A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation - PubMed (original) (raw)

Jun Tang 2, David P Cormode 3, Aneta J Mieszawska 4, David Izquierdo-Garcia 4, Canturk Ozcan 4, Maarten J Otten 4, Neeha Zaidi 4, Mark E Lobatto 5, Sarian M van Rijs 4, Bram Priem 4, Emma L Kuan 6, Catherine Martel 7, Bernd Hewing 8, Hendrik Sager 9, Matthias Nahrendorf 9, Gwendalyn J Randolph 7, Erik S G Stroes 10, Valentin Fuster 11, Edward A Fisher 12, Zahi A Fayad 4, Willem J M Mulder 5

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A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation

Raphaël Duivenvoorden et al. Nat Commun. 2014.

Erratum in

Abstract

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

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Conflict of interest statement

Conflict of interest. There is disclosure for conflict of interest

Figures

Figure 1

Figure 1. Schematic of the study design

(a) The targeting dynamics, targeting mechanism and anti-inflammatory action of [S]-rHDL in apoE-KO mice were investigated by analyzing the dynamics of phospholipids and hydrophobic cargos of [S]-rHDL in the blood by NIRF and flow cytometry. The biodistribution was evaluated in organs with NIRF. (b) Magnetic resonance imaging (MRI), near infrared fluorescence imaging (NIRF), fluorescence microscopy, and flow cytometry were used to validate the plaque macrophage targeting efficiency of [S]-rHDL. The effect of [S]-rHDL on the mRNA levels of inflammatory genes of plaque macrophages were determined in macrophages isolated with laser capture microdissection. Fluorescence molecular tomography and computed tomography was used to assess the effect of [S]-rHDL on inflammatory protease activity in aortic root plaques. (c) The efficacy of low-dose long-term (12 weeks) [S]-rHDL treatment on disease progression was evaluated in the abdominal aortas with MRI and in aortic roots with histology. (d) The efficacy of high-dose short-term (1 week) [S]-rHDL treatment was evaluated in aortic roots with histology.

Figure 2

Figure 2. Schematic representations of the nanoparticle formulations and in vitro efficacy data

(a) Schematic representation of dual gadolinium and fluorescent dye (Cy5.5, DiO, DiR) labeled statin containing reconstituted high density lipoprotein ([Gd-dye-S]-rHDL), statin containing rHDL ([S]-rHDL), and rHDL. Negative staining transmission electron microscopy (TEM) images of each of the aforementioned particles showed the typical disk-like morphology. The circular shapes are nanoparticles viewed en face, while the striped configurations are rouleaux of nanoparticles viewed from the side. Dynamic light scattering measurements showed the average size of [Gd-dye-S]-rHDL to be 28.5 nm, of [S]-rHDL to be 26.0 nm and of rHDL to be 10.5 nm. For larger view TEM also see Supplementary Fig. S1. (b) In vitro cell viability assays of murine macrophages (J774A.1), incubated with combinations of [S]-rHDL (10 μM statin) free simvastatin (10 μM), rHDL plus free statin (10 μM), free statin (10 μM) plus mevalonate (100 μM), [S]-rHDL (10 μM) plus mevalonate (100 μM), and only mevalonate (100 μM). There was also a control group of cells not incubated with anything. Macrophage cell viability is markedly decreased in the [S]-rHDL and free statin group. This effect is abolished by addition of mevalonate, indicating that the effect of HMGR inhibition on cell viability is mediated through the mevalonate pathway. N=6 for all bars. (c) Production of the inflammatory cytokines MCP-1 and TNF-α. LPS and INF-γ challenged macrophages were incubated with the same treatments as mentioned above for 24 hours. MCP-1 and TNF-α levels are markedly reduced by [S]-rHDL and free statin. MCP-1 and TNF-α levels are restored by the addition of mevalonate to [S]-rHDL and free statin incubation. N=6 for all bars. Cell viability in the different groups was not affected under these conditions (Supplementary Fig. S4). All error bars are 95% confidence intervals. P-values are calculated with Mann-Whitney U tests for comparisons with [S]-rHDL, * indicates P < 0.05, ** indicates P < 0.01. Kruskal-Wallis P-values are < 0.0001 for all plots.

Figure 3

Figure 3. Pharmacokinetics and accumulation in plaque of labeled nanoparticles

(a) Cy5.5- and DiO-labelled [S]-rHDL was intravenously injected to apoE-KO mice (N=21, 3 mice per time point) and blood and tissues were analyzed at different time points post-injection. NIRF shows that components from the lipid monolayer (Cy5.5) have much shorter blood half-life than components from hydrophobic core (DiO). It also shows that the majority of [S]-rHDL stays in serum and very little in red blood cells (RBC) and mononuclear cells (MNC). (b) Fluorescence intensity in serum, MNC, and RBC is quantified (N=21, 3 mice per time point). We calculated that plasma half-life of [S]-rHDL to be 21.9 hours for the DiO signal. (c) Flow cytometric analysis of blood cells shows that [S]-rHDL targets Gr-1hi pro-inflammatory monocytes more efficiently than Gr-1lo anti-inflammatory monocytes in blood (N=21, 3 mice per time point). (d) Typical T1-weighted 9.4 Tesla magnetic resonance images of the abdominal aorta of an apoE-KO mouse, made at identical locations, before and 24 hours after injection of [Gd-Dye-S]-rHDL. The lumen is indicated by *. The scale bar in the upper images represent 10 mm, and in the lower images 1 mm. The 24h post-injection image showed signal enhancement in the vessel wall (white arrows), indicative of nanoparticle infiltration and retention in the aortic plaques. (e) [S]-rHDL labeled with Cy5.5 (lipid monolayer) and DiR (hydrophobic core) was intravenously injected to apoE-KO mice. NIRF shows that Cy5.5 and DiR preferentially accumulates in the areas rich with atherosclerotic lesions. The scale bar represent 10 mm. (f) Cy5.5 and DiO both appear in the plaque, until 4 hours post-injection the presence of Cy5.5 declines while DiO remains present. The scale bar represents 500 μm. (g) DiO-labelled [S]-rHDL co-localizes in the plaque with CD68 (macrophages). The scale bar in the inset represent 100μm, and in the overview 400 μm (h) Flow cytometric analysis of cells in aorta walls shows that [S]-rHDL is taken up by plaque macrophages, furthermore, macrophages are targeted more efficiently than monocytes (N=3 per timepoint). All error bars are standard errors of the mean.

Figure 4

Figure 4. In vivo efficacy of 12 weeks biweekly low dose [S]-rHDL infusions

(a) Efficacy of [S]-rHDL on abdominal atherosclerosis quantified by 9.4 Tesla magnetic resonance imaging (9.4T-MRI). Typical T1-weighted MR images of the abdominal aortas of mice in each group at all time points show the thickening of the aorta wall in all groups except in the [S]-rHDL-treated group. The analysis method of the images is shown in Supplementary Fig. S9. The scale bar represents 1 mm. (b) MRI scans of the abdominal aortas of 32 mice (N=8 per group) were performed at three time points during the study. When the apoE-KO mice were 14 weeks on high cholesterol diet the baseline scans were acquired and subsequent scans were performed 6 and 12 weeks after the baseline scan. From baseline onwards the mice received placebo, oral statin therapy, or injections of reconstituted high-density lipoprotein (rHDL) or statin containing rHDL ([S]-rHDL). Thickness of the vessel wall is expressed as the normalized wall index (NWI), which is defined as the ratio between the mean wall area and the outer wall area. (c) Efficacy of [S]-rHDL assessed by histology shows that mean plaque area was lower in the [S]-rHDL treated group (N=15) as compared to placebo (N=16) and rHDL (N=16), and there was a trend towards decreased plaque area compared to statin therapy (N=15). Kruskal-Wallis P-value for plaque area is 0.0011. (d) Plaque macrophage content as measured by the CD68 positive area was decreased in the [S]-rHDL group (N=15) as compared to placebo- (N=16), statin- (N=15) and rHDL-therapy (N=16), indicating decreased plaque inflammation in the [S]-rHDL group. Bars represent the standard error of the mean, P-values were calculated with Mann-Whitney U tests. Kruskal-Wallis P-value for CD68 area is 0.0001. (e) Typical histology images of the aortic sinus area of each group are shown. The hematoxylin phloxine saffron (HPS)-stained images are shown on the left and the cross-sections stained with CD68 antibodies are shown on the right. The analysis method of the histology images is shown in Supplementary Fig. S9. The scale bar represents 400 μm.

Figure 5

Figure 5. In vivo efficacy of a single week high dose [S]-rHDL infusions

(a) Mean plaque area was lower in the high dose [S]-rHDL-treated group (N=10) as compared to placebo (N=15), high dose rHDL (N=8), and low dose [S]-rHDL(N=10). Kruskal-Wallis P-value for plaque area is 0.037. (b) Plaque macrophage content as measured by the CD68 positive area was decreased in the high dose [S]-rHDL (N=10) as compared to placebo (N=15), high dose rHDL (N=8), and low dose [S]-rHDL (N=10), indicative of decreased plaque inflammation in this group. Bars represent the standard error of the mean, P-values were calculated with Mann-Whitney U tests. Kruskal-Wallis P-value for CD68 area is 0.0006. (c) Typical histology images of the aortic sinus area from a mouse in the placebo group and a mouse in the high dose [S]-rHDL-group show that mean plaque area is similar, while the plaque macrophage content is notably smaller in the [S]-rHDL-group. The hematoxylin phloxine saffron (HPS)-stained images are shown on the left and the cross-sections stained with CD68 antibodies are shown on the right. The scale bar represents 400 μm. (d) One-week high dose [S]-rHDL treatment (N=6) significantly reduced the mRNA expression levels of monocyte recruitment genes (composite variable of MCP-1, CCL-3, ICAM-1, VCAM-1, CCL-15, CXCL-12) and pro-inflammatory genes (composite variable of TNF-α, IL-1β, IL-1α, SPP-1) of plaque macrophages in the aortic root when compared to placebo (N=6) and oral statin (N=6). The expression of the anti-inflammatory mRNA level (MR) was increased in the high dose [S]-rHDL group as compared to placebo, but not compared to oral statin therapy. P-values are calculated with Mann-Whitney U tests for comparisons with [S]-rHDL, * indicates P < 0.05, ** indicates P < 0.01. Kruskal-Wallis P-values are shown in the plot. The error bars represent the standard deviations. (e) FMT-CT molecular imaging of protease activity revealed that high dose [S]-rHDL treatment (N=6) significantly reduced the inflammation levels in the aortic roots of live apoE-KO mice with advanced atherosclerosis as compared to placebo (N=5). The yellow circles indicate the aortic root area. The error bars represent the standard error of the mean. P-values were calculated with the Mann-Whitney U test.

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