Korean Circ J.  2020 Jul;50(7):613-624. 10.4070/kcj.2019.0421.

Inhibition of Smooth Muscle Cell Proliferation and Migration by a Talin Modulator Attenuates Neointimal Formation after Femoral Arterial Injury

• Lim IR
• Kim C
• Jung JW
• Kim JH
• Hong SJ

• KMID: 2504221
• DOI: http://doi.org/10.4070/kcj.2019.0421

Abstract

Background and Objectives
Vascular smooth muscle cell (SMC) proliferation and migration play a critical role in neointimal formation. Focal adhesion is involved in cell proliferation and migration, and talin is known to be a key regulator of these processes. We synthesized a new talin modulator that binds to the talin protein, and investigated its effects on SMCs and neointimal formation after vascular injury.
Methods
Human aortic SMCs (HAoSMCs) were treated with a newly synthesized talin modulator. Apolipoprotein E knockout (ApoE KO) mice were subjected to left femoral arterial injury and orally administered with the talin modulator daily. Laser Doppler imager was used to compare the blood flow, and injured femoral arteries and blood serum were analyzed after 28 days.
Results
The talin modulator significantly inhibited cell proliferation in a concentration-dependent manner and suppressed the migration of HAoSMCs. Treatment with a talin modulator resulted in a significant reduction in the phosphorylation of focal adhesion molecules and downstream signaling molecules related to cell proliferation and migration. The effects of the talin modulator in HAoSMCs were found to be reversible, as evidenced by the reactivation of signaling pathways upon its removal. After 28 days of administration of the talin modulator, an improvement in the blood flow and reduction in neointimal formation in the injured femoral arteries were observed.
Conclusions
We demonstrated the inhibitory effects of a talin modulator on SMC proliferation and migration, and that were associated with downregulation of signaling pathways, resulting in the attenuation of neointimal formation in ApoE KO mice.

Keyword

Talin; Focal adhesion; Vascular smooth muscle cell; Vascular injury; Neointima formation

Figure

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  Figure 1 Inhibition of proliferation and migration of HAoSMCs by a talin modulator. (A) Chemical structure of the newly synthesized talin modulator. (B) SPR analysis of the talin modulator showing the interaction with a talin protein. (C) Concentration-dependent decrease in cell proliferation by treating HAoSMCs with the talin modulator. The experimental concentration of the talin modulator was set as 50 μM. (D) Phase-contrast images after treatment of HAoSMCs with the talin modulator for 24 hours. (E, F) Immunofluorescence images and quantification of PHH3-positive cells (white arrowheads) in HAoSMCs. (G, H) Wound healing migration assay to evaluate the effects of the talin modulator on cell migration. Cell migration was assessed by quantifying the area covered by migrated cells across the wounded line. All scale bars represent 100 μm.DAPI = 4′,6-diamidino-2-phenylindole; HAoSMC = human aortic smooth muscle cell; PHH3 = phosphohistone H3; SPR = surface plasmon resonance.*p<0.05 vs. negative control at each time point; †p<0.05 vs. vehicle control at each concentration or each time point. All experiments were repeated at least 3 times independently.

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  Figure 2 Effects of a talin modulator on focal adhesion molecules and related signaling pathways. (A) Western blot analyses of the expression of focal adhesion-associated molecules including talin (B), vinculin (C), and paxillin (D) in HAoSMCs after 24 hours of treatment with the talin modulator. All band intensities were normalized to GAPDH, and the phosphorylated forms were then normalized to the total form of the respective forms. (E) Western blot analyses of downstream signaling pathways indicating decreased phosphorylation of FAK at Y397 and Y925 (F), MEK1/2, ERK1/2 (G), and AKT (H) by treating HAoSMCs with the talin modulator for 4 hours. All band intensities were normalized to GAPDH, and the phosphorylated forms were then normalized to the total form of the respective forms.AKT = protein kinase B; ERK1/2 = extracellular signal-regulated kinase 1/2; FAK = focal adhesion kinase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HAoSMC = human aortic smooth muscle cell; MEK1/2 = mitogen-activated protein kinase kinase 1/2; NS = not significant.*p<0.05 vs. negative control; †p<0.05 vs. vehicle control. All experiments were repeated at least 3 times independently.

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  Figure 3 Reversible effects of a talin modulator. (A) HAoSMCs were treated with a talin modulator for 24 hours and then cultured in fresh media for 24, 48, and 72 hours. Scale bars represent 100 μm. (B, C) Western blot analyses of FAK, MEK1/2, ERK1/2, and AKT phosphorylation after treatment, and at 2 and 4 hours after wash out of the talin modulator. All band intensities were normalized to GAPDH, and the phosphorylated forms were then normalized to the total form of the respective forms.AKT = protein kinase B; ERK1/2 = extracellular signal-regulated kinase 1/2; FAK = focal adhesion kinase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HAoSMC = human aortic smooth muscle cell; MEK1/2 = mitogen-activated protein kinase kinase 1/2.*p<0.05 vs. treatment. All experiments were repeated at least 3 times independently.

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  Figure 4 Improvement of the blood flow and suppression of neointimal formation by a talin modulator after femoral arterial injury. (A) Representative images of the ventral side of a mouse measured by laser Doppler imager post-surgery (day 0) and at day 7, 14, and 28 after femoral arterial injury. (B) Quantitative analysis of the blood flow at day 28 after injury and oral administration of the talin modulator. (C) Histological images of left and right femoral arteries showing the reduced neointima after administration of the talin modulator. (D) Immunohistological staining of α-SMA (DAB, brown) to visualize SMCs within the neointima. (E) Serum levels of inflammatory cytokines such as IL-6 and TNF-α after 28 days of injury and administration as measured by ELISA. All scale bars represent 100 μm.α-SMA = alpha-smooth muscle actin; DAB = 3,3′-diaminobenzidine; ELISA = enzyme-linked immunosorbent assay; H&E = hematoxylin and eosin; IL-6 = interleukin-6; SMC = smooth muscle cell; TNF-α = tumor necrosis factor alpha.*p<0.05 vs. sham; †p<0.05 vs. vehicle control. Sham (n=5); vehicle control (n=5); talin modulator (n=6).

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