Atherosclerosis is well-known as a chronic inflammatory disease and disorder of lipid metabolism involving the retention of atheroprone lipoproteins and accumulation of monocyte-derived macrophages triggering maladaptive immune response and necrotic core formation.1 The oxygen supply of vascular cells in atherosclerosis relies on the luminal blood or the adventitial vasa vasorum, and the distance to the deep layer of the intima exceeds the oxygen diffusion threshold resulting in local hypoxia2, 3, 4, 5. The uptake of modified lipoproteins by macrophages differentiated from recruited monocytes results in the accumulation of macrophage foam cells in the atherosclerotic lesions. Specifically, hypoxia strongly correlates with the macrophage foam cell clusters surrounding the plaque core6. The higher oxygen demand of activated immune cells and insufficient oxygen supply lead to severe hypoxia and tissue acidification in the macrophage-rich regions of atherosclerotic lesions7, 8. Notably, the acidic extracellular environment in macrophage foam cells impairs the expression of ATP-binding cassette transporter ABCA1, resulting in decreased cholesterol efflux and accelerated lipid accumulation9.
Our group and others initially identified microRNA-33 (miR-33) as crucial regulator of cellular lipid homeostasis and lipoprotein metabolism, controlling downstream target genes including ABCA1 and ABCG110, 11, 12, 13, 14, 15, 16. The benefits of miR-33 deficiency on atherosclerosis development are attributed to its protective effects in macrophages12 Therapeutic inhibition of miR-33 in the mice and non-human primates raises plasma high density lipoprotein (HDL) levels and inhibits the progression of atherosclerosis by increasing HDL levels/functionality or enhancing cholesterol efflux through induction of ABCA1 and ABCG1 in macrophages14, 15, 16, 17. Notably, specific disruption of Abca1 targeting by miR-33 is sufficient to mimic the effects of miR-33 deficiency on cholesterol efflux and atherogenesis18. However, long-term silencing of miR-33 increases circulating triglycerides levels and lipid accumulation in the liver through upregulation of genes involved in fatty acid synthesis when mice are fed a high-fat diet, indicating deleterious effects of moderate hepatic steatosis and hypertriglyceridemia19. Moreover, genetic models of miR-33 deficiency showed a strong predisposition to obesity and metabolic dysfunction12, 13, 19, 20 Thus, specific targeting of miR-33 in the macrophages of atherosclerotic lesions could provide an effective therapeutic strategy for atherosclerosis that avoids the deleterious effects in other metabolic tissues.
pH-Low Insertion Peptides (pHLIP) are a novel class of water-soluble membrane molecules that target areas of high acidity at the surface of cells, which have been employed to deliver miRNA inhibitors to the acidic environment of tumors and the kidney21, 22. Given the hypoxia in macrophage foam cells and the acidic environment of the lipid core in atherosclerosis, we explore the utility of anti-miR-33 peptide nucleic acid (PNA) delivery vectors (anti-miR-33pHLIP) for specific targeting of the macrophages in vascular lesions (Fig. 1a). Notably, near-infrared fluorescence imaging showed highly specific uptake of pHLIP variant 3 conjugated with fluorescent Alexa 750 (A750-Var3) in the aortic arch, an atherosclerotic prone area, of hypercholesterolemic mice. Other tissues characterized by the acidic microenvironment such as the kidney also accumulate the peptide. The targeting of VAR3 to atherosclerotic plaques was specific since a similar peptide with an altered amino acid sequence that prevents insertion across the membranes in acidic conditions (A546-5K-Var3) was unable to target vascular lesions (Fig. 1b). The targeting of A546-Var3 into the aortic arch occurred early (4 hours) and was sustained for 24 hours (Supp Fig. 1a and b). Imaging of whole organs indicated that uptake in the liver was diminished with the A546-Var3 compared to the A546-5K-Var3 mutant (Supp Fig. 1a and b), while similar fluorescent density was observed in histologic sections (Supp Fig. 1c). Both techniques demonstrated higher uptake of A546-Var3 in primary renal tubular cells (Supp Fig. 1c). The affinity of A750-Var3 to atherosclerotic aortic arch was confirmed by analyzing aortas in low-density lipoprotein receptor knockout (Ldlr−/−) and WT mice injected with A750-Var3, A750-5K-Var3 or PBS (Fig. 1c). We further demonstrated the internalization of A750-Var3 in isolated macrophages from atherosclerotic aortas by flow cytometry. The results showed a significant uptake of A750-Var3 in macrophages (Lin−CD11bhighF4/80+Ly-6Clow) and less pronounced internalization in monocytes (Lin−CD11bhighF4/80−Ly-6Chigh) from atherosclerotic plaques (Fig. 1d). These findings correlate with our previous studies showing a specific uptake of pHLIP in tumor associated macrophages.23 Similar to the results observed in vivo, acidification of the media (pH = 6.2) promoted a marked increase of A546-Var3 uptake in mouse macrophages compared to macrophages cultured at a neutral pH (Fig. 2a). Assessment of fluorescently labeled constructs (A633-Var3) confirmed the high uptake by in vivo foam cells isolated from Ldlr−/− mice fed a WD for 3 months using both fluorescent microscopy and flow cytometry (Fig. 2b). Next, we tested whether the delivery of anti-miR-33pHLIP was able to enhance the expression of ABC transporters and reduce foam cell formation in vivo. We found that suppressing miR-33 in macrophages using anti-miR-33pHLIP resulted in significant reduction of neutral lipid accumulation compared to macrophages treated with a non-targeting antisense oligonucleotide conjugated with pHLIP (SrcpHLIP) (Fig. 2c, quantified in right panel). The marked reduction in foamy macrophages correlated with an increase of ABCA1 expression in anti-miR-33pHLIP treated macrophages (Fig. 2d, quantified in right panel). Together, these results demonstrate that pHLIP is an effective and highly specific vehicle to target the expression of miRNAs in macrophages accumulated in atherosclerotic plaques.
We next assessed the efficacy of inhibiting miR-33 expression in lesional macrophages during the regression of atherosclerosis. To this end, Ldlr−/− mice were fed a Western diet (WD) for 3 months to establish atherosclerotic plaques, then switched to a chow diet (CD) and injected with SrcpHLIP and anti-miR-33pHLIP (1 mg/Kg) weekly for one month (Fig. 3a). Consistent with previous studies, switching Ldlr−/− mice to CD resulted in a significant decrease of circulating total cholesterol (TC) and triglycerides (TG) level and increase in plasma HDL-C (Supp Fig. 2a). However, there was no difference between mice treated with anti-miR-33pHLIP and SrcpHLIP, suggesting that the hepatic delivery of anti-miR-33 using pHLIP was not sufficient to impact regulation of HDL biogenesis by miR-33 in the liver (Supp Fig. 2a). Similar lipoprotein profiles (Supp Fig. 2b), body weight (Supp Fig. 2c) and circulating leukocytes (Supp Fig. 3a) were observed in mice treated with anti-miR-33pHLIP and SrcpHLIP. Additionally, no changes in serum hepatotoxicity markers, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were found in mice treated with anti-miR-33pHLIP compared with SrcpHLIP-treated mice (Supp Fig. 3b).
We next sought to determine whether delivery of anti-miR-33 by pHLIP peptides could contribute to the regression of established atherosclerotic plaques. Consistent with our previous studies,24 miR-33 antisense oligonucleotide treatment did not influence plaque size as compared with mice treated with SrcpHLIP (Fig. 3b, quantified in right panel). However, we found that anti-miR-33pHLIP treatment promoted a significant decrease of lipid accumulation in atherosclerotic lesions, which correlated with higher expression of Abca1 mRNA in the aorta (Fig. 3c and d). No differences in Abca1 expression was found in the liver of mice injected with anti-miR-33pHLIP and SrcpHLIP, suggesting that pHLIP direct the specific silencing of miR-33 into the arterial wall (Supp Fig. 5). We further characterized the plaques by analyzing markers of inflammation and lesion stability. Quantification of macrophage content in the lesions by CD68+ staining demonstrated a non-significant decrease in anti-miR-33pHLIP treated mice compared to controls (Supp Fig. 5). Notably, there was a significant increase in lesional collagen (Fig. 3e, quantification right panel) without any effect on smooth muscle cell content (Supp Fig. 5) in mice treated with anti-miR-33pHLIP. Together, these results indicate that targeted inhibition of miR-33 in vascular lesions promotes macrophage cholesterol efflux and atherosclerotic plaque remodeling resulting in a more stable phenotype.
To assess the potential mechanisms by which anti-miR-33pHLIP regulates macrophage function and regression of atherosclerosis, we isolated live whole cells from enzyme-digested aortas of anti-miR-33pHLIP- and SrcpHLIP-treated mice and performed single cell transcriptomics (Fig. 4a, Supp Fig. 5). A total of 7,771 SrcpHLIP cells and 13,424 anti-miR-33pHLIP cells were obtained from atherosclerotic plaques in each group and run on the 10x Genomics platform. Unsupervised Seurat-based clustering identified 12 distinct cell clusters based on gene expression of established canonical markers including monocytes/macrophages (Mono/Mac, Cluster 2, 10 and 11), endothelial cells (EC, Cluster 3), dendritic cells (DC, Cluster 8), vascular smooth muscle cells (VSMC, Cluster 0, 1 and 4), T cells (Cluster 5 and 7) and fibroblasts (Cluster 6 and 9) (Fig. 4a, Supp Fig. 6). Given the cellular delivery of anti-miR-33 by pHLIP into macrophages and monocytes in atherosclerotic lesions (Fig. 1d), we further analyzed the phenotype of these Mono/Mac populations during the regression of atherosclerosis (Supp Fig. 6a and b). Specific gene expression profiles differentiated 5 aortic monocyte and macrophage populations from the Mono/Mac clusters (Fig. 4b, Supp Fig. 7a). These included Trem2high Mac (Cluster 0: Trem2, Cd9, Spp1, Lgals3), F10+ Mono (Cluster 2: F10, Ccr2, low H2-Eb1), Inflammatory Mac (Cluster 3: Il1b, Cxcl2, Nfkbiz, S100a9, S100a8) and Stem-like Mac (Cluster 4: Top2a, Ube2c, Cenpf, Stmn1) (Supp Fig. 7b). Interestingly, besides these well-established cell populations found in atherosclerotic lesions (Trem2high Mac, F10+ Mono, Inflammatory Mac and Stem-like Mac),25, 26 we observed one specific macrophage cluster (Cluster 1, herein referred to as “ECMhigh Mac”) that expressed monocyte and macrophage genes (Cd14, Cd68, Adgre1 and Csf1r) (Supp Fig. 7a) and was highly enriched for extracellular matrix (ECM)-associated genes, including Col1a2, Col3a2, Col1a1, Fn1, Eln, Lum, Bgn and Dcn (Fig. 4c, Supp Fig. 7c). The percentage of ECMhigh Mac were increased in the mice treated with anti-miR-33pHLIP as compared to SrcpHLIP (Fig. 4d). This intriguing finding indicate that during the regression of atherosclerosis suppression of miR-33 in macrophages promotes a pro-fibrotic phenotype that favour plaque stabilization. Notably, the population of inflammatory macrophages (Cluster 3) characterized by the high expression of pro-inflammatory cytokines and chemokines was decreased, and the “stem-like macrophages” in cluster 4, enriched for cell cycle genes and highly proliferative, were increased in the mice treated with anti-miR-33pHLIP as compared to SrcpHLIP (Fig. 4d). We next performed pathway enrichment analysis associated with changes in gene expression in Mono/Mac cells. Among all the pathways that were significantly altered in response to anti-miR-33pHLIP treatment, we found upregulated fibrosis, M2 polarization (IL-4 Signaling) and antigen presentation pathways (Fig. 4e). In addition to an increased signature with extracellular matrix (Fig. 4f), we also observed a decrease of inflammatory genes (Cxcl2 and Tnfsf9) and an increase of antigen presentation genes (H2-Eb1, H2-Aa and H2-Ab1) in the mice treated with anti-miR-33pHLIP (Fig. 4g), indicating that anti-miR-33 treatment by pHLIP peptides induces macrophages towards a less inflammatory and more stable phenotype in the atherosclerotic lesions.
In conclusion, we demonstrated the remarkable selectivity of pHLIP peptides to target macrophages in atherosclerotic lesions based on their affinity for acidic microenvironments. The specific delivery of anti-miR-33 to atherosclerotic plaques using pHLIP peptides promotes a more stable phenotype by induction of ABCA1-mediated cholesterol efflux and extracellular matrix deposition. These findings highlight the therapeutic potential of anti-miR-33pHLIP constructs for the regression of atherosclerosis, while avoiding the potential deleterious effects in other organs. The pHLIP technology can also be applied to the selective delivery of other protective miRNAs to the macrophages from atherosclerotic plaques for the therapy of atherosclerosis-associated cardiovascular diseases. One of the most unique aspects of miRNAs is their ability to target many different mRNAs, which allows them exert both very nuanced and extremely pronounced effects in different situations. However, this promiscuity has also raised important concerns for both research on and clinical applications, especially since the target preferences and impact of miRNAs can vary dramatically in different tissues and cell types. Considering the potential for disparate and possibly adverse effects in different organs, targeted delivery systems such as that described in this work may prove incredibly valuable for the development of safe and reliable miRNA-based therapies.