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miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation.

McDonald RA, White KM, Wu J, Cooley BC, Robertson KE, Halliday CA, McClure JD, Francis S, Lu R, Kennedy S, George SJ, Wan S, van Rooij E, Baker AH - Eur. Heart J. (2013)

Bottom Line: Identifying novel strategies to prevent neointimal thickening is important.Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8TA, UK.

ABSTRACT

Aims: The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.

Methods and results: We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation.

Conclusion: This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

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Effect of pharmacological knockdown of miR-21 in HSVs and miR-21 expression in failed human vein grafts. HSV segments were cultured for 7 and 14 days in the presence of 5000 nM anti-miR-ctl or anti-miR-21 (n = 5/group), then RNA was isolated from the vessels, and expression was measured by quantitative real-time PCR. (A) miR-21 expression. (B) Expression of putative miR-21 target genes. (C) Neointimal thickness. (D) Representative images of elastic van Gieson-stained sections from Day 14 samples. (E) In situ hybridization with a scrambled microRNA probe, miR-21 probe, and immunohistochemistry for smooth muscle cell actin in a failed human vein graft. Scale bar represents 100 μm, applicable to all panels. *P < 0.05 vs. anti-miR-ctl; **P < 0.01 vs. anti-miR-ctl, ***P < 0.001 vs. anti-miR-ctl, ##P < 0.01 vs. Day 0, ###P < 0.001 vs. Day 0.
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EHT105F7: Effect of pharmacological knockdown of miR-21 in HSVs and miR-21 expression in failed human vein grafts. HSV segments were cultured for 7 and 14 days in the presence of 5000 nM anti-miR-ctl or anti-miR-21 (n = 5/group), then RNA was isolated from the vessels, and expression was measured by quantitative real-time PCR. (A) miR-21 expression. (B) Expression of putative miR-21 target genes. (C) Neointimal thickness. (D) Representative images of elastic van Gieson-stained sections from Day 14 samples. (E) In situ hybridization with a scrambled microRNA probe, miR-21 probe, and immunohistochemistry for smooth muscle cell actin in a failed human vein graft. Scale bar represents 100 μm, applicable to all panels. *P < 0.05 vs. anti-miR-ctl; **P < 0.01 vs. anti-miR-ctl, ***P < 0.001 vs. anti-miR-ctl, ##P < 0.01 vs. Day 0, ###P < 0.001 vs. Day 0.

Mentions: To investigate the translational application of our findings and evaluate whether it is possible to achieve pharmacological knockdown of miR-21 in HSVs, the ex vivo culture model was utilized. Culturing HSV in the presence of anti-miR-21 for 7 or 14 days resulted in a >95% knockdown in miR-21 expression (Figure 7A). The expression of previously identified miR-21 target genes25,29–31 was analysed by qRT-PCR. Compared with anti-miR-ctl-treated HSVs, anti-miR-21 treatment caused significant de-repression of STAT3, PTEN, and BMPR2 at 14 days (Figure 7B). However, PDCD-4 and TIMP3 expression were not significantly altered (Figure 7B). Neointima formation was significantly reduced in anti-miR-21-treated vessels (Figure 7C and D).Figure 7


miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation.

McDonald RA, White KM, Wu J, Cooley BC, Robertson KE, Halliday CA, McClure JD, Francis S, Lu R, Kennedy S, George SJ, Wan S, van Rooij E, Baker AH - Eur. Heart J. (2013)

Effect of pharmacological knockdown of miR-21 in HSVs and miR-21 expression in failed human vein grafts. HSV segments were cultured for 7 and 14 days in the presence of 5000 nM anti-miR-ctl or anti-miR-21 (n = 5/group), then RNA was isolated from the vessels, and expression was measured by quantitative real-time PCR. (A) miR-21 expression. (B) Expression of putative miR-21 target genes. (C) Neointimal thickness. (D) Representative images of elastic van Gieson-stained sections from Day 14 samples. (E) In situ hybridization with a scrambled microRNA probe, miR-21 probe, and immunohistochemistry for smooth muscle cell actin in a failed human vein graft. Scale bar represents 100 μm, applicable to all panels. *P < 0.05 vs. anti-miR-ctl; **P < 0.01 vs. anti-miR-ctl, ***P < 0.001 vs. anti-miR-ctl, ##P < 0.01 vs. Day 0, ###P < 0.001 vs. Day 0.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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EHT105F7: Effect of pharmacological knockdown of miR-21 in HSVs and miR-21 expression in failed human vein grafts. HSV segments were cultured for 7 and 14 days in the presence of 5000 nM anti-miR-ctl or anti-miR-21 (n = 5/group), then RNA was isolated from the vessels, and expression was measured by quantitative real-time PCR. (A) miR-21 expression. (B) Expression of putative miR-21 target genes. (C) Neointimal thickness. (D) Representative images of elastic van Gieson-stained sections from Day 14 samples. (E) In situ hybridization with a scrambled microRNA probe, miR-21 probe, and immunohistochemistry for smooth muscle cell actin in a failed human vein graft. Scale bar represents 100 μm, applicable to all panels. *P < 0.05 vs. anti-miR-ctl; **P < 0.01 vs. anti-miR-ctl, ***P < 0.001 vs. anti-miR-ctl, ##P < 0.01 vs. Day 0, ###P < 0.001 vs. Day 0.
Mentions: To investigate the translational application of our findings and evaluate whether it is possible to achieve pharmacological knockdown of miR-21 in HSVs, the ex vivo culture model was utilized. Culturing HSV in the presence of anti-miR-21 for 7 or 14 days resulted in a >95% knockdown in miR-21 expression (Figure 7A). The expression of previously identified miR-21 target genes25,29–31 was analysed by qRT-PCR. Compared with anti-miR-ctl-treated HSVs, anti-miR-21 treatment caused significant de-repression of STAT3, PTEN, and BMPR2 at 14 days (Figure 7B). However, PDCD-4 and TIMP3 expression were not significantly altered (Figure 7B). Neointima formation was significantly reduced in anti-miR-21-treated vessels (Figure 7C and D).Figure 7

Bottom Line: Identifying novel strategies to prevent neointimal thickening is important.Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8TA, UK.

ABSTRACT

Aims: The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.

Methods and results: We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation.

Conclusion: This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

Show MeSH