Prevention of neointimal formation using miRNA-126-containing nanoparticle-conjugated stents in a rabbit model

<div><p>Background</p><p>Despite recent progress with drug-eluting stents, restenosis and thrombosis after endovascular intervention are still major limitations in the treatment of cardiovascular diseases. These problems are possibly caused by inappropriate inhibition of neointimal formation and retardation of re-endothelialization on the surface of the stents. miR-126 has been shown to have the potential to enhance vascular endothelial cell proliferation.</p><p>Methods and results</p><p>We designed and constructed a 27-nt double strand RNA (dsRNA) conjugated to cholesterol, which has high membrane permeability, and formed mature miR-126 after transfection. For site-specific induction of miR-126, we utilized poly (DL-lactide-co-glycolide) nanoparticles (NPs). miR-126-dsRNA-containing NPs (miR-126 NPs) significantly reduced the protein expression of a previously identified miR-126 target, SPRED1, in human umbilical vascular endothelial cells (HUVECs), and miR-126 NPs enhanced the proliferation and migration of HUVECs. On the other hand, miR-126 NPs reduced the proliferation and migration of vascular smooth muscle cells, via the suppression of IRS-1. Finally, we developed a stent system that eluted miR-126. This delivery system exhibited significant inhibition of neointimal formation in a rabbit model of restenosis.</p><p>Conclusions</p><p>miR-126 NP-conjugated stents significantly inhibited the development of neointimal hyperplasia in rabbits. The present study may indicate the possibility of a novel therapeutic option to prevent restenosis after angioplasty.</p></div

Effect of miR-126 NPs on HUVECs.

<p>(A) mRNA expression changes of potential target genes of miR-126 in HUVECs determined by real-time PCR analyses. Values are means ± SEM; n = 5 each; *P<0.05, **P<0.01. ***P<0.001. (B) Protein levels of SPRED1 after addition of control RNA NPs and miR-126 NPs. Values are means ± SEM; n = 6 each; *P<0.05. (C) Proliferation of HUVECs determined by MTT assay. Values are means ± SEM; n = 8 each; *P<0.05, **P<0.01. ****P<0.0001. (D) Photograph of scratch assay and serial changes in the migration area determined using Image J. Values are means ± SEM; n = 8 each; *P<0.05, ***P<0.001. ****P<0.0001.</p

Effect of miR-126 NPs on VSMCs.

<p>(A) Proliferation of VSMCs determined by cell count. Values are means ± SEM; n = 6 each; ***P<0.001. (B) Photograph of scratch assay and the serial changes in migration area determined using Image J. Values are means ± SEM; n = 4 each; *P<0.05. (C and D) Proliferation of VSMCs determined using an MTT assay. Values are means ± SEM; n = 6 each; *P<0.05, ***P<0.001. (E and F) Serial changes in migration area determined using Image J. Values are means ± SEM; n = 4 each; *P<0.05, **P<0.01, ***P<0.001.</p

miR-126 NPs target IRS-1 in VSMCs.

<p>(A) miR-126 expression levels after the addition of miR-126 NPs and control NPs to VSMCs. Values are means ± SEM; n = 4 each; **P<0.001. (B) mRNA expression changes of potential target genes of miR-126 in VSMCs determined by real-time PCR analyses. Values are means ± SEM; n = 4 each; **P<0.01. (C) Protein levels of IRS-1 after addition of control RNA NPs and miR-126 NPs. Values are means ± SEM; n = 6 each; **P<0.001. (D) Conservation of the miR-126 target site in the 3’-UTR of IRS-1. (E) 3’-UTR reporter assay used to verify the target. Luciferase reporter activity of rabbit IRS-1 gene 3’-UTR constructs with or without mutation of the miR-126 binding site in 293T cells overexpressing miR-control and miR-126; n = 4 each; *p < 0.05 and ***p < 0.001.</p

Structure of miR-126 dsRNA and miR-126 expression from miR-126 NPs <i>in vitro</i>.

<p>(A) Structure of miR-126 dsRNA and control dsRNA. (B) miR-126 expression after the induction of miR-126 dsRNA and control dsRNA using Lipofectamine 2000 in 293T cells. Values are means ± SEM; n = 5 each; *P<0.05, **P<0.01. (C) Luciferase activity of a reporter gene with miR-126 binding sites. Values are means ± SEM; n = 4 each; *P<0.05, **P<0.01. (D) FITC levels in HUVECs after the addition of FITC-NPs. Fluorescence intensity (left panel) and phase contrast image (right panel). (E) Electron micrograph of miR-126 NPs and control NPs. (F) miR-126 expression levels after the addition of miR-126 NPs and control NPs in HUVECs. Values are means ± SEM; n = 4 each; *P<0.05, **P<0.01. ***P<0.001.</p

Effect of miR-126 NP-conjugated stents on a rabbit model of neointimal formation.

<p>(A) Photograph of an miR-126 NP-conjugated stent. (B) Cumulative release of miR-126 from an miR-126 NP-conjugated stent. (C) Protocol of the <i>in vivo</i> rabbit model of neointimal formation. (D) Representative OCT images of a rabbit iliac artery after the implantation of control RNA NP-conjugated stents or miR-126 NP-conjugated stents. (E) Representative images of HE staining of a rabbit iliac artery after the implantation of a control RNA NP-conjugated or miR-126 NP-conjugated stent. (F) Changes in neointimal area of rabbit iliac arteries after the implantation of a control RNA NP-conjugated or miR-126 NP-conjugated stent; n = 8 each; *P<0.05. (G) Changes in mean neointimal thickness of rabbit iliac arteries after the implantation of a control RNA NP-conjugated or miR-126 NPs-conjugated stent; n = 8 each; *P<0.05.</p