A total of 16 male Sprague-Dawley (SD) rats, 8 month old and weighing 310 to 380 g, were obtained from the Experimental Animal Centre of The General Hospital of Western Theater Command at Chengdu, China. Rats were housed in a room with a 12 light-dark cycle at 23°C and given free access to standard rodent food and tap water. Rats randomized to the sham-operated group, balloon injury group and TMEM66-overexpression group. Balloon denudation was performed as previously described, with a 2-French catheter in the left common carotid artery (8,9). After balloon injury, a 100 µl solution of lenti-TMEM66-GFP (10⁸ IU per rat) or PBS were infused into the injured artery segment and incubated for 15 min. The efficiency of transfection was identified by fluorescence microscopy (Fig. S1). After 4 week balloon injury and lentiviral infection, the rats were euthanized using an overdose of sodium pentobarbital. The vessel tissues were harvested for the subsequent experiments. This study was approved by the Research Council and Animal Care and Use Committee of The General Hospital of Western Theater Command. All experiments conformed to the guidelines of the American Association for the Accreditation of Laboratory Animal Care and conformed to the guidelines of the ethical use of animals, and all efforts were made to minimize animal suffering and to reduce the number of animals used.

Carotid artery tissues were lysed in lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 15,000 × g. The homogenates were separated by SDS-polyacrylamide gel (4% stacking gel with an 8% separating gel) and transferred to nitrocellulose filter membranes. The transblots were probed with the rabbit anti-TMEM66 antibody (1:500; cat. no. ab80890; Abcam), rabbit anti-α-SMA antibody (1:500; cat. no. ab32575; Abcam) and rabbit anti-calponin antibody (1:500; cat. no. ab46794; Abcam) at 4°C overnight, after washing with TBST and blocking with 1% BSA. The membranes were then washed and incubated with goat anti-rabbit-IgG (1:4,000; cat. no. BA1039; Boster Biological Technology) conjugated to horseradish peroxidase at room temperature for 1 h, and the bands were visualized with enhanced chemiluminescence (EMD Millipore). The amount of protein transferred onto the membranes was verified by immunoblotting for GAPDH (rabbit anti-GAPDH; 1:1,000; cat. no. ab181602; Abcam).

RNA from the carotid artery was extracted using Trizol (Thermo Fisher Scientific, Inc.), synthesized to cDNA and served as a template for amplification of TMEM66 using an RNA reverse transcriptase kit (Takara Biotechnology Co., Ltd.) under the following conditions: 37°C for 15 min and 85°C for 5 sec. The cDNA was then stored at 4°C. Primer information is described in a previous study (10). Amplification was performed using quantitative PCR (qPCR) using a SYBR Green Premix (SYBR Real-Time PCR Kit; Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions using the following conditions: 50°C for 2 min, 95°C for 2 min and 35 cycles of 58°C for 30 sec and 72°C for 40 sec. The relative expression of target genes was standardized to GAPDH, evaluated using the 2^−ΔΔCq method and expressed as a ratio to control the experiments (11).

The injured artery tissues from SD rats were cleared of blood with ice-cold PBS and kept in 4% paraformaldehyde for 24 h at 4°C. After embedding in paraffin, sectioning to a thickness of 4 µm and mounting on slides, tissues were deparaffinized and rehydrated by successive incubations in xylene, 100% ethanol, 95% ethanol, 75% ethanol, and PBS. Sections were then stained with hematoxylin and eosin using a standard protocol, or treated with rabbit anti-TMEM66 antibody (1:100, Abcam) for immunohistochemistry.

The artery tissue lysates (400 µg protein/ml supernatant) were incubated with affinity-purified anti-Orai1 receptor antibody (5 µl/ml; cat. no. ab59330; Abcam) for 2 h at room temperature and protein-G agarose at 4°C for 12 h. The immunoprecipitates were subjected to immunoblotting with the Stim1 antibody (1:300; cat. no. ab57834; Abcam).

Embryonic thoracic aortic smooth muscle A10 cells (ATCC, American Type Culture Collection) were cultured at 37°C in a 95% air/5% CO₂ atmosphere in DMEM/F-12. TMEMT66 transduction was performed using the lentivirus-based pLenti6.3-TMEM66 plasmid (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.). Next, the A10 cells were cultured in 3 ml DMEM containing 3% FBS, 10 µg/ml polybrene and virus (MOI=100). The medium was replaced 48 h after transfection, and then 5 µg/ml blasticidin was incubated in the medium for the next 48 h. The cells were incubated with platelet-derived growth factor-BB (PDGF-BB (30 ng/ml) (R&D Systems) for 24 h.

Cell proliferation was assessed using a 5-bromo-2′-deoxyuridine (BrdU) staining kit (BioVision, Inc.) and the Cell Counting Kit-8 assay (CCK-8; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, for BrdU staining, cells were pulsed with BrdU for 2 h after 24 h PDGF-BB stimulation. BrdU incorporation was visualized by immunocytochemical staining with the anti-BrdU monoclonal antibody (BioVision, Inc.). The cells were then fixed with 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI). The number of BrdU-positive cells were counted using fluorescent microscopy from three randomly chosen high power fields (magnification, ×20). Cells were also seeded into 96-well plates and incubated with CCK-8 for 2 h at 37°C. Absorbance was determined using a spectrophotometer (Model 680; Bio-Rad Laboratories, Inc.) at 450 nm. Moreover, a wound scratch assay was used to determine cell migration. A10 cells were cultured in a 6-well dish until >90% confluent was achieved. The scratching was performed with a 200-µl sterile pipette tip by creating a line. The cells were then incubated with PDGF-BB for 24 h. Images were obtained by phase contrast microscopy at ×40, and the number of migrated cells were counted under a light microscope.

Intracellular calcium was measured using a Thermo Varioskan flash instrument (Thermo Fisher Scientific, Inc.) as previously described (12) with various modifications. Briefly, A10 cells were seeded in 96-well cell culture plates together with 5 µM calcium-dependent fluorescent indicator Fura-2 in DMEM at 37°C. The cells were then washed twice with Ca²⁺-free Krebs buffer (140 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO₄, 0.3 mM NaH₂PO₄, 10 mM HEPES, 5 mM glucose, pH 7.4 with Tris base) and measured by the flash instrument with every 5 sec alternating between 340 and 380 nm excitation (2 nm slit size) at 510 nm emission (5-nm slit size). The free Ca²⁺ concentration [Ca²⁺]_free was calculated from the equation (13): [Ca²⁺]_free=K_d[(R-R_min)/(R_max-R)](F380_max/F380_min); The K_d is the dissociation constant of Fura-2 to calcium. R is the ratio of each 340/380 nm. For the positive control, the cells were incubated with calcium channel blocker, verapamil, for 2 h, to reduce the Ca²⁺ concentration. Minimum and maximum are the fluorescence values of cells treated by Triton X-100 (saturating Ca²⁺ concentration) or by EGTA (zero Ca²⁺ concentration).

Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). Comparisons between two groups was carried out using dependent paired t-test, and comparisons among more than two groups was carried out by repeated measures one-way ANOVA. Data are expressed as mean ± SEM. A probability value P<0.05 was considered as indicative of statistical significance.

We first determined the expression of TMEM66 after carotid artery injury by immunostaining, immunoblotting and qPCR. Immunostaining of the arterial sections revealed expression of TMEM66 in the medial and neointimal layers of the carotid artery after balloon injury (Fig. 1A). TMEM66 mRNA from the carotid artery tissue was significantly decreased after balloon injury (Fig. 1B). Moreover, TMEM66 protein was lower in the balloon-injured artery than that noted in the sham control (Fig. 1C), indicating that the impairment of TMEM66 expression by balloon injury occurred at both the post-translational and transcriptional levels.

To investigate whether or not TMEM66 is involved in protection against neointimal hyperplasia, the carotid artery thickness in TMEM66-overexpression mice was determined after balloon injury. The histological analysis showed that balloon injury significantly increased the ratio of the intima to media thickness in the carotid artery, whereas the ratio of intima to media thickness was reversed in the TMEM66-overexpression mice (Fig. 2A).

Moreover, A10 cell proliferation was determined by BrdU labeling (Fig. 2B) and Cell Counting Kit-8 (CCK-8, Fig. 2C). A10 cells with TMEM66 overexpression exhibited a significant decrease in cell proliferation in response to PDGF-BB. The effect of lentivirus overexpression of TMEM66 on PDGF-BB-induced A10 cell migration was also assessed by scratch assay. It was demonstrated that TMEM66 also reduced the cell migration induced by the PDGF-BB (Fig. 2D). In addition, expression levels of α-smooth muscle actin (α-SMA) and calponin were decreased following PDGF-BB treatment, while TMEM66 overexpression significantly restored these VSMC phenotypic markers at the mRNA (Fig. 3A) and protein (Fig. 3B) expression levels.

Stim1 and Orai1 are critical molecules involved in the modulation of SOCE activation in VSMCs (14). Therefore, our further experiments assessed the expression of Stim1 and Orai1 in balloon injured carotid artery with or without TMEM66 transduction. The data revealed that TMEM66 overexpression reduced the increased expression of Stim1 and Orai1 after balloon injury (Fig. 4A). In addition, TMEM66 destabilized the SOCE signalplexes: Interaction of Stim1 and Orai1 was increased in the PDGF-BB-treated A10 cells, while it was deceased by TMEM66 overexpression (Fig. 4B). Due to the role of TMEM66 in SOCE modulators, the intracellular free [Ca²⁺] was assessed. It was demonstrated that TMEM66 overexpression reduced the PDGF-BB treatment-enhanced [Ca²⁺]_i (Fig. 5).