Kaplan-Meier plot of cardiac failure-free fraction and cardiac function of WT and α-MHC CaMKKβ^kd TG mice after TAC.

<p>A. α-MHC CaMKKβ^kd TG mice had a lower cardiac failure-free fraction compared with WT mice. Log-rank test indicates the significant difference between WT and α-MHC CaMKKβ^kd TG mice after TAC (p = 0.0002) (WT; n = 26, TG; n = 25). B. Representative echocardiograph pictures of WT and α-MHC CaMKKβ^kd TG hearts 3 weeks after TAC. Arrows indicate left ventricular cavities of diastolic and systolic phase. C. Serial changes in echocardiographic parameters of WT and α-MHC CaMKKβ^kd TG mice after TAC. *p<0.05 vs WT (n = 3–5 for each group).</p

Left ventricular dilatation, hypertrophy, and fibrosis is prominent in α-MHC CaMKKβ^kd TG mice compared with WT mice after TAC.

<p>A. Low-power magnifications of Masson Trichrome stained images of the left ventricle of WT and α-MHC CaMKKβ^kd TG mice with or without TAC. B. Heart weight (HW) and lung weight (LW) of WT and α-MHC CaMKKβ^kd TG mice with or without TAC. Each value was normalized to tibial length (TL). Values are the means ± S.E. of 5 mice (*p<0.05 vs sham control, †p<0.05 vs WT after TAC). C. Relative expression levels of hypertrophy-associated genes in WT and α-MHC CaMKKβ^kd TG mice with or without TAC. Values are the means ± S.E. of four mice (*p<0.05 vs sham control, †p<0.05 vs WT after TAC). D. Picrosirius staining of the left ventricle in WT and α-MHC CaMKKβ^kd TG mice with or without TAC and measurement the area of fibrosis. Values are the means ± S.E. of three to five mice (*p<0.05 vs sham control, †p<0.05 vs WT after TAC).</p

Phosphorylation of downstream targets of CaMKKβ in WT and α-MHC CaMKKβ^kd TG mice with or without TAC.

<p>Representative pictures of immunoblotting for phosphorylated form and total amount of adenosine monophosphate (AMP)-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinase (CaMK) I, and CaMKIV (A–C). Results of densitometric analysis are indicated. Values are the means ± S.E. of five mice (*p<0.05 vs sham control, †p<0.05 vs WT after TAC).</p

Myocardial energy reserve measured by in situ ³¹P magnetic resonance (MR) spectroscopy.

<p>A. A ¹H MR image to define the region of interest to measure a ³¹P MR spectrum of the left ventricular anterior wall. B. In vivo cardiac ³¹P MR spectra from WT and α-MHC CaMKKβ^kd TG mice 3 weeks after TAC. C. The creatine phosphate/ATP ratio of the left ventricle in WT and α-MHC CaMKKβ^kd TG after TAC. Values are mean±standard error of the mean. *p<0.05 vs WT at same time point, n = 3–8 for each group.</p

Generation of α-MHC CaMKKβ^kd TG mice.

<p>A. Immunoblotting and densitometry for calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) after transverse aortic binding (TAC) in wild-type (WT) mice. Values are the means ± standard error (S.E.) of four independent experiments (*p<0.05 vs 0 week). B. Immunoblotting for CaMKKβ in WT mice and two lines of α-MHC CaMKKβ^kd TG mice. C. Immunoblotting for flag tag in two lines of α-MHC CaMKKβ^kd TG mice indicating the heart-specific overexpression of the transgene. D. Activities of CaMKKβ in the hearts of two lines of α-MHC CaMKKβ^kd TG mice by immunoprecipitate kinase assays.</p

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