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. 2022 Nov 30;8(2):155-170.
doi: 10.1016/j.jacbts.2022.07.012. eCollection 2023 Feb.

TCF7L1 Accelerates Smooth Muscle Cell Phenotypic Switching and Aggravates Abdominal Aortic Aneurysms

Jing Wang  1 , Xiaoxiang Tian  1 , Chenghui Yan  1 , Hanlin Wu  1 , Yuxin Bu  1 , Jia Li  1 , Dan Liu  1 , Yaling Han  1
Affiliations

TCF7L1 Accelerates Smooth Muscle Cell Phenotypic Switching and Aggravates Abdominal Aortic Aneurysms

Jing Wang et al. JACC Basic Transl Sci. .

Abstract

Phenotypic switching of vascular smooth muscle cells is a central process in abdominal aortic aneurysm (AAA) pathology. We found that knockdown TCF7L1 (transcription factor 7-like 1), a member of the TCF/LEF (T cell factor/lymphoid enhancer factor) family of transcription factors, inhibits vascular smooth muscle cell differentiation. This study hints at potential interventions to maintain a normal, differentiated smooth muscle cell state, thereby eliminating the pathogenesis of AAA. In addition, our study provides insights into the potential use of TCF7L1 as a biomarker for AAA.

Keywords: AAA, abdominal aortic aneurysm; AAV, adeno-associated virus; Ang II, angiotensin II; CVF, collagen volume fraction; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SM22α, smooth muscle protein 22-α; SMA, smooth muscle actin; SRF, serum response factor; TCF7L1; TCF7L1, transcription factor 7-like 1; VSMC, vascular smooth muscle cell; abdominal aortic aneurysms; cDNA, complementary DNA; mRNA, messenger RNA; phenotypic switching; siRNA, small interfering RNA; smooth muscle cell.

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Conflict of interest statement

This work was supported by the National Natural Science Foundation of China (81870553, 82070308, 82070300, and 82070875), the Excellent Youth Fund of Liaoning Natural Science Foundation (2021-YQ-03), and the Shenyang Science and Technology Project (20-205-4-001). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

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Graphical abstract
Figure 1
Figure 1
TCF7L1 Is Upregulated in Ang II–Induced AAAs In Vivo and During Phenotypic Switching Induced by Ang II In Vitro (A) Real-time polymerase chain reaction of transcription factor 7-like 1 (TCF7L1), α-smooth muscle actin (SMA), and smooth muscle protein 22-α (SM22α) messenger RNA (mRNA) expression in the aortas of abdominal aortic aneurysms (AAAs) induced by angiotensin (Ang) II (n = 3). (B, C) Western blotting of TCF7L1, αSMA, SM22α, and matrix metalloproteinase (MMP)-2 protein expression in in the aortas of AAAs induced by Ang II (n = 3). (D, E) Immunohistochemical staining of TCF7L1, αSMA, and SM22α in aortic vascular smooth muscle cells (VSMCs) (n = 3). (F) Real-time polymerase chain reaction for TCF7L1, αSMA, and SM22α in Ang II–induced VSMCs (n = 3). (G, H) Western blotting of TCF7L1, αSMA, SM22α, and MMP-2 protein in Ang II–induced VSMCs (n = 3). (I) Immunofluorescence staining of TCF7L1 (green), αSMA (green), SM22α (green), and DAPI (blue) in VSMCs (n = 3). Data are presented as mean ± SEM. ∗∗∗P < 0.01 vs the saline/phosphate-buffered saline (PBS) group.
Figure 2
Figure 2
TCF7L1 Deficiency Prevents Ang II–Induced AAAs Formation in ApoE-/- Mice (A) Representative images showing the macroscopic features of aortic aneurysms at 28 days after Ang II infusion in male ApoE-/- mice. (B) The incidence of Ang II–induced AAAs in ApoE-/- mice with TCF7L1 deficiency (n = 10). (C, D) Representative ultrasound images showing the abdominal aortas and the maximal diameter of the abdominal aorta in ApoE-/- mice with TCF7L1 deficiency. (E, F) Representative hematoxylin and eosin (HE), Masson trichrome (collagen), and Van Gieson (elastin) (EVG) staining and representative immunohistochemistry for TCF7L1, αSMA, and SM22α in the aortas of mice with TCF7L1 deficiency. (G, H) Western blotting of aortic MMP-2, αSMA, and SM22α in ApoE-/- mice with TCF7L1 deficiency (n = 3). Data are presented as mean ± SEM. ∗∗∗P < 0.01, ∗∗P < 0.05 vs AAV-sh-RNA-saline group. ##P < 0.05 vs AAV-sh-RNA-Ang II group. Abbreviations as in Figure 1.
Figure 3
Figure 3
TCF7L1 Overexpression Promotes AAA Formation in Ang II–Infused ApoE-/- Mice (A) Representative images showing the macroscopic features of aortic aneurysms at 28 days after Ang II infusion in male ApoE-/- mice with TCF7L1 overexpression. (B) The incidence of Ang II–induced AAAs in ApoE-/- mice with TCF7L1 overexpression. n = 5 in the AAV-cmv-GFP or AAV-cmv-TCF7L1 groups (mice treated with saline). n = 15 in the AAV-cmv-GFP groups or AAV-cmv-TCF7L1 (mice treated with Ang II). (C, D) Representative ultrasound images showing the abdominal aortas and the maximal diameter of the abdominal aorta in ApoE-/- mice with TCF7L1 overexpression. (E, F) Representative HE, Masson trichrome (collagen), and EVG staining and representative immunohistochemistry for TCF7L1, αSMA, and SM22α in the aortas of mice with TCF7L1 overexpression. (G, H) Western blotting of aortic MMP-2, αSMA, and SM22α in ApoE-/- mice with TCF7L1 overexpression (n = 3). Data are presented as mean ± SEM. ∗∗∗P <0.01, ∗∗P <0.05 vs AAV-cmv-GFP-saline group. ##P < 0.05 vs AAV-cmv-GFP-Ang II group. Abbreviations as in Figures 1 and 2.
Figure 4
Figure 4
TCF7L1 Knockdown Inhibits VSMC Phenotypic Transformation and Migration, While TCF7L1 Overexpression Promotes VSMC Phenotypic Transformation and Migration (A, B) Western blotting of MMP-2, αSMA, and SM22α in VSMCs with TCF7L1 knockdown (n = 3). (C) Real-time polymerase chain reaction of TCF7L1, αSMA, and SM22αin VSMCs with TCF7L1 knockdown (n = 3). (D, E) Transwell assays were used to assess cell migration. (F, G) Western blotting of MMP-2, αSMA, and SM22α in VSMCs with TCF7L1 overexpression (n = 3). (H) Real-time polymerase chain reaction of TCF7L1, αSMA, and SM22α in VSMCs with TCF7L1 overexpression (n = 3). (I, J) Transwell assays were used to assess cell migration. Data are presented as mean ± SEM. ∗∗∗P < 0.01, ∗∗P < 0.05 vs the siControl or the pcDNA3.1 FLAG group; ##P < 0.05 vs the siControl+Ang II or pcDNA3.1 FLAG+Ang II group; &&P < 0.05 vs siTCF7L1 or pcDNA3.1TCF7L1 group. HPF = high-power field; other abbreviations as in Figures 1 and 2.
Figure 5
Figure 5
TCF7L1 Inhibits SRF Expression and Activity In Vivo and In Vitro (A to D) Western blotting of serum response factor (SRF) protein in aortas from Ang II–infused AAAs (n = 3). Male ApoE-/- mice were transfected with AAV-cmv-GFP, AAV-cmv-TCF7L1, AAV-sh-RNA, or AAV-sh-TCF7L1 followed by treated with Ang II for 28 days (n = 3). (E, F) Western blotting of SRF protein in VSMCs. VSMCs were transfected with pcDNA3.1 FLAG or pcDNA3.1 TCF7L1 for 24 hours and treated with Ang II (1 μM) or vehicle for 24 hours. (G) Real-time polymerase chain reaction of SRF mRNA in VSMCs with TCF7L1 overexpression and Ang II (1 μM) stimulation. (H, I) Western blotting of SRF protein in VSMCs. VSMCs were transfected with siControl or siTCF7L1 for 24 hours and treated with Ang II (1 μM) or vehicle for 24 hours. (J) Real-time polymerase chain reaction of SRF mRNA in VSMCs with TCF7L1 knockdown. (K) Putative TCF7L1 binding sites in the promoter of SRF. TCF7L1 overexpression suppressed the activity of the wild-type SRF promoter. Values are mean ±SEM (n = 3). ∗∗∗P <0.01, ∗∗P <0.05 vs the AAV-cmv-GFP /AAV-sh-RNA/siControl/pcDNA3.1 FLAG group. ##P < 0.01 vs AAV-cmv-GFP-Ang II/AAV-sh-RNA-Ang II/siControl-Ang II/pcDNA3.1 FLAG-Ang II group. LUC = luciferase; PBS = phosphate-buffered saline; other abbreviations as in Figures 1 and 2.
Figure 6
Figure 6
Knocking Down SRF Weakens the Effect of TCF7L1 Overexpression on VSMCs Phenotypic Switching and Migration (A, B) Western blotting of the relative protein expression of TCF7L, αSMA, SM22α, SRF, and MMP-2 in VSMCs with SRF or TCF7L1 knockdown (n = 3). (C) Real-time polymerase chain reaction of TCF7L1, αSMA, SM22α, and SRF mRNA in VSMCs with SRF or TCF7L1 knockdown (n = 3). (D, E) Transwell assays of VSMCs were used to assess cell migration (n = 3). (F, G) Western blotting of the relative protein expression of TCF7L, αSMA, SM22α, SRF, and MMP-2 in VSMCs with SRF or TCF7L1 overexpression (n = 3). (H) Real-time polymerase chain reaction of TCF7L1, αSMA, SM22α, and SRF mRNA in VSMCs with SRF or TCF7L1 overexpression (n = 3). (I, J) Transwell assays of VSMCs were used to assess cell migration. (K) Schematic illustration of the proposed mechanism of TCF7L1 in the formation of AAAs. Data are presented as mean ± SEM. ∗∗∗P < 0.01, ∗∗P <0.05 vs the siControl or pcDNA3.1FLAG groups. ##P < 0.05 vs the siTCF7L1 or pcDNA3.1 TCF7L1 groups. &&P < 0.05 vs the siSRF or pcDNA3.1 SRF group. Abbreviations as in Figures 1, 2, 4, and 5.
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