Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts

Rationale: Neointimal hyperplasia caused by dedifferentiation and proliferation of venous smooth muscle cells (SMCs) is the major challenge for restenosis after coronary artery bypass graft. Herein, we investigated the role of Lamtor1 in neointimal formation and the regulatory mechanism of non-coding RNA underlying this process. Methods: Using a “cuff” model, veins were grafted into arterial system and Lamtor1 expression which was correlated with the activation of mTORC1 signaling and dedifferentiation of SMCs, were measured by Western blot. Whole transcriptome deep sequencing (RNA-seq) of the grafted veins combined with bioinformatic analysis identified highly conserved circSlc8a1 and its interaction with miR-20a-5p, which may target Lamtor1. CircSlc8a1 was biochemically characterized by Sanger sequencing and resistant to RNase R digestion. The cytoplasmic location of circSlc8a1 was shown by fluorescence in situ hybridization (FISH). RNA pull-down, luciferase assays and RNA immunoprecipitation (RIP) with Ago2 assays were used to identify the interaction circSlc8a1 with miR-20a-5p. Furthermore, arterial mechanical stretch (10% elongation) was applied in vitro. Results: In vivo, Lamtor1 was significantly enhanced in grafted vein and activated mTORC1 signaling to promote dedifferentiation of SMCs. Arterial mechanical stretch (10% elongation) induced circSlc8a1 expression and positively regulated Lamtor1, activated mTORC1 and promoted SMC dedifferentiation and proliferation. Local injection of circSlc8a1 siRNA or SMC-specific Lamtor1 knockout mice prevented neointimal hyperplasia in vein grafts in vivo. Conclusions: Our study reveals a novel mechanobiological mechanism underlying the dedifferentiation and proliferation of venous SMCs in neointimal hyperplasia. CircSlc81/miR-20a-5p/Lamtor1 axis induced by arterial cyclic stretch may be a potential clinical target that attenuates neointimal hyperplasia in grafted vessels.


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All images were quantified with Image J, and the immunofluorescence intensity value of each 6 image was divided by the area of immunostaining region to obtain the average fluorescence 7 intensity. 8 For each data, 3 independent biological repeats, each with one respective image, were analyzed. 9 10 Fluorescence in Situ Hybridization (FISH). Sections were permeabilized with 0.3% Triton X-11 100 for 30 min at room temperature and washed 3 times with RNase-Free PBS. Sections were 12 treated with Proteinase K (5 μg/mL) for 5 min at 37 °C and then washed with RNase-Free PBS. 13 Specific biotinylated circSlc8a1 probe or scrambled RNA probe was hybridized at 55 °C overnight. 14 Probe detection was performed using Tyramide SuperBoost TM Kit with Alexa Fluor TM Tyramides 15 (TSATM) signal Amplification kit (Invitrogen, B40933) following the manufacturer's instructions. 16 Briefly, samples were incubated at 4 °C overnight in streptavidin-HRP and 3 h in tyramide solution.

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Nuclei were stained with DAPI and cytoskeleton was stained with α-smooth muscle actin-FITC 18 antibody (sigma, 1:500). The images were obtained using a confocal laser scanning microscope 19 (Fluoview 1000, Olympus), and the immunofluorescence intensity was analyzed with Image J.

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The sequences of specific circSlc8a1probe and scrambled control probe were list in Table S1.

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Housekeeping gene GAPDH was used as internal control.

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For miRNA, reverse transcription and PCR were performed using the Hairpin-it TM miRNA 13 qPCR Quantitation Kit (Gene pharma, Shanghai, China), and U6 was used as internal control.
14 For circRNA, cDNA was reverse transcribed with SuperScript Ⅱ reverse transcriptase 15 (Invitrogen) using random primers, and PCR was performed using convergent primers and 16 divergent primers. GAPDH was used as internal control.

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All assays were performed on the StepOnePlus TM Real-Time PCR Instrument. Relative gene 18 expression was analyzed with 2 -ΔΔCt method. The gene specific primers were listed in Table S7.  For circSlc8a1 siRNA treatment in vivo, circSlc8a1-specific siRNA or the corresponding 10 nonsilencing siRNA control (Shanghai GenePharma Co., Ltd.) was subcutaneously injected after 11 vein graft in vivo. For the first injection, 20 nmol circSlc8a1-specific siRNA or nonsilencing 12 control was subcutaneously injected around the grafted veins, and then, 15 nmol was administered 13 every day for 1 wk.