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

Abstract

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. 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. Simvastatin was tested for its effect on exosome secretion under various in-vitro and in-vivo settings and was found to reduce the secretion of exosome from various cell-types. It was also found to alter the levels of various proteins important for exosome production. Murine model of Acute Airway Inflammation was used for further validation of our findings. We believe that the knowledge acquired in this study holds potential for extension to other exosome dominated pathologies and model systems.

Figure 1

Simvastatin reduces exosomes secretion. (A,B), Cells at a concentration of 2 × 10⁶/well of a 6-well plate were treated with indicated concentrations of simvastatin in 2 ml of media for a period of 24 hours, after which the culture supernatant was harvested and 1 ml from it was used for measuring exosomes. Secreted exosome levels in culture supernatant from simvastatin treated epithelial cells (A) and THP-1 monocytes (B), measured as in Fig. S4 in the online supplement. (C), Levels of exosome associated Alix, Tsg-101 and β-actin in pelleted exosome fraction from supernatant of 10⁷ simvastatin treated cells. (D) Effect of simvastatin treatment on exosome associated CD9/CD81 and Annexin V in cell culture supernatant from IL13 (25 ng/ml) and simvastatin treated epithelial cells. Data in (A,B,D) represent the mean ± SE from three independent experiments. Data in C is representative image from one of the two independent experiments. (_*p < 0.05 vs Control and ^¥p < 0.05 vs rIL13). Sim: Simvastatin.

Figure 2

Simvastatin directly alters the level of various exosome associated proteins. (A) Western blots for Alix and CD63 levels in total cell protein treated with different doses of simvastatin. (B) Cell surface levels of CD63 and CD81 were measured by flow cytometry after treatment with various doses of simvastatin, E-cadherin was used as control surface marker. (C) Immunocytochemistry for CD63 on cells treated with 0.31 um of simvastatin for 24 hours. Sim: Simvastatin.

Figure 3

Effect of simvastatin and mevalonate cotreatment on inflammatory parameters. (A) Secreted exosome levels in BAL supernatant of mice from indicated groups. (B,C) Lung sections stained with hematoxylin and eosin (H&E, B) showing leukocyte infiltration, periodic acid–Schiff (PAS, C) for collagen deposition. (D) Airway resistance with increasing concentrations of methacholine 12 h after the last challenge. (E,F) Effect of indicated treatments on total leukocyte count (E) and differential leukocyte count enumerated by morphological criteria (F). (G) Ova specific serum IgE levels measured by ELISA. (H) Cytokines IL-13 and IL-4 measured in pulmonary homogenate. Sections in (B,C) shown at 20X magnification. Br, Bronchus. Results (A,D,E,F,G,H) are the mean ± SE for each group from two experiments with 4–6 mice in each group, (*p < 0.05 vs SHAM; ^¥p < 0.05 vs OVA; ^¶p < 0.05 vs Sim), Sim: Simvastatin (40 µmg/kg/dose), Mev: Mevalonate (20 mg/kg/dose).

Figure 4

Simvastatin reduces exosome production from monocytes and attenuates exosome-enclosed mir-150 mediated endothelial cell migration. 1 × 10⁶ of THP-1 cells were seeded and treated with 0.3 µM of simvastatin and 1 µM GW4869 for a period of 24 hours. Cell pellet and supernatant was harvested and used separately for RNA isolation. Presence of indicated micro-RNAs was determined using qRT-PCR. Simvastatin mediated reduction of exosomes secretion from THP-1 monocytes results in lower levels of secretory miRNAs (A) but not intracellular miRNAs (B). (C) Uptake of DIO-labeled THP-1 derived exosomes (10 μg/mL) by HUVECs. (D) Relative mir-150 levels in HUVECs with indicated treatment (E), Simvastatin mediated reduction in exosome secretion by THP-1 monocytes results in lower mir-150 levels in HUVECs incubated with exosomes from simvastatin treated THP-1 in comparison to exosomes from same number of untreated THP-1 control cells, and consequent reduction in migration of endothelial cells. (*p < 0.05 vs Control in A, D and E2. ^¥p < 0.05 vs Ctrl + MV in D and E2). Sim: Simvastatin, Control: Control HUVEC.

Figure 5

Simvastatin alters localization of CD63-positive compartments in cells. Representative Immunohistochemistry images for CD63 from lung tissue sections of OVA and OVA/Simvastatin treated mice in (A) epithelial cells and macrophages (B). (C), Representative TIRF images of CD-63 levels and localization post treatment with simvastatin in CD63-EGFP transfected cells. Arrows indicates CD63 localization pattern. (D) Distribution of CD63 in Rab-27b esiRNA treated cells (D1) and subplasmalemmal region associated CD-63 signals (D2), as visualized by confocal microscopy. (E) Representative images showing CD63-EGFP distribution and its association with linear beeline like structures (E1-E2, inset) in control and simvastatin treated cells in subplasmalemmal region, detected by TIRF microscopy. (F) Localization of CD63 with actin in absence and presence of actin polymerization enhancer Jasplakinolide (100 nM). Images in (E,F) shown at 63X while (A–D) shown at 100 X magnification. Sim: Simvastatin.