A mixture of 2-chloroacetamide, N,N-dimethylformamide, K₂CO₃, and 2-methoxyphenol was stirred overnight. The residue was diluted with ethyl acetate and dried with anhydrous MgSO₄. After separation using SiO₂ column chromatography with ethyl acetate/n-hexane (1:1), the compound (0.2207 mM) and oxalyl chloride (0.509 mM) in dichloroethane were stirred at 85°C for 5 hours and concentrated under reduced pressure. Then, 4-bromo-3-methoxyaniline (0.221 mM) and methanol were added and stirred for 30 minutes at 0°C, and then overnight at room temperature (RT). The mixture was dried over MgSO₄ and concentrated under reduced pressure. The final compound (N-((4-bromo-3-methoxyphenyl)carbamoyl)-2-(2-methoxyphenoxy)acetamide; talin modulator) was subjected to SiO₂ column chromatography with ethyl acetate/n-hexane (1:3); ¹H-NMR (dimethyl sulfoxide [DMSO], 500 MHz) δ 10.72 (s, 1H), 10.31 (s, 1H), 7.49 (d, 1H, J = 7.5Hz), 7.31 (s, 1H), 7.16 (d, 1H, J = 9.0Hz), 7.01 (d, 1H, J = 7.5Hz), 6.96–6.85 (m, 3H), 4.81 (s, 2H), 3.82 (s, 3H), 3.78 (s, 3H); LC-MS ([M+Na]+431), and was finally dissolved in DMSO (D8418, Sigma-Aldrich, St. Louis, MO, USA).

Surface plasmon resonance (SPR) was performed to confirm the interaction between the talin modulator and talin protein as previously reported.16) Briefly, the response curves were obtained using a ProteOn XPR36 system equipped with a sensor chip and ProteOn XPR36 control software ProteOn Manager v.3.1.0.6 (both from Bio-Rad, Hercules, CA, USA). Serially diluted samples were injected through the channels of the HTE chip for 1 minute at 100 μL/min, and stopped followed by injection with phosphate buffered saline (PBS) containing 0.005% Tween-20 to obtain the dissociation curves. The response unit of the empty channel was used for normalization.

Human aortic SMCs (HAoSMCs; CC-2571) were cultured in SMC growth medium-2 (CC-3182, both from Lonza, Basel, Switzerland) supplemented with 100 U/mL penicillin/streptomycin (#15140, Gibco) at 37°C in a 5% CO₂ humidified incubator. After 24 hours of attachement, HAoSMCs were treated with the talin modulator or with an equivalent amount of DMSO (vehicle control). All phase-contrast images were obtained using an upright microscope (DMI 300B, Leica Microsystems, Wetzlar, Germany).

Cell proliferation was assessed by water-soluble tetrazolium salt assay (WST-1 assay; 05015944001, Roche, Basel, Switzerland). HAoSMCs were plated and treated with varying concentrations of the talin modulator or equivalent volume of DMSO. After 24 hours, the cells were incubated with the WST-1 reagent for 2 hours, and absorbance at 450 nm and 655 nm (reference wavelength) was detected using a microplate reader (iMark, Bio-Rad).

HAoSMCs were plated in 6-well plates and allowed to grow to be confluent for 24 hours. A scratch wound was made across the midline of each well using a sterile pipette tip and washed with PBS. The cells were then treated with the talin modulator or DMSO, and phase-contrast images of the wounded area were acquired at 0, 8, 12, and 24 hours after scratching and treatment. The area covered by migrated cells across the wounded line was quantified using Image J software (v1.32, National Institutes of Health, Bethesda, MD, USA).

Total protein from HAoSMCs was extracted using cell lysis buffer (#9803, Cell Signaling Technology, Danvers, MA, USA). Equal amounts of the lysate were resolved on 8% or 10% gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (10600023, GE Healthcare, Chicago, IL, USA). The membranes were blocked with 5% skim milk in 0.1% tris-buffered saline and 0.1% Tween-20 for 1 hour at RT, and incubated with the following primary antibodies overnight at 4°C: talin (T3287), vinculin (V9131, both from Sigma-Aldrich), paxillin (#610051, BD Biosciences, San Jose, CA, USA), AKT (sc-1618, Santa Cruz, Dallas, TX, USA), p-vinculin (#44-1078G, Thermo Fisher Scientific, Waltham, MA, USA), p-paxillin (#2541), p-FAK^Y397 (#8556), p-FAK^Y925 (#3284), FAK (#13009), p-AKT (#9271), p-mitogen-activated protein kinase kinase 1/2 (MEK1/2; #9121), MEK1/2 (#9122), p-extracellular signal-regulated kinase 1/2 (ERK1/2; #9106), and ERK1/2 (#9102, all from Cell Signaling Technology). The membranes were washed and incubated with anti-mouse (#7076), anti-rabbit (#7074, both from Cell Signaling Technology), or anti-goat (HAF109, R&D systems, Minneapolis, MN, USA) HRP-conjugated secondary antibodies for 1 hour at RT. The protein bands were visualized by Clarity Western ECL Substrate (#1705061) and ChemiDoc Touch Imaging System (both from Bio-Rad). All band intensities were normalized to glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma-Aldrich) and analyzed using Quantity One software (Bio-Rad).

HAoSMCs were fixed with 2% paraformaldehyde (PFA; P6148) and permeablized using 0.1% Triton X-100 (×100, both from Sigma-Aldrich). The cells were blocked with 5% normal goat serum (NGS; #16210, Gibco), and incubated with phosphohistone H3 (PHH3) antibody (06-570, Merck Millipore, Burlington, MA, USA) and Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (IgG) (A11012, Thermo Fisher Scientific). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (D9542, Sigma-Aldrich). Immunofluorescent images were acquired using a BX61 motorized system microscope (Olympus, Tokyo, Japan).

All animal experiments were approved by the Institutional Animal Care and Use Committee of Korea University College of Medicine (KOREA-2017-0126). Male, 10-week-old ApoE KO mice (#002052, The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized by intraperitoneal (IP) injection of ketamine (80 mg/kg; Yuhan, Seoul, Korea) and xylazine (8 mg/kg; Bayer, Leverkusen, Germany). After a groin incision, the left femoral arteries were injured by passing a sterile wire followed by ligation. ApoE KO mice were randomly assigned to the following 3 groups; sham (subjected to the procedure alone; n=5), vehicle control (DMSO; n=5), and talin modulator (5 mg/kg; n=6). The talin modulator and an equivalent volume of DMSO were prepared in 0.5% carboxymethylcellulose sodium salt (C5678, Sigma-Aldrich) for administration via oral gavage daily for 28 days. The mice were anesthetized at 7, 14, and 28 days post-procedure and the blood flow in the ventral side was analyzed using a laser Doppler imager (Moor Instruments, Devon, UK).

At day 28, all mice were euthanized by IP injection of ketamine and xylazine, and femoral arteries were harvested. PFA-fixed paraffin-embedded arteries were sectioned in a 5 μm of thickness and stained with hematoxylin and eosin or elastic staining solutions.

After de-paraffinization, the tissue sections were incubated with proteinase K solution (#21627, Merck Millipore) for antigen retrieval and blocked with 5% NGS in 0.1% PBST. The sections were then incubated with α-smooth muscle actin (α-SMA; A2547, Sigma Aldrich) antibodies for 1 hour at RT and then with biotinylated anti-mouse IgG antibodies (BA-2000, Vector, Burlingame, CA, USA) for 30 minutes at RT. Subsequently, the sections were incubated with the ABC reagent (PK-6100) and peroxidase substrate (SK-4100), and mounted using VectaMount (H-5000, all from Vector). A BX61 motorized system microscope was used to obtain bright-field images.

Blood serum collected from the abdominal vein of mice was separated using BD Vacutainer SST tubes (367955, BD Diagnostics, Franklin Lakes, NJ, USA). Mouse interleukin-6 (IL-6; M6000B) and tumor necrosis factor alpha (TNF-α; MTA00B, both from R&D Systems) enzyme-linked immunosorbent assay (ELISA) kits were used to evaluate serum levels after 28 days of femoral arterial injury and oral administration of the talin modulator. The ELISA reaction was measured using a microplate reader following the manufacturer's protocol.

Data are represented as the mean value±standard deviation. Significance of differences was analyzed by 1-way analysis of variance and Student-Newman Keuls test, and results were considered statistically significant when p<0.05. All experiments were repeated at least 3 times independently.

In this study, a new talin modulator (N-((4-bromo-3-methoxyphenyl)carbamoyl)-2-(2-methoxyphenoxy)acetamide) designed to bind to the talin protein was synthesized (Figure 1A). Interaction between the talin protein and the talin modulator was confirmed using SPR analysis as shown in Figure 1B. The talin modulator bound to the talin protein at all tested concentrations and the binding was reversible.

The effect of various concentrations (5–100 μM) of the talin modulator on the proliferation of HAoSMCs was examined by WST-1 assay (Figure 1C). The talin modulator induced a concentration-dependent inhibition of cell proliferation, and the half maximal inhibitory concentration of the talin modulator on cell proliferation was 50 μM (0.612±0.033) relative to the untreated condition (0 μM; 1.0±0.041). After 24 hours of treatment at the concentration of 50 μM, the talin modulator decreased the attached cell density compared to the negative and vehicle controls (Figure 1D). To confirm these results, we also performed immunofluorescence staining for PHH3, a marker for mitosis (Figure 1E). Consistently, the talin modulator significantly decreased the percentage of PHH3-positive cells (11.69±8.83%) compared to the negative and vehicle controls (32.96±5.03% and 25.85±5.93%; Figure 1F). Moreover, the anti-migratory effect of the talin modulator was evaluated by wound healing migration assay (Figure 1G). In the negative and vehicle controls, continuous migration of HAoSMCs was observed in a time-dependent manner, however, the talin modulator significantly suppressed cell migration at 8, 12, and 24 hours (Figure 1H). Therefore, the talin modulator exhibited significant inhibitory effects on cell proliferation and migration of HAoSMCs.

We examined the effects of the talin modulator on focal adhesion complexes that mainly consist of integrin, talin, vinculin, paxillin, and FAK. Western blot analyses in Figure 2A and B revealed that the talin modulator did not alter the expression of the talin protein in spite of binding to it (Figure 1B). Intriguingly, the talin modulator dramatically reduced the phosphorylation of both vinculin and paxillin after 24 hours of treatment compared to the negative and vehicle controls (Figure 2C and D). Moreover, paxillin expression was also significantly decreased upon treatment of HAoSMCs with the talin modulator. As FAK is well known as a key downstream molecule in integrin-talin interaction, we next performed experiments to elucidate the underlying mechanisms of inhibition of the talin modulator in HAoSMCs (Figure 2E). The integrin-induced auto-phosphorylation of FAK at Y397 was significantly decreased at 4 hours of treatment with the talin modulator compared to that in the negative and vehicle controls (Figure 2F). The talin modulator also markedly reduced FAK phosphorylation at Y925 in HAoSMCs. Subsequently, the phosphorylation of MEK1/2 and ERK1/2 in the MAPK pathway was significantly decreased in talin modulator-treated cells after 4 hours compared to that in the negative and vehicle controls (Figure 2G), due to decreased FAK phosphorylation at Y925. In addition, a significant reduction in AKT phosphorylation was also observed when HAoSMCs were treated with the talin modulator (Figure 2H). Taken together, these findings suggest that the talin modulator inhibits cell proliferation and migration of HAoSMCs possibly via suppression of focal adhesion molecules and downstream signaling pathways of talin.

To investigate the reversibility of downstream molecules that were suppressed upon treatment with the talin modulator in HAoSMCs, cells were treated with the talin modulator and then the culture medium was replaced with fresh medium. As shown in Figure 3A, the number of viable and attached cells was decreased after treatment with the talin modulator; however, the proliferative capacity of the cells was restored with no morphological changes after 72 hours of wash out of the talin modulator. Furthermore, the auto-phosphorylation of FAK at Y397 was completely reversed at 4 hours after removal of the talin modulator. The reversible effect of the talin modulator at 4 hours of treatment significantly restored the downregulated phosphorylated levels of MEK1/2, ERK1/2, and AKT in HAoSMCs (Figure 3B and C). These reversible effects are due to the reversible binding of the talin modulator to the talin protein as shown in Figure 1B.

ApoE KO mice were subjected to left femoral arterial injury to induce abnormal proliferation and migration of SMCs. The mice were daily administered with the talin modulator or vehicle control for 28 days. There was no significant change in body weight among groups throughout the duration of the experiment (data not shown). The blood flow on the ventral side of mice was examined using laser Doppler imager post-surgery (day 0), and at day 7, 14, and 28 (Figure 4A). At 28 days, oral administration of the talin modulator significantly improved the blood flow compared to that in the sham and vehicle control groups (Figure 4B). Moreover, the wire injury induced increased neointimal formation in the left femoral arteries of the sham group compared to right arteries. Interestingly, mice that had been administered the talin modulator exhibited markedly reduced neointima in femoral arteries compared to that in the sham and vehicle control groups (Figure 4C). Decreased expression of α-SMA-positive SMCs was observed by administration of the talin modulator compared to that in the sham and vehicle control groups (Figure 4D). In addition, at 28 days after administration of the talin modulator changes in the expression of proinflammatory cytokines including IL-6 and TNF-α were observed. Talin modulator-administered mice exhibited significantly lower serum levels of IL-6 and TNF-α than the sham and vehicle control groups (Figure 4E).