Plaque-targeted, proteolysis-resistant, activatable and MRI-visible nano-GLP-1 receptor agonist targets smooth muscle cell differentiation in atherosclerosis

Abstract

Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists are powerful glycemia-lowering agents, which have systematically been shown to lower cardiovascular events and mortality. These beneficial effects were difficult to pinpoint within atherosclerotic plaque due to lack of particular specificity of such agonists to the vascular cells and an inadequate understanding of the GLP-1R expression in atherosclerosis. Here, we hypothesized that the direct engagement of the GLP-1R in atherosclerosis by targeted agonists will alleviate vascular inflammation and plaque burden, even at a very low dose. Methods: The expression of GLP-1 receptor (GLP-1R, Glp1r mRNA) in human lesions with pathologic intimal thickening, Apoe^-/- mouse atheroma and cultured immune/non-immune cells was investigated using genetic lineage tracing, Southern blotting and validated antisera against human GLP-1R. Protease-resistant and "activatable" nanoparticles (NPs) carrying GLP-1R agonist liraglutide (GlpNP) were engineered and synthesized. Inclusion of gadolinium chelates into GlpNP allowed for imaging by MRI. Atherosclerotic Apoe^-/- mice were treated intravenously with a single dose (30 µg/kg of liraglutide) or chronically (1 µg/kg, 6 weeks, 2x/week) with GlpNP, liraglutide or control NPs, followed by assessment of metabolic parameters, atheroma burden, inflammation and vascular function. Results: Humal plaque specimens expressed high levels of GLP-1R within the locus of de-differentiated smooth muscle cells that also expressed myeloid marker CD68. However, innate immune cells under a variety of conditions expressed very low levels of Glp1r, as seen in lineage tracing and Southern blotting experiments examining full-length open reading frame mRNA transcripts. Importantly, de-differentiated vascular smooth muscle cells demonstrated significant Glp1r expression levels, suggesting that these could represent the cells with predominant Glp1r-positivity in atherosclerosis. GlpNP resisted proteolysis and demonstrated biological activity including in vivo glycemia lowering at 30 µg/kg and in vitro cholesterol efflux. Activatable properties of GlpNP were confirmed in vitro by imaging cytometry and in vivo using whole organ imaging. GlpNP targeted CD11b⁺/CD11c⁺ cells in circulation and smooth muscle cells in aortic plaque in Apoe^-/- mice when assessed by MRI and fluorescence imaging. At a very low dose of 1 µg/kg, previously known to have little effect on glycemia and weight loss, GlpNP delivered i.v. for six weeks reduced triglyceride-rich lipoproteins in plasma, plaque burden and plaque cholesterol without significant effects on weight, glycemia and plasma cholesterol levels. Conclusions: GlpNP improves atherosclerosis at weight-neutral doses as low as 1 µg/kg with the effects independent from the pancreas or the central nervous system. Our study underlines the importance of direct actions of GLP-1 analogs on atherosclerosis, involving cholesterol efflux and inflammation. Our findings are the first to suggest the therapeutic modulation of vascular targets by GlpNP, especially in the context of smooth muscle cell inflammation.

Keywords: GLP-1; MRI; atherosclerosis; nanomedicine; smooth muscle cells.

Figure 1

GLP-1R is expressed in human and mouse atherosclerosis. A-H) Human coronary artery pathology obtained from a 35 years old white male autopsy case with severe coronary mutivessel atherosclerotic disease and a history of prior myocardial infarction. A) Low power H&E staining section of atherosclerotic coronary artery (early fibroatheroma) in proximal LAD. B) Middle power H&E image of black rectangular field in A. C) IF staining image of αSMA (green) and GLP-1R (red) with DAPI nuclear staining (blue) which is adjacent to section A. (D-E) Middle power (D) and high power (E) image of white rectangular field in C and D. GLP1R (red) positive but αSMA (green) negative cells (likely macrophage) were observed in the field. F) High power image of adventitial lesion at white rectangular field in C. Multiple GLP1-R positive cells were observed. (G-H) IHC image of GLP1R positive cell (brown) in the fields of intra-plaque (G) and adventitia (H) which is obtained from adjacent fields of E and F. I) Atherosclerosis-resident macrophage-like (CD11b) and pan-negative fraction but not dendritic cell-like (CD11c) cells expressed Glp1r as determined via magnetic-bead sorting followed by real-time quantitative PCR. J) Full-length Glp1r mRNA transcripts in whole aortas from Apoe^-/- mice (fed as indicated) as analyzed by Southern blotting. Hprt served as a housekeeping control. Neg.ctrl - Glp1r^-/- lung, Pos.ctrl - Glp1r^+/+ lung. K) Ward's hierarchical cluster analysis allowed for clustering of tissues expressing high, low and medium levels of Glp1r. L) mRNA from B was analyzed by RNA sequencing that demonstrated differentially regulated top canonical pathways comparing tissues expressing high vs. low levels of Glp1r. M) Results of the correlation analysis between Glp1r expression found in (J-L) and the levels of various myeloid and smooth muscle-related gene mRNA transcripts. Confidence belt is 85% and is shown in grey. Abbreviations: H&E - hematoxylin and eosin; LAD - left anterior descending artery; NC - necrotic core; DAPI - 4′,6-diamidino-2-phenylindole; N.d. - not detected.

Figure 2

Glp1r is highly expressed in mouse aortic smooth muscle cells (MASMCs). A) Southern blotting analysis of Glp1r expression in MASMCs from Apoe^-/- mice treated as indicated for 48 h. Hprt served as a housekeeping control. Neg.ctrl - Glp1r^-/- lung, Pos.ctrl - Glp1r^+/+ lung. B) Markers characteristic of VSMCs (blue) or macrophages (red) as examined by qPCR. Fold change (FC) vs. DMSO control is shown as a bubble chart, with the bubbles sized by their p value, as analyzed by ANOVA with the Tukey post hoc test. The complete list of markers is presented in the Supplementary Figure 6A and the detailed statistical analysis is presented in Table S4. C) Glp1r expression in BMDMs treated as indicated and analyzed by Southern blotting as in (A). D) Glp1r expression in the murine endothelial cell (EC) line 3B11 and in the primary ECs isolated from carotid artery, treated as indicated (and in the Supplementary material online, methods) and analyzed by Southern blotting as in (A, B). E) Immunofluorescence staining of VSMC and macrophage markers (red and green respectively) in MASMCs subjected to the treatments as indicated. Nuclei were counterstained with Hoescht (blue).

Figure 3

GlpNP carrying the GLP-1R agonist liraglutide are bioactive and resistant to proteolysis. A) GlpNP were synthesized from a fluorescent core, Lira, gadolinium-DTPA and PEG lipids. Inset shows DLS and cryo-TEM characterization of GlpNP. B) GlpNP are resistant to proteolysis by neutral endopeptidase (NEP) in cell-free assays as determined by HPLC. Average of two independent experiments is shown. C) GlpNP but not CtrlNP improved oral glucose tolerance (OGTT) after a single I.V. injection 2 h before the glucose challenge and followed by a glucose load. D) GlpNP promoted insulin release in the same set of experiments. E) Area under the curve (AUC) quantification derived from OGTT and insulin release in C and D, respectively. F) Plasma from fasting Apoe^-/- mice injected with NPs was subjected to fast protein liquid chromatography (FPLC) analysis at baseline and 24 hrs after injection, and triglyceride levels were measured in the eluted fractions. G) Area under the curve analysis of FPLC chromatograms quantified the levels of cholesterol and triglycerides. H) ApoB-48 concentrations in plasma measured over time after NP administration. I) qPCR analysis of the lipid metabolism-related genes in the liver of animals treated with NPs for 24 h. *p<0.05, †p<0.01 via two-way ANOVA, Tukey's post-hoc correction, n=5-7 animals (C, D) and pairwise T-test with Holm post-hoc correction (F, G, H). n=3-4 animals.

Figure 4

GlpNP target myeloid and SMCs in atherosclerosis. A, B) GlpNP preferentially target myeloid and smooth muscle cells in atherosclerosis as shown by flow cytometry. Apoe-/- mice were bolus-injected with saline or Cy-labeled GlpNP and the aortas were extracted 24 h later. Single-cell suspensions from enzymatically-digested whole aortas were stained with antibodies against CD45, F4/80 and αSMA. C) Myeloid (CD45⁺) and non-myeloid (CD45^-) cells in atherosclerotic plaque demonstrated GlpNP positivity, with the highest expression seen in αSMA-positive cells, as shown in the pie diagram demonstrating total GlpNP positivity among indicated cell types. D) MRI of atherosclerotic plaque after administration of gadolinium-loaded GlpNP or PtdSer^negCtrlNP as indicated. E) Signal-to-noise measurements from (D) represented as signal intensities (SI) in aorta vs. SI in muscle. Significance was determined by repeated measures ANOVA. F) Whole-organ fluorescence imaging 24 h after GlpNP administration. Red-yellow signal is indicative of GlpNP accumulation in the given organ as annotated. G, H) Localization of GlpNP in aortic root tissue sections as seen by fluorescence microscopy. GlpNP fluorescence (pseudo-colored green, arrows) was observed in close proximity with the cells expressing macrophage (LGALS3, cyan) and smooth muscle (αSMA, red) markers. Nuclei (blue) were counterstained with DAPI. L - indicates plaque lumen.

Figure 5

GlpNP reduced plaque burden without changes in body weight. A) Apoe^-/- mice were fed Western diet (WD) for 24 weeks to induce atherosclerosis. Animals were then fasted as indicated and baseline metrics were taken as described in Methods and the text. Mice were continued on WD and received GlpNP, CtrlNP or Lira (1 μg/kg based on Lira or 100 µg of particles in CtrlNP) I.V. for 6 weeks twice weekly. B) Body weight measurements over time before and after randomization. C) Morphometric assessment of plaque burden with representative sections of aortic root after indicated staining. Sections were stained with Hematoxylin Eosin (H&E, general tissue morphology), Collagen I, F4/80 (macrophage-like cells), alpha smooth muscle actin (ɑSMA). D) Quantification of IHC staining indicated 15% reduction in aortic sinus lesion area and 13% decrease in macrophage content, while Collagen I expression increased by 18% (GlpNP vs. Lira) via pairwise T-test with Holm post-hoc correction. n= 7-9/group. E) Concentration-response curves recorded in the presence of acetylcholine (Ach) of aortic rings from animals injected with NPs. Pre-contraction was induced with 10^-5 M phenylephrine (Phe). F) Aortic sections from mice injected with NPs were subjected to laser capture microdissection, after which targeted cells were identified by characteristic NP fluorescence (arrows). G) Captured cells were analyzed according to mRNA expression of Icam, Tnf and Arg1. *p<0.05, †p<0.01 vs CtrlNP via pairwise T-test with Holm correction in G and ANOVA with Tukey post-hoc test in E. n= 3-4/group.