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Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.

Turunen MP, Husso T, Musthafa H, Laidinen S, Dragneva G, Laham-Karam N, Honkanen S, Paakinaho A, Laakkonen JP, Gao E, Vihinen-Ranta M, Liimatainen T, Ylä-Herttuala S - PLoS ONE (2014)

Bottom Line: Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts.Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity.In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Molecular Medicine, A.I.Virtanen Institute, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
"Epigenetherapy" alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

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Related in: MedlinePlus

ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.
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pone-0089979-g003: ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.

Mentions: The use of MRI techniques for cardiovascular applications has recently become available in mice, such as measuring infarct size and functional parameters under increased workload [9], [10]. In the current work, we applied cine imaging to follow up infarct size and heart functional parameters in the infarcted myocardium. After the therapy the infarct size diminished from day 4 to day 14 by over 5%, whereas in the control group the infarct size increased resulting in a significant difference between the study groups at day 14 (p<0.05, Two-way ANOVA, Fig. 2). We also analyzed murine VEGF-A protein expression from whole-protein lysates of the transduced hearts using ELISA assay and found that VEGF-A was upregulated in the treated hearts as compared to the shRNA control (Fig. 3, a). The result indicates a therapeutic effect of epigenetic VEGF-A upregulation especially in the infarcted areas where cardiomyocytes are suffering from insufficient circulation and hypoxia.


Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.

Turunen MP, Husso T, Musthafa H, Laidinen S, Dragneva G, Laham-Karam N, Honkanen S, Paakinaho A, Laakkonen JP, Gao E, Vihinen-Ranta M, Liimatainen T, Ylä-Herttuala S - PLoS ONE (2014)

ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935957&req=5

pone-0089979-g003: ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.
Mentions: The use of MRI techniques for cardiovascular applications has recently become available in mice, such as measuring infarct size and functional parameters under increased workload [9], [10]. In the current work, we applied cine imaging to follow up infarct size and heart functional parameters in the infarcted myocardium. After the therapy the infarct size diminished from day 4 to day 14 by over 5%, whereas in the control group the infarct size increased resulting in a significant difference between the study groups at day 14 (p<0.05, Two-way ANOVA, Fig. 2). We also analyzed murine VEGF-A protein expression from whole-protein lysates of the transduced hearts using ELISA assay and found that VEGF-A was upregulated in the treated hearts as compared to the shRNA control (Fig. 3, a). The result indicates a therapeutic effect of epigenetic VEGF-A upregulation especially in the infarcted areas where cardiomyocytes are suffering from insufficient circulation and hypoxia.

Bottom Line: Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts.Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity.In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Molecular Medicine, A.I.Virtanen Institute, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
"Epigenetherapy" alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

Show MeSH
Related in: MedlinePlus