Cardiac function-related AAs revealed unique correlations with concomitant systemic factors in the HF group.

<p>A. Concentrations of hepatic-related AAs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117325#pone.0117325.g002" target="_blank">Fig. 2A</a> were significantly associated with serum NO concentration and the NO:hydroperoxide ratio (p < 0.05). B. Concentration of histidine, hepatic- and skeletal muscle-related AA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117325#pone.0117325.g002" target="_blank">Fig. 2A</a>, was associated with serum albumin concentration and GNRI (p < 0.01). C. Concentration of 1-Me-His, skeletal muscle- and renal-related AA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117325#pone.0117325.g002" target="_blank">Fig. 2A</a>, was negatively associated with flow-mediated dilatation (p < 0.05). %FMD, flow-mediated dilatation.</p

Specific AAs and Fischer ratio were significantly correlated with cardiac function in the HF group.

<p>Of 41 AAs examined, the amounts of five AAs and Fischer ratio were significantly correlated with cardiac function by univariate and stepwise multivariate analyses (p < 0.05). Their amounts, except that of methionine, significantly changed between the control and HF groups. r, correlation coefficient.</p

Plasma AAs and ratios—amounts significantly changed in the HF group.

<p>Plasma AAs and ratios—amounts significantly changed in the HF group.</p

Baseline clinical characteristics in the Control and HF groups.

<p>Baseline clinical characteristics in the Control and HF groups.</p

Exploratory factor analysis categorized cardiac function-related AAs by two components in the HF group.

<p>A. A two-dimensional plot of factor loading. Cardiac function-related AAs and Fischer ratio in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117325#pone.0117325.g001" target="_blank">Fig. 1</a> were subjected to exploratory factor analysis that identified two potential factors by which AAs and ratio could be categorized: factor 1 (MEA, tyrosine, and Fischer ratio) and factor 2 (histidine, methionine, and 1-Me-His). The percentages in parentheses and values of the axes denote a contribution ratio of the factors and a factor loading, respectively. B and C. The correlations between AAs and Fischer ratio in A. Correlations of MEA with methionine and histidine and that of histidine and 1-Me-His were relatively weak.</p

Summary of cardiac function-related AAs on the cardio-hepatic-skeletal muscle axis in patients with systolic HF.

<p>Plasma AA profiling revealed that five AAs and Fischer ratio specifically correlated with cardiac function in patients with systolic HF. These AAs can be categorized based on the cardio-hepatic-skeletal muscle axis and had specific correlations with serum NO and albumin concentrations, GNRI, and %FMD.</p

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

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 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