Associate Professor University of Southern California, United States
Introduction:
Introduction: Atherosclerosis is the primary contributor to cardiovascular disease which is the leading cause of death worldwide. Vascular smooth muscle cells (VSMCs) play a significant role in atherosclerosis progression by transdifferentiating from a healthy contractile phenotype into atherogenic cell types such as synthetic VSMCs and osteoblast-like VSMCs. Synthetic VSMCs propagate plaque formation and osteoblast-like VSMCs drive vascular calcification. However, extracellular vesicles (EVs) secreted from contractile VSMCs have been shown to therapeutic cargo including calcification inhibitors (i.e. matrix gla protein (MGP)) and atheroprotective microRNA-145 (miR-145) which rescues the contractile VSMC phenotype and miR-133 which inhibits osteoblast differentiation. Additionally, VSMCs can be engineered to overexpress and load specific microRNA cargo into their EVs. We hypothesize that sEVs secreted from engineered contractile VSMCs contain endogenous atheroprotective and anti-calcifying cargo that can be used to inhibit both plaque formation and late stage vascular calcification. To test our hypothesis, herein, we isolate, characterize, and engineer contractile VSMC-sEVs loaded with miR-145/133 and surface modified with a CCR2 binding peptide (MCP1-EVs, MCP1 sequence: YNFTNRKISVQRLASYRRITSSK) and hydroxyapatite (HA) binding peptide (HABP-EVs, HABP sequence: SVSVGMKPSPRP) to actively target synthetic and osteoblast-like VSMCs. In addition, we evaluate the phenotype modulation potential of HABP-EVs and MCP1-EVs.
Materials and
Methods: VSMC Transduction: Plasmids encoding for pre-miR-145 and pre-miR-133 with CAUG substitutions were transfected in 293FT packaging cells (Thermofisher, R70007), and the lentiviruses released by these cells were applied to MOVAS cells. EV Isolation: Media from cultured MOVAS, miR-145/133-loaded MOVAS (miR-133 MOV, miR-145 MOV) was subject to differential ultracentrifugation to isolate EVs. EV Characterization: EV size and concentration were determined using Nanoparticle Tracking Analysis (NTA) and TEM. Exosomal markers (CD9, CD63, and TSG101) were characterized via Western Blot. miR-145/133 content was determined using qRT-PCR. EV Surface Modification: EVs were modified with DSPE-PEG(2000)-HABP (.25mg/mL) and DSPE-PEG(2000)-MCP1 by sonication for 3 minutes to form HABP-EVs and MCP1-EVs. EV binding and cell internalization: Calcified MOVAS cells were incubated with DIO-labeled HABP-, MOVAS-EVs for 1 and 4 hours. Cells stained (alizarin red for calcium, DAPI for cell nuclei) and imaged on a fluorescent microscope. Cholesterol treated MOVAS cells were incubated with MCP1- and MOVAS-EVs and Cells were imaged on a fluorescent microscope. In vivo calcification binding: 9 month old APOE-/- mice were fed a high fat diet for 10 weeks followed by a single dose of DIR-labeled HABP-EVs, MOVAS-EVs, or Osteoscence. Aortas were harvested after 24 hours, imaged with a IVIS, and sectioned and immunostained for calcification (alizarin red). Treatment of calcified MOVAS: Calcified MOVAS cells were treated with miR-145 HABP-EVs and miR-133 HABP-EVs (1x1011 EV/mL). qRT-PCR was performed for the contractile markers (myocardin, alpha smooth muscle actin, myosin heavy chain 11, calponin) and synthetic and osteochondrogenic markers (kruppel like factor 4/5 and RUNX2).
Results, Conclusions, and Discussions: To evaluate confirm successful isolation of sEVs we performed NTA, TEM, and western blot for exosomal markers CD9, CD63, and TSG101 and found sEVs of mean and mode sizes 100-200 nm with enrichment of exosomal markers (Fig 1). Next, we evaluated the atheroprotective cargo of miR-145/133 MOV-, MOVAS- and HABP-EVs and found that MGP and miR-145 were enriched in MOVAS-EVs compared to calcified MOVAS-EVs by 26- and 5.6-fold, respectively (Fig 1). miR-145/133 transduction led to a ~60 and ~40 fold increase in EV loading compared to MOVAS-EVs (Fig 1). Importantly, modification of MOVAS-EVs with DSPE-PEG(2000)-HABP did not result in a statistically significant loss of therapeutic cargo. After modification of MOVAS-EVs with DSPE-PEG(2000)-HABP, we evaluated the binding capacity of HABP-EVs to calcified MOVAS cells and MCP1-EVs to CCR2 expressing VSMCs. HABP-EVs showed increased colocalization to calcium in the calcified MOVAS cell model compared to MOVAS-EVs with a Pearson’s Colocalization Coefficient of 0.77±.07 and 0.64±.06, respectively (Fig 2). We found MCP1-EVs showed an increased binding to CCR2 expressing VSMCs compared to unmodified MOVAS-EVs (Fig 2). Additionally, HABP-EVs bound to aortic calcification at levels similar to that of the commercial calcification tracer Osteosense (Fig 3). Confirming enhanced binding of HABP-EVs to calcified MOVAS cells, we treated calcified MOVAS cell with miR-145/133-HABP-EVs and MOVAS-EVs to evaluate their gene modulatory ability. We found treatment of calcified MOVAS cells with miR-145/133-HABP-EVs show statistically significant upregulation of the contractile markers and down regulation of the osteoblast compared to treatment with MOVAS-EVs (Fig 3).
