MicroRNA-145 Micelles for Reverting Endothelium Activation and Monocyte Recruitment in Atherosclerosis

Introduction: Atherosclerosis is a chronic, inflammatory disease characterized by plaque buildup on arterial walls and remains the largest contributor to cardiovascular disease, the leading cause of death globally. Pharmacological management of atherosclerosis is limited to statins, which aim to decrease circulating lipids. However, disease progression is only partially determined by serum lipid profile and many patients are unresponsive to statins. After vascular cells are activated by lipids in circulation, they take on migratory, synthetic, and pro-inflammatory phenotypes. Specifically, endothelial cells (EC) become permeable to circulating lipids, migratory and recruit monocytes into the plaque by expression of adhesion molecules, exasperating disease. MicroRNA-145 (miR-145) is highly expressed in healthy vasculature, decreased in atherosclerosis, and plays an important role in maintaining vascular health. Specifically, after cells in the vessel wall are activated by lipids, miR-145 can alter gene expression post-transcriptionally to inhibit an atherogenic, synthetic phenotype and promote a healthy, senescent phenotype. Thus, it is a promising alternative therapeutic. To deliver miR-145, it must be protected from circulating nucleases and target atherosclerotic lesions. Thus, miR-145 is incorporated into peptide-amphiphile micelles incorporating targeting moiety, MCP-1 peptide which binds chemokine-receptor 2 (CCR2) as CCR2 is upregulated in endothelial cells and monocytes in lesions. Here, micelle formulation will be optimized for EC targeting and EC-level regulation of plaque formation will be evaluated by in vitro models of EC barrier integrity, migration and monocyte recruitment. Thus, the effects of miR-145 micelles on atherogenic EC and monocytes will be elucidated for the first time, prior to in vivo studies.

Materials and

Methods: miR-145 mimic (IDT Technologies) and MCP-1 peptide (CYNFTNRKISVQRLASYRRITSSK) were conjugated to DSPE-PEG(2000)-Maleimide (Avanti Lipids), purified by high performance liquid chromatography and incorporated into micelles by thin film formation and self - assembly due to hydrophobic interactions in solution. Micelle formulations were altered by MCP-1 molar ratio and were characterized by dynamic light scattering, zetasizer and transmission electron microscopy (TEM). miR-145 incorporation into micelles was confirmed by a gel shift assay stained for nucleic acids with ethidium bromide. Primary human aortic endothelial cells (HAEC) were purchased from Lonza and cultured under recommended conditions. To induce a diseased phenotype in vitro, HAEC were stimulated with 100ng/mL of TNF-α for 24 hours. To assess transendothelial integrity, HAEC were cultured in transwell systems and transendothelial resistance (TER) was measured with an EndOhm. HAEC were grown to confluence then activated with TNF-α and activation was confirmed by TER and Vascular Cell Adhesion Molecule 1 (VCAM-1) and CCR2 expression by fluorescence microscopy. HAEC treated with 25 uM micelles in media conditions for 4 hours. Varying MCP-1 targeting peptide molar ratio in micelle formation was assessed for binding in activated HAEC stimulated with TNF- α compared to healthy controls by fluorescence microscopy. To assess miR-145 micelle efficacy, HAEC were treated with micelles for 4 hours and media was replenished for 20 hours prior to assessment. miR-145 transfection and target gene expression were assessed by qPCR (Qiagen), compared to activated only controls. Further, VCAM-1 expression and TER were assessed after micelle treatment as described above.

Results, Conclusions, and Discussions: The mean miR-145 micelle diameter was 14.2 ± 2.9 nm with zeta potential -6.71 ± 0.33 mV and TEM showed a uniform population of spherical nanoparticles which are stable over 30 days (Fig. 1 A-D). Micelle protection of miR-145 was confirmed via a gel shift assay as micellar miR-145 remained in the well with or without RNAse treatment and free miR-145 amphiphiles migrated down the gel or degraded under RNAse treatment (Fig. 1 E). HAEC activation was confirmed by decreased transendothelial resistance (TER), increased VCAM-1 and CCR2 expression (Fig 2 A-B). A significant (p < 0.05) difference in micelle binding between activated and healthy HAEC was observed with 50% molar MCP-1 amphiphile formulation, which was used for future studies (Fig. 2C). HAEC miR-145 expression increased after treatment with miR-145 micelles by 30-fold compared to activated only control (Fig 3 A). Further, miR-145 target atheroprotective gene Kruppel-like Factor 2 (KLF2) increased over 2-fold while atherogenic genes CCR2 and alpha smooth muscle actin (ACTA2) decreased over 4-fold compared to activated only controls (Fig 3 A). Additionally, HAEC vascular cell adhesion molecule 1 (VCAM-1) expression decreased after micelle treatment by 3-fold with no significant difference between healthy HAEC VCAM-1 expression, and transendothelial resistance (TER) increased 18.9 ± 6.86 % compared to control micelles (Fig 3 B). In summary, our studies confirm the synthesis of micelles that contain MCP-1 and protect miR-145 from nuclease degradation. Micelles with 50% molar MCP-1 amphiphile showed the most selective binding to activated versus untreated HAEC. When miR-145 was incorporated into this formulation, HAEC atheroprotective KLF2 and atherogenic CCR2, ACTA2 and monocyte adhesion molecule, VCAM-1, expression decreased and transendothelial resistance increased, suggesting the therapeutic value of miR-145 micelles on reverting HAEC activation and monocyte recruitment. Thus, miR-145 micelles have multi-faceted effects in cell-level control of plaque formation. Future experiments will determine the dose-response relationship in plaque reduction in mouse models of atherosclerosis. The ultimate goal of this project aims to test miR-145 micelles in animal studies, as a vital step towards testing in Phase 1 clinical trials.

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