medium is plotted against(C) plasmid dose and(D) FLuc activity at the corresponding plasmid dose.(E) Cell proliferation, expressed as a percentage of that at 0 μg plasmid, is plotted for the plasmid dose shown. The error bars represent SEM for triplicate determinations. SEM, standard error of mean.
HIF-1α-VP2 expression and HIF-1α signaling after intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc. Experimental mice co-injected with pcTnT-HIF-1α-VP2-TSTA-fluc and p5X-HRE-hRluc underwent serial BLI imaging for 4 weeks. Mice co-injected with pcDNA3.1(+) (empty vector) and p5X-HRE-hRluc served as negative controls.(A) The mean FLuc signal (surrogate for HIF-1α-VP2 expression) and(C) the mean RLuc signal (indicator of HIF-1α/HRE pathway activation) of the experimental (black solid line; EXP) and control (gray solid line; CNTRL) mouse groups are plotted for the days indicated. The error bars represent SEM of five mice. Representative(B) FLuc and(D) RLuc images of an experimental mouse (EXP) and a control mouse (CNTRL) are displayed for the selected days indicated, with the heart signal and the background hepatic RLuc signal indicated by the red and black arrows, respectively.(E) Mice that received intramyocardial co-injections of varying amounts of pcTnT-HIF-1α-VP2-TSTA-fluc and a fixed amount of p5X-HRE-hRluc were sacrificed on day 2, with their hearts assayed for both FLuc and RLuc enzyme activities, which are plotted against each other. Color images available online atwww.liebertpub.com/hgtb
Sustained HIF-1α-VP2 expression failed to improve long-term microvessel density. Mice that underwent experimental MI, followed by intramyocardial injection of either pcTnT-HIF-1α-VP2-TSTA-fluc (experimental vector) or pcTnT-EGFP-TSTA-fluc (control vector), were serially imaged with BLI for 4 weeks to assess vector expression by inferring from the FLuc-mediated BLI signal.(A) The mean FLuc signal over time is plotted for both the experimental (solid black line; EXP) and the control (solid gray line; CNTRL) mouse groups. The error bars represent SEM for 11 experimental mice and 9 control mice.(B) Representative bioluminescence images of a control mouse (CNTRL) and an experimental mouse (EXP) are displayed for the selected days indicated, with the heart signals indicated by the red arrows.(C) For both mouse groups, the postmortem CD31 immunohistochemical staining of the peri-infarct regions at day 28 highlighted the microvessels (bright red) within the host myocardium (darker red background). (D) The mean microvessel density quantified using ImageJ is plotted for both mouse groups. The error bars represent SEM for five mice. Color images available online atwww.liebertpub.com/hgtb
Serial echocardiographic assessment of cardiac function pre- and post-MI or pcTnT-HIF-1α-VP2-TSTA-fluc delivery.(A) The LVFS% (top graph) and the LVEF% (bottom graph) of the experimental (black bar; EXP) and the control (gray bar; CNTRL) MI mouse groups injected with pcTnT-HIF-1α-VP2-TSTA-fluc and pcTnT-EGFP-TSTA-fluc, respectively, are shown for baseline (3 days before MI) and days 3, 14, and 28 post-MI or vector injection. Error bars represent SEM for 11 experimental mice and 9 control mice.(B) Representative M-mode strips acquired at the levels indicated by the short-axis images displayed above are shown for both a control mouse (top row, CNTRL) and an experimental mouse (bottom row, EXP) at 3 days (left column) and 4 weeks (right column) after MI. Color images available online atwww.liebertpub.com/hgtb
References
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