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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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Multiphoton microscopy and histology analysis of myocardial infarction animals.(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.
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pone-0089979-g001: Multiphoton microscopy and histology analysis of myocardial infarction animals.(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.

Mentions: As a treatment strategy, we delivered a lentiviral vector (LV) expressing shRNA that is targeted to the promoter area of the murine VEGF-A and upregulates its expression by an epigenetic mechanism (LV-451). Both the treatment vector and the scrambled shRNA vector contained a GFP marker gene (shRNA control). Immunohistological analysis showed a strong GFP expression localized mostly around the needle track in the transduced hearts (Fig. 1, b), with some signal also under pericardium. Multiphoton microscopy confirmed the 3D expression pattern (Fig. 1, a and Movie S1). Masson’s Trichrome staining (Fig. 1, d, e, h, k, l, o) was made to analyze the infarct area in VEGF-A upregulated (d, e, h) and control group (k, l, o). The upper insert box in both Fig. 1, d and k is from the infarcted area and the lower insert box is from area with borderline infarction. The three images on the right are from that same location, for example Fig. 1, e-g are from the area in upper box in Fig. 1, d. Smooth muscle cells were detected using Alpha-SMA staining (Fig. 1, f, i, m, p) and the formation of arterioles, especially in VEGF-A upregulated group (f and i), was seen. Staining for endothelial cells (CD31, Fig. 1, g, j, n, q) showed their localization in the arterioles.


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)

Multiphoton microscopy and histology analysis of myocardial infarction animals.(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089979-g001: Multiphoton microscopy and histology analysis of myocardial infarction animals.(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.
Mentions: As a treatment strategy, we delivered a lentiviral vector (LV) expressing shRNA that is targeted to the promoter area of the murine VEGF-A and upregulates its expression by an epigenetic mechanism (LV-451). Both the treatment vector and the scrambled shRNA vector contained a GFP marker gene (shRNA control). Immunohistological analysis showed a strong GFP expression localized mostly around the needle track in the transduced hearts (Fig. 1, b), with some signal also under pericardium. Multiphoton microscopy confirmed the 3D expression pattern (Fig. 1, a and Movie S1). Masson’s Trichrome staining (Fig. 1, d, e, h, k, l, o) was made to analyze the infarct area in VEGF-A upregulated (d, e, h) and control group (k, l, o). The upper insert box in both Fig. 1, d and k is from the infarcted area and the lower insert box is from area with borderline infarction. The three images on the right are from that same location, for example Fig. 1, e-g are from the area in upper box in Fig. 1, d. Smooth muscle cells were detected using Alpha-SMA staining (Fig. 1, f, i, m, p) and the formation of arterioles, especially in VEGF-A upregulated group (f and i), was seen. Staining for endothelial cells (CD31, Fig. 1, g, j, n, q) showed their localization in the arterioles.

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