Simvastatin mediates inhibition of exosome synthesis, localization and secretion via multicomponent interventions

Discovery of exosomes as modulator of cellular communication has added a new dimension to our understanding of biological processes. Exosomes influence the biological systems by mediating trans-communication across tissues and cells, which has important implication for health and disease. Identification of strategies for exosome modulation may pave the way towards better understanding of exosome biology and development of novel therapeutics. In absence of well-characterized modulators of exosome biogenesis, an alternative option is to target pathways generating important exosomal components. Cholesterol represents one such essential component required for exosomal biogenesis. We initiated this study to test the hypothesis that owing to its cholesterol lowering effect, simvastatin, a HMG CoA inhibitor, might be able to alter exosome formation and secretion. Using previously established protocols for detecting secreted exosomes in biological fluids, simvastatin was tested for its effect on exosome secretion under various in-vitro and in-vivo settings. Murine model of AAI was used for further validation of our findings. Utilizing aforementioned systems, we demonstrate exosome-lowering potential of simvastatin in various in-vivo and in-vitro models, of AAI and atherosclerosis. We believe that the knowledge acquired in this study holds potential for extension to other exosome dominated pathologies and model systems.


Introduction
( Figure S1 in the online supplement). Characterization of these particles as exosomes using 89 EM, western blotting, DLS and density gradient centrifugation has already been described in an 90 earlier report from our group, but we still validated the size and morphology using TEM ( Figure   91 S2 in the online supplement). 10 Epithelial cells and monocytes treated with increasing 92 concentration of simvastatin for a period of 24 hours exhibited a significant reduction in the level 93 of secreted exosomes, as measured by the bead-based assay A-B). A significant reduction of 94 about 40% was noted at the 0.3 µM dose of simvastatin, which corresponds to non-toxic 95 maximal plasma concentration associated with simvastatin therapy in humans, 19 confirming the 96 plausibility of this effect at usual clinical dosing. The lack of toxicity was also confirmed by 97 MTT staining (data not shown) and visible inspection ( Figure S3 in the online supplement). 98 The efficacy of bead-based assay was validated by confirming linearly increased detection of The effect of simvastatin on exosome production and inflammation is only partially related 132 to cholesterol reduction: 133 We (and others) have previously reported elevated levels of pro-inflammatory exosomes in 134 asthmatic lung as well as in BALF from mice of experimental model of allergic airway 135 inflammation (AAI). Pharmacological inhibition of these exosomes was found to provide 136 protective effect in experimental asthma. 10 To determine whether simvastatin treatment would Atherosclerotic plaque formation is a process whereby deposition of excess lipid and cholesterol 160 in coronary artery leads to narrowing of blood vessels, thereby causing a reduction in blood flow 161 to heart, resulting in heart failure. Atherosclerotic lesions are usually characterized by increased 162 endothelial migration. In a study exploring this phenomenon, 24 authors implicated the role of 163 exosomes (referred to as microvesicles in this paper) secreted by plaque-associated monocytes in 164 endothelial migration. Microvesicle (MV) associated mir-150 was identified as the key driver of 165 this process. 24 Since simvastatin has long been prescribed to patients of cardiovascular 166 disorders, we wondered if one of the mechanisms by which it renders its protective effects could 167 be by inhibiting microvesicle secretion from accumulated monocytes at plaque surface. For 168 testing this hypothesis, we adopted the model previously described, 24 wherein monocytic 169 microvesicles were shown to promote endothelial migration, and in turn atherosclerosis. These 170 microvesicles contained several micro-RNA species including mir-150, mir-16 and mir-181a, 171 however the pro-atherogenic nature of these vesicles was attributed majorly to mir-150, which 172 caused reduction of c-myb in nearby endothelial cells, hence promoting their migration from the 173 site of plaque formation.

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Simvastatin treatment of monocytic cell line, THP-1, led to reduction in exosome secretion and mir-181b were used as positive controls for exosomes-associated micro-RNA content.

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Simvastatin treatment however did not significantly alter the intracellular levels of any of these 178 miRNAs ( Figure 4B). Incubation of THP-1 derived DIO labeled MVs with HUVECs led to 179 rapid uptake of these vesicles by HUVECs ( Figure 4C

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Once the exosome inhibitory role of simvastatin was established using various model systems, 190 we furthered our study to investigate the putative underlying molecular mechanism.

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Simvastatin treatment alters MVB trafficking and results in their accumulation near the 193 plasma membrane. 194 We found notable reduction in cellular CD-63 levels upon simvastatin treatment in in vitro 195 systems ( Figure 2C), that led us to test if this observation extends to in vivo conditions as well.

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For this purpose, lung tissue sections from our mouse model of AAI were stained for  Under these conditions, lungs are known to have elevated exosome-associated proteins in epithelial cells and macrophages. 10 While inspecting lung-tissue sections of simvastatin treated 199 mice, we observed an interesting phenomenon, wherein simvastatin treatment led to   We had previously reported that exosomes actively play a proinflammatory role in asthma 232 pathogenesis and speculated that molecules capable of reducing exosome secretion might play a 233 protective role in asthma. 10 In this study, we report that simvastatin mediated exosome reduction 234 indeed result in protective phenotype in murine model of asthmatic airway inflammation, which

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For semi-quantitative detection of exosomes, antibody coated beads were used as described 300 earlier. 14 Briefly, 20,000 anti-CD-63 antibody coated beads were washed in 2% BSA and then 301 incubated with 10,000g supernatant of BALF or culture supernatant overnight. Next day, the 302 bead bound exosomes were detected using surface proteins for exosomes or phosphatidylserine 303 on their surface, using flow cytometer.

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For surface labeling, cells were incubated with the antibodies diluted 1:25 in staining buffer for 307 30 minutes on ice, followed by a PBS wash, after which the cells were fixed with 2% 308 paraformaldehyde. For intracellular labeling, cells were fixed and permeabilized, followed by 309 staining. For total (surface+intracellular) CD-63 staining, initially the surface labeling was 310 carried out as mentioned above. After the antibody incubation, cells were fixed and 311 permeabilized and then the protocol for intracellular labeling was carried out.

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Western blot 313 Total cell protein was extracted and was resolved onto a polyacrylamide gel, which was then 314 transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk 315 and then probed for proteins of interest.

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AHR in the form of airway resistance was estimated in anesthetized mice using the FlexiVent 318 system (Scireq, Canada) which uses a computer-controlled mouse ventilator and integrates with 319 respiratory mechanics as described previously. 25 Final results were expressed as airway 320 resistance with increasing concentrations of methacholine. The migration ability of HUVEC was tested in a Transwell Boyden Chamber (6.5 mm, Costar).   Figure D3). Sim: Simvastatin