Perivascular delivery of Notch 1 siRNA inhibits injury-induced arterial remodeling - PubMed (original) (raw)

Perivascular delivery of Notch 1 siRNA inhibits injury-induced arterial remodeling

Eileen M Redmond et al. PLoS One. 2014.

Abstract

Objectives: To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling.

Methods and results: Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown.

Conclusion: These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Perivascular delivery of Notch 1 siRNA in Injured Vessel inhibits Notch Signaling Component Expression.

(A) Representative Western Blot and (B) cumulative protein data for Notch 1 IC and Notch target gene (Hrt-1 and Hrt-2) expression 14 days after carotid ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. (C) qRT-PCR analysis of Notch 1, Hrt-1 and Hrt-2 mRNA levels 14 days after carotid ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. Data are normalized to GAPDH and represent the mean ± SEM, n = 6.

Figure 2

Figure 2. Perivascular delivery of Notch 1 siRNA inhibits Medial SMC Notch Signaling Component Expression.

(A) Representative image of medial SMC layer dissected by Laser Capture Microdissection (LCM) for mRNA analysis. (B) qRT-PCR analysis of medial SMC Notch 1, Hrt-1 and Hrt-2 mRNA levels 14 days after carotid ligation in sham, ligated (scrambled siRNA) and Ligated+Notch 1 siRNA vessels. Data are normalized to GAPDH and represent the mean ± SEM, n = 6.

Figure 3

Figure 3. Localized Notch 1 inhibition attenuates injury-induced increases in Vascular Cell Growth.

(A) Representative Western Blot and cumulative data of proliferating cell nuclear antigen (PCNA) protein expression (B) qRT-PCR analysis of pro-apoptotic Bax and anti-apoptotic BcL-XL (C) Representative Western Blot and cumulative data of pro-apoptotic Bax and anti-apoptotic BcL-XL and (D) Bax/Bcl-XL ratios 14 days after carotid ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. Data are normalized to GAPDH and represent the mean ± SEM, n = 6.

Figure 4

Figure 4. Perivascular delivery of Notch 1 siRNA prevents injury-induced Vascular Remodeling.

(A) Representative Verhoeff-van Gieson staining of carotid artery from C57BI6/J mice 14 days after ligation in the absence or presence of scrambled siRNA (ligated) or Ligated+Notch 1 siRNA. White bars mark media area from internal elastic lamina to the external elastic lamina. (B) Carotid artery vessel media, neointima and lumen volumes (evaluated over a 1 mm carotid length) for sham, ligated and Ligated+Notch 1 siRNA groups. (C) Carotid artery Intima/Media ratios for ligated and Ligated+Notch 1 siRNA groups. Data are mean ± SEM, 4 sections analyzed/animal, n = 6 animals for each group.

Figure 5

Figure 5. Perivascular delivery of Notch 1 siRNA prevents injury-induced Vascular Remodeling.

(A) Photomicrographs of confocal immunofluorescence staining for Notch 1, SMC α-actin or dual staining for α-actin/Notch 1 in carotid arteries 14 d after ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. (B) Photomicrographs of confocal immunofluorescence (60X) staining for Bax, Bcl-XL and Caspase-3 expression in carotid arteries 14 d after ligation in sham, ligated control (scrambled siRNA) and ligated Notch 1 siRNA vessels. Data are mean ± SEM, 4 sections analyzed/animal, n = 6 animals for each group.

References

    1. Acampora KB, Nagatomi J, Langan EM 3rd, LaBerge M (2010) Increased synthetic phenotype behavior of smooth muscle cells in response to in vitro balloon angioplasty injury model. Annals of vascular surgery 24: 116–126 10.1016/j.avsg.2009.07.019 - DOI - PubMed
    1. Anderson LM, Gibbons GH (2007) Notch: a mastermind of vascular morphogenesis. The Journal of clinical investigation 117: 299–302 10.1172/JCI31288 - DOI - PMC - PubMed
    1. Andres V (1998) Control of vascular smooth muscle cell growth and its implication in atherosclerosis and restenosis (review). International journal of molecular medicine 2: 81–89. - PubMed
    1. Brown LA, Rodaway AR, Schilling TF, Jowett T, Ingham PW, et al. (2000) Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos. Mech Dev 90: 237–252 doi:[]S0925477399002567 [pii] - PubMed
    1. Dangas G, Kuepper F (2002) Cardiology patient page. Restenosis: repeat narrowing of a coronary artery: prevention and treatment. Circulation 105: 2586–2587. - PubMed

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