Abstract

   Atherosclerotic plaque rupture mainly contributes to acute coronary
   syndrome (ACS). Insufficient repair of these plaques leads to
   thrombosis and subsequent ACS. Central to this process is the
   modulation of vascular smooth muscle cells (VSMCs) phenotypes,
   emphasizing their pivotal role in atherosclerotic plaque stability and
   healing post‐disruption. Here, an expansion of FSP1^+ cells in a tandem
   stenosis (TS) model of atherosclerotic mice is unveiled, predominantly
   originating from VSMCs through single‐cell RNA sequencing (scRNA‐seq)
   analyses and VSMC lineage tracing studies. Further investigation
   identified TEA domain transcription factor 1 (TEAD1) as the key
   transcription factor driving the trans‐differentiation of VSMCs into
   fibroblast‐like cells. In vivo experiments using a TS model of plaque
   rupture demonstrated that TEAD1 played a crucial role in promoting
   plaque stability and healing post‐rupture through pharmacological or
   TEAD1‐AAV treatments. Mechanistically, it is found that TEAD1 promoted
   the expression of fibroblast markers through the Wnt4/β‐Catenin
   pathway, facilitating the trans‐differentiation. Thus, this study
   illustrated that TEAD1 played a critical role in promoting the
   trans‐differentiation of VSMCs into fibroblast‐like cells and
   subsequent extracellular matrix production through the Wnt4/β‐Catenin
   pathway. Consequently, this process enhanced the healing mechanisms
   following plaque rupture, elucidating potential therapeutic avenues for
   managing atherosclerotic instability.

   Keywords: ACS, atherosclerotic plaque, plaque rupture, TEAD1,
   thrombosis
     __________________________________________________________________

   This study investigates the crucial role of VSMC trans‐differentiation
   into fibroblast‐like cells in stabilizing atherosclerotic plaques after
   rupture. It identifies TEAD1 as a key regulator of this process through
   the Wnt4/β‐Catenin signaling pathway, highlighting novel therapeutic
   targets for enhancing plaque healing and stability amidst inflammatory
   challenges. Discover how these insights can transform atherosclerosis
   treatment.

   graphic file with name ADVS-12-2407408-g005.jpg

1. Introduction

   Vulnerable plaques, with their hallmark thin fibrous caps and necrotic
   lipid cores, predispose individuals to thrombosis and acute coronary
   syndrome (ACS) when they rupture or erode.^[ [44]^1 , [45]^2 , [46]^3
   ^] Nonetheless, advancements in intravascular imaging technologies,
   such as intravascular ultrasound (IVUS) and optical coherence
   tomography (OCT), have revealed that a reparative process often
   coincides with the rupture of these plaques, particularly when the
   associated thrombi are small.^[ [47]^4 , [48]^5 , [49]^6 , [50]^7 ,
   [51]^8 , [52]^9 ^] This repair response may help counteract the
   destabilization of the plaques, supported by clinical evidence that
   some patients with vulnerable plaques exhibit resistance to developing
   ACS.^[ [53]^10 , [54]^11 ^] However, the balance is precarious: a shift
   toward larger thrombotic events and significant myocardial infarction
   can abruptly disrupt this reparative process, culminating in the onset
   of ACS.^[ [55]^12 ^] Therefore, timely and effective repair of ruptured
   plaques is essential to reduce the risk of ACS; yet the underlying
   mechanisms of these reparative processes are not well elucidated.

   In healthy blood vessels, vascular smooth muscle cells (VSMCs) maintain
   a stable, contractile state that is essential for normal vessel
   function. However, in response to injury, aging, or conditions such as
   atherosclerosis, VSMCs lose their contractile characteristics and
   undergo de‐differentiation. During this process, Shankman et al. found
   that Kruppel‐like factor 4 (KLF4) acted as a molecular fate switch,
   promoting the de‐differentiation of contractile VSMCs.^[ [56]^13 ^]
   Specifically, KLF4 repressed the expression of Myocardin, providing a
   transcriptional mechanism for the fate‐switching of contractile VSMCs
   to a de‐differentiated state.^[ [57]^14 , [58]^15 ^] These changes
   resulted in the cells adopting a synthetic phenotype, characterized by
   increased proliferation and migration ability.^[ [59]^16 ^] Due to
   their remarkable phenotypic plasticity, VSMCs could adopt various
   cellular phenotypes, including those resembling foam cells,
   macrophages, mesenchymal stem cells, fibroblasts, and osteochondrogenic
   cells.^[ [60]^17 , [61]^18 , [62]^19 ^] This adaptability could
   influence the trajectory of plaque development and stabilization in
   both beneficial and detrimental ways. For instance, our previous study
   demonstrated that the deletion of peptidyl arginine deiminase 4 (PAD4)
   increased the proportion of fibroblast‐like cells derived from VSMCs.^[
   [63]^17 ^] Additionally, VSMCs could trans‐differentiate into
   inflammatory macrophage‐like cells and calcified osteochondrogenic
   cells, contributing to the progression of vascular diseases.^[ [64]^20
   , [65]^21 ^] Qing Zhu et al. found that the carotid artery balloon
   injury model induced VSMCs de‐differentiation and neointima formation
   in rats.^[ [66]^22 ^] Sun‐Hwa Song et al. also provided evidence of
   VSMCs de‐differentiation in injured human atherosclerotic plaque.^[
   [67]^23 ^]

   However, it remains unclear whether de‐differentiated VSMCs can
   trans‐differentiate into fibroblast‐like cells that contribute to the
   repair of injured plaques or vessels. Additionally, the extent to which
   these fibroblast‐like cells facilitate the repair of disrupted plaques,
   as well as the signaling pathways regulating the trans‐differentiation
   of VSMCs into these cells, require further investigation.

   To address this question, we utilized VSMCs lineage tracing mice,
   previously reported in our studies, and subjected them to a model of
   spontaneous plaque rupture.^[ [68]^17 ^] By integrating histology,
   single‐cell RNA sequencing (scRNA‐seq), and molecular analyses, we
   identified that TEAD1 could promote the trans‐differentiation of VSMCs
   into fibroblast‐like cells through the Wnt family member 4
   (Wnt4)/β‐Catenin pathway, thereby promoting the healing process after
   plaque rupture.

2. Results

2.1. FSP1^+ Cells were Expanded Along with Atherosclerosis Progression

   To assess the involvement of fibroblast‐like cells in arterial
   remodeling, we first examined the fibroblast populations from the
   scRNA‐seq datasets of atherosclerotic plaques ([69]GSE197073)^[ [70]^17
   ^] and ligated carotid arteries ([71]GSE174098).^[ [72]^24 ^] Our
   analysis revealed that cells expressing fibroblast markers Fibroblast
   Specific Protein 1 (FSP1), Collagen Type III Alpha 1 Chain (COL3A1)
   were abundant during the process of vascular injury (Figure [73] 1A,B).
   Furthermore, we stained FSP1 in plaques to distinguish their
   distribution and enrichment patterns during the development of
   atherosclerosis. Intriguingly, we observed an increased in FSP1^+
   (Fibroblast Specific Protein 1 positive) cells as plaque progression
   occurred, particularly in the fibrous cap region, which was identified
   using Masson staining (Figure [74]1C,D; Figure [75]S1A, Supporting
   Information). Next, we analyzed carotid plaques from patients
   undergoing endarterectomy ([76]GSE21545)^[ [77]^25 ^] and found that
   higher FSP1 expression within atherosclerotic plaque correlated with a
   better prognosis (Figure [78]S1B, Supporting Information). These data
   indicated that the expansion of fibroblasts within advanced plaques
   might contribute to plaque stabilization.

Figure 1.

   Figure 1
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   FSP1^+ cells were expanded along with atherosclerosis progression. A,B)
   Uniform manifold approximation and projection (UMAP) visualization and
   violinplot of single cell RNA sequencing (scRNA‐seq) data from
   atherosclerotic plaques ([80]GSE197073) and ligated carotid arteries
   ([81]GSE174098). C) Immunofluorescence staining (IF) of FSP1 was
   performed on plaques from brachiocephalic artery of mice fed an HFD for
   10, 15, and 20 weeks. The white dot line showed the plaque area, the
   white star represented lumen area. Scale bar = 100 µm. D)
   Quantification the percentage of FSP1^+ cells/total cells in C (n = 5
   per group). For all panels, error bars represented standard error of
   the mean (SE). P‐value was determined by one‐way ANOVA with Bonferroni
   post‐test (D). ns no significance, *P < 0.05, **P < 0.01, ***P < 0.001,
   and ****P < 0.0001.

2.2. VSMCs were the Primary Source of FSP1^+ Cells in Atherosclerotic Plaques

   Previous studies suggested that VSMCs, endothelial cells, and resident
   fibroblasts could give rise to FSP1^+ cells within atherosclerotic
   plaques.^[ [82]^26 , [83]^27 , [84]^28 ^] However, the specific cell
   type that predominantly contributes to this population remains unknown.
   Given that VSMCs are the most abundant cell type within arteries and
   exhibit remarkable phenotypic plasticity during atherogenesis. We
   hypothesized that they might account for the majority of FSP1^+ cells
   in atherosclerotic plaques.^[ [85]^29 ^] To test this hypothesis, we
   subjected VSMCs lineage tracing (Myh11^Cre B6G/R Ldlr^−/− ) mice to a
   high‐fat diet (HFD) and monitored the changes in FSP1^+tdTomato^+ cells
   within atherosclerotic plaques over 20 weeks. FSP1 staining
   demonstrated a progressive increase in the number of FSP1^+tdTomato^+
   cells as plaque developed, with a primary localization within the
   fibrous cap region (Figure [86] 2A,B). Next, analysis of our previous
   scRNA‐seq dataset also confirmed that VSMCs were the predominant cell
   type capable of giving rise to fibroblast‐like cells in advanced
   plaques (Figure [87]2C,D). We next sought to explore whether
   FSP1^+tdTomato^+ cells were associated with plaque stability. The
   plaques of VSMCs lineage tracing mice were categorized into unstable
   (high vulnerability index) and stable (low vulnerability index) groups
   based on the median of vulnerability index, as previously described.^[
   [88]^30 , [89]^31 ^] Briefly, the vulnerability index was calculated as
   a ratio of the sum of lipids (Oil red O, % plaque area), macrophages
   (CD68, % plaque area), and intra‐plaque hemorrhage (TER119, % plaque
   area) to the sum of smooth muscle cells (tdTomato^+, % plaque area) and
   collagen (Masson Staining, % plaque area) (Figure [90] 3E–H). We then
   compared the percentage of VSMCs‐derived FSP1^+ cells
   (FSP1^+tdTomato^+). We found that the percentage was decreased in
   plaques with a high vulnerability index compared to those with a low
   vulnerability index (Figure [91]3I–K). However, VSMCs could hardly
   trans‐differentiate into FSP1^+ cells in the carotid arteries at 14‐
   and 21‐days post‐ligation (Figure [92]S2A,B, Supporting Information).

Figure 2.

   Figure 2
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   VSMCs were the primary source of FSP1^+ cells in atherosclerotic
   plaques. A) IF staining of FSP1 on plaques from brachiocephalic artery
   of Myh11^Cre B6G/R Ldlr^−/− mice fed an HFD for 10, 15, and 20 weeks.
   Scale bar = 100 µm. B) (Left) Quantification of the percentage of
   plaque area % (n = 5 per group). (Right) Quantification of the number
   of tdTomato^+FSP1^+ cells within the plaques harvested from three‐time
   points HFD‐fed Myh11^Cre B6G/R Ldlr^−/− mice (n = 5 per group). C) UMAP
   visualization of our previous scRNA‐seq data. D) Featureplot of FSP1
   (S100A4) on zsGreen^+ fibroblasts and tdTomato^+ fibroblasts from C. E)
   Representative images of CD68 on stable and unstable aortic root
   plaques. Scale bar = 100 µm. F) Representative images of Oil Red O
   staining on stable and unstable aortic root plaques. Scale bar =
   100 µm. G) Representative images of TER119 on stable and unstable
   aortic root plaques. Scale bar = 100 µm. H) Representative images of
   Masson staining on stable and unstable aortic root plaques. Scale bar =
   100 µm. I) Representative images of tdTomato^+FSP1^+ area on stable and
   unstable aortic root plaques. Scale bar = 100 µm. J) the quantification
   of vulnerability index between unstable (high index) and stable (low
   index) groups (n = 8 per group). K) the quantification of the
   percentage of tdTomato^+ FSP1^+ cells within plaque area between
   unstable (high index) and stable (low index) group (n = 8 per group).
   For all panels, error bars represented SE. P‐value was determined by
   unpaired two‐tailed Student's t‐test (J and K) or one‐way ANOVA with
   Bonferroni post‐test (B). ns no significance, *P < 0.05, **P < 0.01,
   ***P < 0.001, and ****P < 0.0001.

Figure 3.

   Figure 3
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   VSMCs‐derived FSP1^+ fibroblast‐like cells contributed to healing after
   plaque rupture. A) Representative images of TER119 IF staining on
   brachiocephalic artery plaques harvested from Myh11^Cre B6G/R Ldlr^−/−
   mice treated with either injury or sham surgery. Scale bar = 50 µm. B)
   Quantification of the percentage of TER119^+ area/plaque area in A
   (n = 5 per group). C) Representative images of FSP1 IF staining on
   brachiocephalic artery plaques harvested from Myh11^Cre B6G/R Ldlr^−/−
   mice treated with either injury or sham surgery. Scale bar = 50 µm. D)
   Quantification of the percentage of tdTomato^+FSP1^+ cells/tdTomato^+
   cells in C (n = 5 per group). E) Representative images of MMP9 IF
   staining on brachiocephalic artery plaques harvested from Myh11^Cre
   B6G/R Ldlr^−/− mice treated with either injury or sham surgery. Scale
   bar = 50 µm. F) Quantification of the percentage of MMP9^+ area/plaque
   area in E (n = 5 per group). G) Representative images of CD68 IF
   staining on brachiocephalic artery plaques harvested from Myh11^Cre
   B6G/R Ldlr^−/− mice treated with either injury or sham surgery. Scale
   bar = 50 µm. H) Quantification of the percentage of CD68^+ area/plaque
   area in G (n = 5 per group). I) Quantification of the percentage of
   plaque area % between injury and sham group. For all panels, error bars
   represented SE. P‐value was determined by unpaired two‐tailed Student's
   t‐test (B, D, F, H, and I). ns no significance, *P < 0.05, **P < 0.01,
   ***P < 0.001, and ****P < 0.0001.

   Next, we conducted a comparative analysis of VSMCs‐derived FSP1^+ cells
   and fibromyocytes as characterized in Wirka's research.^[ [95]^27 ^]
   Utilizing scRNA‐seq from tdTomato^+ VSMCs, we found that fibromyocytes
   exhibited higher expression of inflammatory markers, including Tumor
   Necrosis Factor Receptor Superfamily 11B（TNFRSF11B), Secreted
   Phosphorylation Protein 1 (SPP1), Tissue Inhibitor of Metallopeptidase
   1 (TIMP1), and Lumican (LUM), compared to FSP1^+ tdTomato^+ cells.
   Despite this difference, both cell types could express fibroblast‐like
   markers such as Fibronectin 1 (FN1), and COL3A1 (Figure [96]S2C–G,
   Supporting Information). In vitro investigations further confirmed that
   VSMCs‐derived FSP1^+ cells induced by FGF21 showed low expression
   levels of LUM, TIMP1, and TNFRSF11b, while maintaining high levels of
   FN1, COL3A1, and FSP1 (Figure [97]S2H, Supporting Information).
   Ingenuity Pathway Analysis (IPA) indicated that fibromyocytes might be
   linked to the secretion of inflammatory cytokines, whereas the genes
   activated in VSMCs‐derived FSP1^+ cells were predominantly associated
   with signaling pathways related to plaque stability, particularly those
   involved in collagen secreting and ECM‐receptor interaction (Figure
   [98]S2I, Supporting Information). So, we named this cell type as
   VSMCs‐derived FSP1^+ fibroblast‐like cells. Furthermore, our
   investigation into their distribution during atheroprogression revealed
   that VSMCs‐derived FSP1^+ fibroblast‐like cells (FSP1^+tdTomato^+
   cells) were significantly more abundant than fibromyocytes
   (LUM^+tdTomato^+ cells) in atherosclerotic plaques of mice fed an High
   Fat Diets (HFD) for 16 weeks (Figure [99]S3A,B, Supporting
   Information). Notably, VSMCs‐derived FSP1^+ fibroblast‐like cells were
   predominantly located on the plaque surface, contrasting with the
   distribution of fibromyocytes (Figure [100]S3A,C, Supporting
   Information). Additionally, we compared VSMCs‐derived FSP1^+
   fibroblast‐like cells with VSMC‐derived GLI1^+ (GLI‐Kruppel family
   member GLI1 positive) adventitial progenitor cells recently
   (AdvSca1‐SMCs) described by Dubner.^[ [101]^32 ^] Our data showed a
   significantly lower number of AdvSca1‐SMCs, which were mainly localized
   within or at the base of atherosclerotic plaques, differing from the
   distribution pattern of VSMCs‐derived FSP1^+ fibroblast‐like cells
   (Figure [102]S3D–F, Supporting Information). This suggested that
   AdvSca1‐SMCs might not have a significant role in plaque stabilization
   and fibrous cap development. Taken together, our results indicated
   VSMCs served as the primary source of FSP1^+ cells in atherosclerotic
   plaques, implicating these cells’ function in stabilizing
   atherosclerotic plaques.

2.3. VSMC‐Derived FSP1^+ Fibroblast‐Like Cells Contributed to Healing after
Plaque Rupture

   Proteoglycans and type III collagen fibers are essential for the
   healing of plaques following rupture.^[ [103]^33 ^] Given that
   FSP1^+tdTomato^+ cells showed upregulation of the pathways related to
   collagen secretion (Figure [104]S2I, Supporting Information), we
   hypothesized that these cells might be involved in the repair process
   after plaque rupture. To test this hypothesis, we simulated plaque
   rupture in ACS by externally clamping the plaques in the
   brachiocephalic artery of VSMC lineage tracing mice. Structural damage
   and increased TER119 staining in injured plaques, compared with sham
   plaques, provided evidence for the success of our plaque injury model
   (Figure [105]3A,B). Intriguingly, we observed a significant increase in
   FSP1^+tdTomato^+ cells within the injured plaques relative to the
   uninjured plaques (Figure [106]3C,D). Although the injury resulted in
   lager plaques with heightened inflammatory features, as indicated by
   elevated MMP9^+ (Matrix Metalloproteinase‐9 positive) and CD68^+
   (Cluster of Differentiation 68 positive) areas (Figure [107]3E–I), the
   proportion of FSP1^+tdTomato^+ cells also increased significantly,
   indicating that plaque rupture coexisted with cellular repair
   processes. Collectively, these findings implied that VSMCs‐derived
   FSP1^+ fibroblast‐like cells might contribute to healing after plaque
   rupture.

2.4. TEAD1 Promoted the Trans‐Differentiation of VSMCs into Fibroblast‐Like
Cells and the Production of Extracellular Matrix

   After challenging rat aortic smooth muscle cells (RASMCs) with
   different stimulants,^[ [108]^34 , [109]^35 , [110]^36 , [111]^37 ^] we
   found that Fibroblast Growth Factor 21 (FGF21) had the strongest effect
   on inducing the expression of FSP1 (Figure [112] 4A,B). FGF21 treatment
   also upregulated the expression of FSP1, FN1, and COL3A1 in RASMCs
   (Figure [113]4C–E). Moreover, FGF21 induced a dramatic morphological
   shift in RASMCs, transitioning from the flattened, typical contractile
   phenotype of VSMCs to a spindle‐like, fibroblast‐like phenotype, along
   with increased FSP1 protein level (Figure [114]4F). Analysis of
   critical transcription factors involved in the trans‐differentiation of
   VSMCs into fibroblast‐like cells through Single‐cell Regulatory Network
   Inference and Clustering (SCENIC) analysis revealed TEAD1 expression in
   VSMCs‐derived fibroblast‐like cells was higher than other cell types
   (Figure [115]4G). Following FGF21 treatment, the messenger RNA (mRNA)
   expression of both TEAD1 and FSP1 was sharply upregulated
   (Figure [116]4H). Knockdown of TEAD1 via specific small interfering RNA
   (siRNA) in the presence of FGF21 stimulation resulted in a reduction of
   FSP1 protein expression by over 70% (Figure [117]4I). Furthermore,
   using adjacent series sections of plaques for TEAD1 and FSP1 staining,
   we noted that TEAD1 was localized in the same region of the fibrous cap
   where VSMCs‐derived FSP1^+ cells were detected (Figure [118]4J,K). In
   human RNA‐sequencing (RNA‐seq) data from [119]GSE21545, we also
   identified a positive correlation between TEAD1 and fibroblast markers
   (FSP1, FN1, and COL3A1). Conversely, TEAD1 expression was negatively
   associated with plaque instability‐related genes (MMP9 and Nod‐like
   Receptor Pyrin Domain Containing 3 (NLRP3)) (Figure [120]S4A,
   Supporting Information). These findings implied a protective role for
   TEAD1 in promoting the trans‐differentiation of VSMCs into
   fibroblast‐like cells and stabilizing plaques. Several publications
   highlighted the critical role of TEAD1 in cellular function through
   interaction with its coactivators Yes‐Associated Protein (YAP) or
   Transcriptional Coactivator with PDZ‐binding motif (TAZ).^[ [121]^38 ,
   [122]^39 ^] To explore their potential contribution to the
   TEAD1‐mediated mechanisms in VSMCs, we first assessed the expression
   changes of YAP or TAZ following FGF21 stimulation. We found that both
   YAP and TAZ were upregulated in RASMCs post‐stimulation. Moreover, both
   YAP and TAZ translocated into the nucleus and co‐localized with TEAD1
   (Figure [123]S4B,C, Supporting Information). Additionally, both the YAP
   inhibitor (Vertrporfin) and TAZ inhibitor (AR42) significantly
   inhibited the trans‐differentiation process in‐vitro, as evidenced by
   decreased the expression of fibroblast markers (COL3A1 and FSP1) in
   FGF21‐stimulated RASMCs (Figure [124]S4D,E, Supporting Information).
   These results illustrated that TEAD1 was a putative critical regulator
   of VSMCs trans‐differentiation into fibroblast‐like cells, dependent
   upon the coactivators YAP and TAZ.

Figure 4.

   Figure 4
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   TEAD1 promoted the trans‐differentiation of VSMCs into fibroblast‐like
   cells and production of extracellular matrix. A) Representative WB
   images demonstrated that FGF21 exhibited the highest efficiency in
   inducing the trans‐differentiation of RASMCs into fibroblast‐like
   cells. B) Quantification of the WB results of A (n = 3 per group). C)
   The relative mRNA expression levels of FSP1, FN1, COL3A1, and MYH11
   were compared between RASMCs and FGF21‐stimulated RASMCs (n = 3 per
   group). D) FGF21 promoted the expression of FSP1, FN1, and COL3A1 in
   RASMCs. E. Quantification of the WB results of D (n = 3 per group). F.
   The morphology changes in RASMCs following FGF21 stimulation by IF
   staining of FSP1 and α‐SMA (n = 5 per group). Scale bar = 20 µm. G)
   SCENIC analysis displayed the key transcriptional factors of different
   cell subtypes. H) The relative mRNA expression levels of TEAD1 and FSP1
   were compared between RASMCs and FGF21‐stimulated RASMCs (n = 3 per
   group). I) After transfection with TEAD1 siRNA, the
   trans‐differentiation of RASMCs into fibroblast‐like cells was
   inhibited (n = 3 per group). J) IF staining of FSP1 and TEAD1 on the
   series plaques from brachiocephalic artery of Myh11^Cre B6G/R Ldlr^−/−
   mice fed an HFD for 10, 15, and 20 weeks. The white arrows indicated
   tdTomato^+FSP1^+ cells or tdTomato^+TEAD1^+ cells. Scale bar = 100 and
   20 µm. K) Quantification of the percentages of tdTomato^+FSP1^+ cells/
   tdTomato^+ cells and tdTomato^+TEAD1^+ cells/tdTomato^+ cells in J
   (n = 5 per group). For all panels, error bars represented SE. P‐value
   was determined by unpaired two‐tailed Student's t‐test (C, E, F, H, and
   I) or one‐way ANOVA with Bonferroni post‐test (B and K). ns no
   significance, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

2.5. TEAD1 Promoted Plaque Stability and Healing Following Plaque Rupture via
Mediating the Trans‐Differentiation of VMSCs into Fibroblast‐Like Cells

   To explore the role of TEAD1 in the healing process after plaque
   rupture, we first used different concentrations of VT103 (0, 0.1, 1.0,
   and 10.0 µm) to inhibit its function in vitro. The results indicated
   that a concentration of 1.0 µm of VT‐103 effectively inhibited the
   trans‐differentiation of VSMCs into fibroblast‐like cells without
   adversely affecting the survival and apoptosis of other cell types
   (Figure [126]S4F–L, Supporting Information). To further investigate its
   role in vivo, we induced spontaneous plaque rupture in atherosclerotic
   mice using a tandem stenosis (TS) model.^[ [127]^40 ^] Given that not
   all segments in the TS model could yield unstable plaques or experience
   plaque rupture, we employed MOVAT's Pentachrome staining, along with IF
   staining for Platelet Glycoprotein IIb (CD41), TER119, and MMP9 to
   identify the most appropriate segment for further analyses. As shown in
   Figure [128]S5A–C (Supporting Information), all segments developed
   significant atherosclerotic lesions. After TS operations,
   atherosclerotic lesions in Segment 2 exhibited the largest necrotic
   area among these segments, as well as a higher density of type I
   collagen and a more loosely arranged type III collagen compared with
   the others. This implied increased plaque instability and a greater
   likelihood of rupture, followed by collagen generation during the
   repair process (Figure [129]S5A,D–F, Supporting Information).
   Furthermore, the positive area of CD41, TER119, and MMP9 in Segment 2
   exceeded those in other segments, suggesting intraplaque hemorrhage,
   thrombosis, and plaque instability, respectively (Figure [130]S5G–I,
   Supporting Information). Therefore, we conducted further analyses based
   on lesions in Segment 2.

   Next, the mice that underwent TS surgery were intraperitoneally
   injected with VT‐103 or control. Post‐surgery, TER119 staining was
   positive within the plaques, highlighting intraplaque hemorrhage, while
   it was hardly detected in plaques without surgical intervention
   (Figure [131] 5A,B; Figure [132]S5J,K, Supporting Information). This
   finding confirmed that TS surgery successfully induced plaque injury.
   No significant differences in lipid profiles were observed between
   VT‐103 and control groups (Figure [133]S5L, Supporting Information).
   However, the CD41^+ area in ruptured plaques of mice treated with
   VT‐103 was significantly larger than that in control group, suggesting
   increased thrombosis and potentially repeated plaque rupture‐repair
   after TEAD1 inhibition (Figure [134]5C,D). Moreover, VT‐103 treatment
   decreased the percentage of VSMCs‐derived FSP1^+ cells within lesions
   after TS surgery (Figure [135]5E,F), but did not impact the apoptosis
   of VSMCs (Figure [136]S6A,B, Supporting Information). Additionally,
   plaque inflammation, assessed by MMP9 staining, was markedly elevated
   following VT‐103 treatment (Figure [137]5G,H). MOVAT pentachrome
   staining demonstrated that TEAD1 inhibition destabilized plaques and
   impeded their repair after rupture, as evidenced by an increased
   necrotic area and reduced collagen content within lesions after VT‐103
   treatments (Figure [138]5I–L). As the function of TEAD1 was dependent
   upon the coactivators YAP and TAZ, we further explored their role in
   modulating healing after plaque rupture through intraperitoneal
   injection of YAP/TAZ enhancer GA‐017. We found that YAP/TAZ activation
   promoted the trans‐differentiation of VSMCs into FSP1^+ fibroblast‐like
   cells (Figure [139]S6C, Supporting Information), accompanied by reduced
   local inflammation and improved stability after plaque rupture, as
   demonstrated by decreased areas of MMP9, CD41, and TER119 following TS
   operations (Figure [140]S6D–F, Supporting Information). These results
   illustrated that the TEAD1 could drive the trans‐differentiation of
   VSMCs into fibroblast‐like cells, thereby promoting plaque stability
   and plaque healing after rupture. We further observed the inhibition of
   trans‐differentiation of VSMCs into fibroblast‐like cells in mice
   without TS surgery (Figure [141]S7A,B, Supporting Information)
   destabilized the plaques, resulting in thinner fibrous caps and
   decreased COL3A1 content (Figure [142]S7C,D, Supporting Information).
   Additionally, VT‐103 increased the incidence of plaque erosion, as
   evidenced by heightened endothelial apoptosis and endothelial cell
   discontinuity (Figure [143]S7E,F, Supporting Information). These
   results hinted to us that TEAD1 played a crucial role in facilitating
   plaque stability and healing following plaque rupture by mediating the
   trans‐differentiation of VMSCs into fibroblast‐like cells.

Figure 5.

   Figure 5
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   TEAD1 inhibitor VT‐103 attenuated plaque stability and hindered plaque
   healing after rupture. A,B) IF staining of TER119 on plaques from
   Myh11^Cre B6G/R Ldlr^−/− mice that underwent TS surgery treated with
   either control or VT‐103 (n = 5 per group). Scale bar = 100 µm. C,D) IF
   staining of CD41 on plaques from Myh11^Cre B6G/R Ldlr^−/− mice that
   underwent TS surgery treated with either control or VT‐103 (n = 5 per
   group). Scale bar = 100 µm. E,F) IF staining of FSP1 on plaques from
   Myh11^Cre B6G/R Ldlr^−/− mice that underwent TS surgery treated with
   either control or VT‐103 (n = 5 per group). Scale bar = 100 µm. G,H) IF
   staining of MMP9 on plaques from Myh11^Cre B6G/R Ldlr^−/− mice that
   underwent TS surgery treated with either control or VT‐103 (n = 5 per
   group). Scale bar = 100 µm. I) MOVAT pentachrome staining on plaques
   from Myh11^Cre B6G/R Ldlr^−/− mice that underwent TS surgery treated
   with either control or VT‐103 (n = 5 per group). Scale bar = 100 µm.
   J–L) Quantification of the percentages of necrotic area/plaque area,
   type I collagen area/plaque area and type III collagen area/plaque area
   in I. For all panels, error bars represented SE. P‐value was determined
   by unpaired two‐tailed Student's t‐test (B, D, F, H, J, K, and L). ns
   no significance, *P < 0.05, **P < 0.01, ***P < 0.001, and
   ****P < 0.0001.

2.6. TEAD1 Promoted the Trans‐Differentiation of VSMCs into Fibroblast‐Like
Cells via the Wnt4/β‐Catenin Signaling Pathway

   To elucidate the mechanisms by which TEAD1 regulates the
   trans‐differentiation of VSMCs into fibroblast‐like cells, we analyzed
   the differentially expressed genes (DEGs) in the aortic media of the
   TEAD1 knockout group compared with control group through using a
   previously published RNA‐seq database (PRJNA492803). Gene Set
   Enrichment Analysis (GSEA) of the DEGs revealed significant enrichment
   in the Wnt signaling pathway (Figure [145] 6A,B). Based on these
   findings, we hypothesized that TEAD1 might regulate genes related to
   the Wnt4 signaling pathway. Further analysis of the RNA‐seq dataset
   identified specific DEGs regulated by TEAD1, indicating that TEAD1
   primarily regulated genes of the Wnt4 signaling pathways
   (Figure [146]6C,D). Using the Just Another Sequence PASSword Archive
   (JASPAR) prediction algorithm ([147]http://jaspar.genereg.net/), we
   identified a TEAD1 binding site in the promoter region of Wnt4
   (Figure [148]6E,F). Overexpression studies of TEAD1 in RASMCs confirmed
   high protein expression of TEAD1 by WB (Figure [149]6G,H). Pulldown
   assays of the TEAD1‐DNA complex using CUT & Tag (Cleavage Under Targets
   and Tagmentation) qPCR, revealed that TEAD1 could bind to the promoter
   region of Wnt4 (Figure [150]6I,J). Therefore, TEAD1 might enhance Wnt4
   expression by occupying its promoter region. Furthermore, stimulation
   with FGF21 in RASMCs significantly increased the expression of the
   Wnt4/β‐Catenin signaling pathway, which was accompanied by the
   trans‐differentiation of VSMCs into fibroblast‐like cells
   (Figure [151]6K–M). To evaluate the dependency of TEAD1 in mediating
   the activation of the Wnt4/β‐Catenin signaling pathway, siRNA knockdown
   studies demonstrated that TEAD1 deficiency reduced FGF21‐induced
   expression of Wnt4, β‐catenin, COL3A1, and FSP1 (Figure [152]6N,O).
   Taken together, these findings highlighted that TEAD1 promoted the
   trans‐differentiation of VSMCs into fibroblast‐like cells through the
   Wnt4/β‐Catenin signaling pathway.

Figure 6.

   Figure 6
   [153]Open in a new tab

   TEAD1 promoted the trans‐differentiation of VSMCs into fibroblast‐like
   cells via the Wnt4/β‐Catenin signaling pathway. A) Volcano plots of
   DEGs screened by comparing aortic media from TEAD1 KO and control
   group. B) GSEA was performed using the DEGs of A. C) The Venn diagram
   showed the DEGs from RNA‐seq and TEAD1‐targeted genes. D) The heatmap
   displayed the expression of the Wnt signaling pathway related genes.
   E,F) The predicted binding sites of TEAD1 on the Wnt4 promoter. G) WB
   results showed that RASMCs highly expressed TEAD1 and Flag protein
   after transfection with plasmid overexpression of TEAD1. H)
   Quantification of WB results of G (n = 4 per group). I) Gel
   electrophoresis analysis showed TEAD1 bound to the promoter region of
   Wnt4 after the TEAD1‐DNA complex was pulled using anti‐Flag antibodies.
   J) Quantification of results of I (n = 3 per group). K) The relative
   mRNA expression levels of Wnt4, β‐Catenin, COL3A1, and FSP1 were
   compared between RASMCs and RASMCs stimulated with FGF21 at
   concentrations of 1.5 and 3 µg ml^−1 (n = 3 per group). L) WB results
   showed increased protein expression of Wnt4, β‐Catenin and FSP1 in
   RASMCs after FGF21 stimulation. M) Quantification of WB results of L
   (n = 4 per group). N) WB results indicated that the protein expression
   levels of TEAD1, Wnt4, β‐Catenin, FSP1, and COL3A1 were inhibited
   following transfection with TEAD1 siRNA in FGF21 stimulated RASMCs. O)
   Quantification of WB results of N (n = 3 per group). For all panels,
   error bars represented SE. P‐value was determined by unpaired
   two‐tailed Student's t‐test (H,M) or one‐way ANOVA with Bonferroni
   post‐test (J, K, and O). ns no significance, *P < 0.05, **P < 0.01,
   ***P < 0.001, and ****P < 0.0001.

2.7. Wnt4/β‐Catenin Signaling Pathway Mediated the Protective Role of TEAD1
in Healing Process after Plaque Rupture

   We further investigated whether TEAD1's role in facilitating the
   trans‐differentiation of VSMCs into fibroblast‐like cells via the
   Wnt4/β‐catenin pathway in vitro similarly supported the reparative
   process after plaque rupture in vivo. After performing TS surgery, we
   injected the SMCs‐targeting TEAD1‐AAV three times to specifically
   overexpress TEAD1 in VSMCs of mouse arteries. No difference in plaque
   load was observed after TEAD1 overexpression (Figure [154] 7A,B).
   However, TEAD1 overexpression in VSMCs significantly enhanced the
   stability and healing after plaque rupture (Figure [155]7A,C–F),
   accompanied by an increase in VSMCs‐derived fibroblast‐like cells
   (α‐SMA^+FSP1^+ cells) (Figure [156]7G,H). As expected, the areas of
   TER119, CD41, and MMP9 were significantly reduced in mice injected with
   SMCs‐targeting TEAD1‐AAV (Figure [157]7I–N), indicating improved plaque
   repair and decreased plaque inflammation following rupture. Notably,
   the protective effects of TEAD1 overexpression in facilitating
   trans‐differentiation of VSMCs into fibroblast‐like cells and
   subsequent healing after plaque rupture were reversed by inhibition of
   Wnt4/β‐Catenin with IWP4 (Figure [158]7A–N). These results suggested
   that the Wnt4/β‐Catenin signaling pathway mediated the protective role
   of TEAD1 in the stabilization and repair of disrupted plaques. To
   further validate these findings, VSMC lineage tracing mice that
   underwent TS operations were locally injected with a Wnt4/β‐Catenin
   enhancer Norrin or PBS. Norrin nearly replicated the protective effects
   of SMCs‐targeting TEAD1‐AAV, as indicated by elevated tdTomato^+FSP1^+
   cells and decreased areas of CD41, TER119, and MMP9 in disrupted
   plaques (Figure [159]S8A–H, Supporting Information). Collectively,
   these results strongly suggested that TEAD1 promoted the
   trans‐differentiation of VSMCs into fibroblast‐like cells and the
   repair following plaque rupture through the Wnt4/β‐catenin signaling
   pathway.

Figure 7.

   Figure 7
   [160]Open in a new tab

   Wnt4/β‐Catenin inhibitor IWP4 could reverse the positive effect of
   SMC‐specific TEAD1‐AAV. A) Representative images of MOVAT pentachrome
   staining on plaques harvested from Ad‐control, Ad‐TEAD1 and
   Ad‐TEAD1+IWP4 groups. Scale Bar = 100 µm. B–F) Quantification the
   percentages of plaque area%, fibrous area%, necrotic area%, type I
   collagen area%, and type III collagen area% in A (n = 5 per group). G)
   IF staining of α‐SMA and FSP1 on plaques harvested from Ad‐control,
   Ad‐TEAD1 and Ad‐TEAD1+IWP4 groups. Scale bar = 100 µm. H)
   Quantification the percentage of α‐SMA^+FSP1^+ cells/α‐SMA^+ cells in G
   (n = 5 per group). I–K) Representative IF staining of TER119, CD41 and
   MMP9 on plaques harvested from Ad‐control, Ad‐TEAD1 and Ad‐TEAD1+IWP4
   groups. Scale bar = 100 µm. L–N) Quantification the percentages of
   TER119^+ area/plaque area, CD41^+ area/plaque area and MMP9^+
   area/plaque area in I–K, respectively (n = 5 per group). For all
   panels, error bars represented SE. P‐value was determined by one‐way
   ANOVA with Bonferroni post‐test. ns no significance, *P < 0.05,
   **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3. Discussion

   Plaque healing is crucial for maintaining the structure and stability
   of atherosclerotic plaques. Our study demonstrated that VSMCs could
   trans‐differentiate into fibroblast‐like cells, thereby contributing to
   plaque stabilization and healing following rupture. Inhibition of this
   transition destabilized plaques and impeded the healing process after
   rupture. Furthermore, we elucidated the role of TEAD1 in regulating the
   trans‐differentiation of VSMCs into fibroblast‐like cells via the
   Wnt4/β‐Catenin signaling pathway, which promoted plaque repair during
   the dynamic process of atherosclerosis. Our findings also provided
   insights into how the trans‐differentiation of VSMCs into
   fibroblast‐like cells contributed to maintaining a stable plaque
   structure and highlighted novel therapeutic targets for promoting
   atherosclerotic plaque healing after rupture.

   Although plaque injury led to aggravated plaque inflammation, there was
   a rapid mobilization of VSMCs‐derived fibroblast‐like cells, suggesting
   that plaque rupture coexisted with the healing process. Our results
   might help explain the findings from a clinical study published by Yu
   et al.,^[ [161]^41 ^] which indicated that multilayered healing plaques
   reflected a larger scope and severity of the lesions. If plaque healing
   is insufficient to repair the rupture, the presence of local
   microthrombi and intraplaque hemorrhage (“first hit”) can increase
   plaque instability, resulting in a “second hit” as positive feedback.

   Previous studies underscored the critical role of a range of immune
   cell types in plaque healing. For example, Back et al. demonstrated
   that the resolution of inflammation in ruptured plaques relied on M2
   macrophages during the healing process.^[ [162]^42 ^] Similarly, the
   modulation of T‐cell activation and macrophages' phenotypic conversion
   also contributed to the plaque healing.^[ [163]^43 ^] Additionally,
   bone marrow‐derived endothelial cells could be recruited to participate
   in this process.^[ [164]^44 ^] While these studies mainly focused on
   exogenous and myeloid cells, the role of vascular resident cells in
   plaque repair remained poorly defined. Accumulating evidence showed
   that VSMCs in atherosclerotic plaques could alter their phenotype in
   response to different microenvironments, playing crucial roles in
   determining the pathological features of plaques.^[ [165]^19 ^] Our
   findings unveiled a novel role for VSMCs‐derived fibroblast‐like cells
   in promoting plaque stability and the healing process after rupture. We
   compared the VSMCs‐derived fibroblast‐like cells described here with
   the fibromyocytes reported by Wirka, et al.^[ [166]^27 ^] Compared with
   VSMCs‐derived fibroblast‐like cells, fibromyocytes exhibited greater
   proinflammatory properties and participated minimally in the healing
   process following plaque rupture (Figure [167]S9A,B, Supporting
   Information). Regarding AdvSca1‐SMCs, as recently described by
   Dubner,^[ [168]^32 ^] we found that these cells were barely detectable
   after plaque rupture (Figure [169]S9A,B, Supporting Information). This
   finding aligned with Zhou et al.’s conclusion that adventitial
   progenitor cells were mobilized for repair primarily in response to
   severe injuries (such as angiotomy), while minimal injuries (such as
   wire injury and atherosclerosis) yielded few detectable progenitor
   cells.^[ [170]^45 ^] Collectively, our finding supported that the
   VSMCs‐derived FSP1^+ fibroblast‐like cells played a more significant
   role in plaque stabilization and repair process following plaque
   rupture.

   As for mechanisms that regulated the trans‐differentiation of VSMCs
   into fibroblast‐like cells, we identified a key role of TEAD1 in this
   process. Several studies have investigated the function of TEAD1 in
   VSMCs in vascular injury models, such as femoral artery wire injury.^[
   [171]^46 , [172]^47 ^] However, these studies primarily focused on its
   function in promoting VSMCs development and contractility. We also
   found that TEAD1 primarily regulated the transition of dedifferentiated
   VSMCs to fibroblast‐like cells rather than the de‐differentiation
   process (Figure [173]S9C,D, Supporting Information). Our study revealed
   a novel mechanism by which TEAD1, coactivated by YAP/TAZ, upregulated
   the Wnt/β‐Catenin pathway, facilitating the trans‐differentiation into
   fibroblast‐like cells and the secretion of key extracellular matrix
   proteins, specifically COL3A1 and FN1.^[ [174]^48 , [175]^49 ^] These
   proteins were essential for initial repair process following plaque
   rupture.^[ [176]^50 ^] These results further substantiated the need for
   screening factors and mechanisms that promoted beneficial VSMCs
   phenotypic trans‐differentiation as potential therapeutic targets for
   improving plaque stability and post‐injury repair.

   Previous studies have indicated that VSMCs can dedifferentiate into
   intermediate cells, and subsequently trans‐differentiated into multiple
   cell types, including macrophages, mesenchymal stem cells, fibroblasts,
   and osteochondrogenic cells, affecting the progression and
   stabilization of atherosclerotic plaques.^[ [177]^51 ^] Consequently,
   it has been proposed that therapeutic intervention for atherosclerosis
   should primarily focus on the inhibition of VSMCs dedifferentiation.^[
   [178]^52 , [179]^53 ^] However, these studies ignored the potential
   benefits of promoting the trans‐differentiation of VSMCs into
   beneficial type cells, which could aid in plaque repair and
   stabilization. Our findings underscored the significance of this
   trans‐differentiation process, demonstrating that VSMCs converting into
   fibroblast‐like cells significantly contributed to stabilization by
   secreting extracellular matrix proteins such as COL3A1 and FN1. Our
   findings were consistent with previous studies showing VSMCs plasticity
   and its impact on plaque stability and repair. Furthermore, our study
   suggested that targeted modulation of these pathways could offer new
   therapeutic strategies for promoting stabilization and healing after
   plaque rupture.

   We also recognized serval limitations in this study. Although the TEAD1
   inhibitor, VT‐103, has been shown to effectively inhibit TEAD1‐mediated
   gene transcription, other cell types beyond VSMCs‐derived
   fibroblast‐like cells might also contribute to improving plaque
   stability and repair. To address this concern, we utilized a
   VSMCs‐specific AAV‐overexpression virus and observed improved repair
   following the specific overexpression of TEAD1 in VSMCs. While the
   dosage of the TEAD1 inhibitor could theoretically influence VSMCs
   trans‐differentiation and cell survival, we determined an optimal
   concentration of TEAD1 inhibitor in vitro that effectively inhibited
   the trans‐differentiation of VSMCs into fibroblast‐like cells without
   adversely affecting the survival or apoptosis of other cell types
   (Figure [180]S4F–L, Supporting Information). Additionally, we noted
   clear differences and translational gaps in studying plaque rupture
   between mice and humans. These disparities were evident in hemodynamic
   alterations, shear stress, recruited immune and non‐immune cell types,
   inflammatory changes, and vessel integrity. The mouse TS plaque rupture
   model used in this study has been extensively characterized to
   elucidate how biological signaling pathways could impact plaque
   rupture.^[ [181]^54 , [182]^55 ^] Future studies that validate our
   findings under alternative experimental conditions or in larger animal
   models will be valuable.

   In conclusion, the findings of this study established a foundation for
   innovative approaches aimed at promoting the repair and stability of
   injured atherosclerotic plaques through targeting VSMCs
   trans‐differentiation. Future research should focus on the development
   of targeted therapies, the validation of cell‐cell interaction, and the
   exploration of the broader implications of these mechanisms for
   clinical practice. This comprehensive approach will deepen our
   understanding of plaque stabilization and repair, potentially leading
   to effective new treatments for atherosclerosis.

4. Experimental Section

Animals

   All mice were housed in a controlled environment with a temperature
   maintained between 18 and 25 °C and a 12 h light/dark cycle. Male
   Ldlr^−/− , Myh11‐CreER^T2 (Myh11^Cre) and
   CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐Tdtomato (B6G/R) mice (purchased from
   Shanghai Model Organisms) were used in this study. Ldlr^−/− , Myh11^Cre
   , and B6G/R mice were genotyped by PCR based on the protocol provided
   by the company. In the Myh11^Cre B6G/R Ldlr^−/− mouse models, Cre
   recombinase was activated in male mice with 75 mg kg^−1 tamoxifen
   (T‐5648, Sigma, USA) intraperitoneal injections for 5 consecutive days
   at the age of 8 weeks. Male littermate controls were used for all
   studies. ZsGreen, located on the ROSA locus, was expressed in every
   single cell prior to tamoxifen induction. VSMCs and their progenies,
   which expressed Myh11, permanently expressed tdTomato following
   tamoxifen induction, resulting in a color change from green to red
   under fluorescent light. Ldlr^−/− and Myh11^Cre B6G/R Ldlr^−/− mice
   were fed a Clinton/Cybulsky High Fat Rodent Diet (HFD, 40% calories
   from fat; and 1.25% cholesterol, D12108C, Research Diets, USA) to
   induce atherosclerosis. Mice were sacrificed by cervical dislocation
   and perfused sequentially with 5 ml of PBS, followed by 10 ml of 4%
   paraformaldehyde, and finishing with another 5 ml of PBS via the left
   ventricle. Mouse aortas, brachiocephalic arteries, and hearts were
   carefully dissected, embedded in the Optimal Cutting Temperature
   compound, frozen, sectioned, and subjected to further analysis. For the
   tissue immunohistochemistry (IHC) analysis, the paraformaldehyde‐fixed
   samples were embedded in paraffin blocks and sectioned into 6 µm‐thick
   sections for subsequent experiments.

Carotid Artery Ligation

   Mice were anesthetized with intraperitoneal injection ketamine
   (80 mg kg^−1) and xylazine (5 mg kg^−1). The left carotid artery was
   completely ligated with a 6–0 silk suture proximal to the carotid
   bifurcation. The left carotid arteries were harvested for further
   analysis at 14‐ and 21‐day post‐ligation.

Plaque Injury Model

   The plaque injury model was developed to simulate the plaque rupture in
   patients with ACS, as initially described by Robert L et al.^[ [183]^56
   ^] This method involves clipping the atherosclerotic plaque in the
   abdominal aorta of Apoe^−/− mice using round‐tipped forceps to induce
   plaque rupture. Minor modifications were implemented to standardize and
   enhance the reproducibility of this method in experiments. After
   anesthetizing Myh11^Cre B6G/R Ldlr^−/− mice that had fed an HFD for 20
   weeks, a partial sternotomy was performed to expose the brachiocephalic
   artery. Mechanical force was then applied to the brachiocephalic artery
   at the site of the plaque using hemostat forceps, maintaining pressure
   for 5 s intervals followed by 5 s intermissions until signs of plaque
   congestion were evident. Specifically, a resistive membrane pressure
   sensor was integrated at the force application point of the hemostat
   (Precision Kelly Hemostat, 5.5 Inches), allowing for precise
   maintenance of the pressure exerted on the blood vessel at ≈1000 g (as
   shown in Figure [184]S9E, Supporting Information). A dedicated data
   acquisition device seamlessly collected the data from this sensor,
   enabling consistent monitoring of the pressure applied to the blood
   vessel, thereby enhancing the reproducibility and standardization of
   these experiments. After this procedure, the mice were fed with an HFD
   for an additional four weeks before sacrificed, at which point the
   artery plaque was harvested for further analysis.

Tandem Stenosis (TS) Model of Plaque Rupture

   The TS model was employed to induce spontaneous plaque rupture, as
   described previously.^[ [185]^54 , [186]^55 ^] Eight‐week‐old male mice
   were fed an HFD for 13 weeks prior to undergoing TS surgery. Following
   anesthetization, a small incision was made on the right common carotid
   artery, after which the tissue underneath was bluntly dissected. Two
   stenosis points were positioned 1 and 4 mm below the carotid
   bifurcation. A 150 µm needle was placed on the top of the right common
   carotid artery, and sutures were tightly tied at the two points to
   occlude blood flow. Subsequently, the needle was carefully removed to
   restore blood flow. After an additional seven weeks on HFD, the artery
   was harvested for further analysis.

Cell Counting Kit‐8 (CCK‐8) Experiments

   Cell viability was assessed by the CCK‐8 assay. Briefly, cells were
   seeded into a 96‐cell plate at a density of 2 × 10^3 cells per well,
   with six replicates for each group. The cells were treated with
   different concentration of VT‐103. After 24 h, a 10 µl CCK8 reagent
   (HY‐K0301, MCE, USA) was added according to the manufacturer's
   instructions, followed by a 2 h incubation. The absorbance was measured
   at 450 nm using a microplate reader.

Atherosclerotic Lesion Analyses

   The plaques from aortic root, right common carotid arteries, and
   brachiocephalic artery were processed for histology. The fibrous cap
   was identified using Masson staining. MOVAT pentachrome staining was
   performed using a commercial staining kit (S191078, Pinuofei
   Biological, China) according to the manufacturer's instructions.
   Briefly, sections were incubated in Weigert's iron hematoxylin for
   10 min to stain nuclei. After differentiation in acid alcohol, collagen
   appeared pink. Subsequently, sections were stained in Biebrich
   Scarlet‐Acid Fuchsin solution for 15 min to highlight elastic fibers
   and muscle. Differentiation in phosphotungstic/phosphomolybdic acid
   solution for 10 min emphasized the distinction between collagen and
   ground substance. Aniline Blue staining for 10 min highlighted
   glycosaminoglycans. Stained sections were examined under a light
   microscope equipped with polarizing filters to optimize visualization
   of collagen and elastic fibers. Digital micrographs were captured at
   representative magnifications for subsequent analysis. Plaque area,
   necrotic core, type I and type III collagen content, and other relevant
   parameters were quantified using image J software (Version 2.10, NIH,
   USA). As for the plaque area% calculation, it was accurately assessed
   through dividing the plaque area by the internal elastic lamina (IEL)
   area.

Cell Culture and Stimulation

   Rat aortic smooth muscle cells (A7r5, RASMCs) were purchased from
   Shanghai Zhongqiaoxinzhou Biotechnology. The cells were cultured in
   DMEM (SH30243.01, HyClone, USA) supplemented with 10% fetal bovine
   serum (FBS) (10 099 141, Gibco, USA) and 1% penicillin‐streptomycin at
   37 °C in an atmosphere of 95% O[2] and 5% CO[2]. To induce the
   trans‐differentiation of RASMCs into fibroblast‐like cells, the RASMCs
   were starved in DMEM with 0.2% FBS overnight and subsequently
   stimulated with FGF21 (1.5 ng ml^−1, HY‐P7173, MCE, USA) for 48 h. In
   some experiments, the RASMCs were stimulated with water‐soluble
   cholesterol at concentration of 10 µg ml^−1 for 72 h (C4951‐30MG,
   Sigma, USA), ox‐LDL at concentration of 100 µg ml^−1 for 48 h
   ([187]P00794, Solarbio, USA), palmitic acid at concentration of
   300 µmol L^−1 for 12 h (HY‐N0830, MCE, USA), lactate at concentration
   of 10 mm for 12 h (HY‐B227, MCE, USA). To further investigate the roles
   of YAP and TAZ in the trans‐differentiation of VSMCs, the YAP inhibitor
   Verteporfin (10 µg ml^−1, SML0534, Sigma, USA), and the TAZ inhibitor
   AR42 (600 nm, T6392, Target Molecule Corporation, USA) were used for
   treatment. The specific procedure was as follows: under FGF21
   stimulation, RASMCs were treated with either Verteporfin or AR42 for
   12 h. The experimental groups included: control group (Ctr), FGF21‐only
   stimulation group (FGF21), FGF21 stimulation with Verteporfin treatment
   group, and FGF21 stimulation with AR42 treatment group. The effects of
   YAP and TAZ inhibition on the trans‐differentiation of RASMCs into
   fibroblast‐like cells induced by FGF21 were evaluated by examining the
   expression changes of trans‐differentiation related markers in the
   different treatment groups. Human umbilical vein endothelial cells
   (HUVECs) were cultured in Ham's Kaighn's Modification F12K medium
   (Invitrogen, USA) supplemented with 10% FBS, 1%
   penicillin‐streptomycin, heparin (15 IU ml^−1, LEO Pharma, Denmark) and
   vascular endothelial growth factor (30 ng ml^−1, Sigma, USA). NIH‐3T3
   fibroblast cells were grown in DMEM supplemented with 10% FBS and 1%
   penicillin‐streptomycin. After stimulations, the cells were harvested
   for downstream experiments.

In Vivo Treatments with TEAD1 Inhibitor (VT‐103), YAP/TAZ Enhancer (GA‐017),
and Wnt4/β‐Catenin Enhancer (Norrin)

   VT‐103 was administered intraperitoneally into mice at a dosage of
   0.3 mg kg^−1 three times per week for seven weeks following TS surgery,
   until the animals were sacrificed. In the in vivo injection experiments
   involving the YAP/TAZ enhancer (GA‐017), GA‐017 was injected
   intraperitoneally at a dosage of 0.1 mg kg^−1 three times per week
   after TS surgery for seven weeks until sacrifice. For the local
   injection of Wnt4/β‐Catenin enhancer Norrin, atherosclerotic mice
   underwent TS operation, followed by a local injection of either the
   Wnt4/β‐Catenin enhancer Norrin (250 ng, 3497‐NR, Biotechne, USA) or PBS
   as a negative control prior to the closure of the incision. After seven
   weeks, both groups of mice were sacrificed for further experiments and
   analysis.

Intravenous Injection of Adeno‐Associated Virus (AAV) and IWP4 Treatments

   AAV targeting mouse TEAD1 (Ad‐TEAD1) and an empty vector (Ad‐Control)
   were obtained from GENECHEM Incorporation (Shanghai, China). At the
   time of TS surgery, the Sm22ap‐MCS‐SV40 AAV virus was intravenously
   injected at a dosage of 0.5 × 10^12 viral genomes (v.g) per mouse. Two
   days after AAV virus injection, a group of mice received Ad‐TEAD1 were
   administered with the Wnt4/β‐Catenin inhibitor IWP4 (5214, Biotechne,
   USA) intraperitoneally at a dosage of 4 mg kg^−1 per day for 10 days.
   After seven weeks on an HFD, the mice were sacrificed for subsequent
   analysis.

Small Interfering RNA (siRNA) Transfection

   siRNA targeting TEAD1 (siTEAD1) and scramble siRNA (siNC) were
   purchased from GenePharma (China). The RASMCs were transfected with
   50 nm siRNA using Lipofectamine iMAX reagent (13 778 030, Thermo
   Fisher, USA) for 8 h before downstream experiments were performed. The
   sequences of siRNAs are shown in Table [188]S1 (Supporting
   Information).

Plasmid Construction and Transfection

   TEAD1‐Flag was constructed and purchased from Shanghai Genechem Co,
   Ltd. The plasmids were transfected into RASMCs at a concentration of
   20 µg ml^−1 using Lipofectamine iMAX reagent (13 778 030, Thermo
   Fisher, USA) for 6 h.

Immunofluorescence (IF) Staining and Laser Confocal Fluorescence Microscopy
Analysis

   Cells were seeded at 1 × 10^6 cells per well on a glass‐bottomed
   culture dish and fixed in 4% paraformaldehyde for 15 min, followed by
   permeabilization in 0.2% Triton X‐100 (X100RS, Sigma, USA) in PBS for
   5 min. Cross sections were also fixed in 4% paraformaldehyde for 15 min
   and permeabilized in 0.2% Triton X‐100 in PBS for 5 min. Permeabilized
   cells or tissue sections were washed three times with PBS and then
   blocked with 5% goat serum. The sections or cells were incubated with
   primary antibodies diluted in a blocking buffer, including anti‐FSP1
   (1:100, ab124805, Abcam, USA), anti‐CD31 (1:100, ab222783, Abcam, USA),
   anti‐TER119 (1:100, 116 202, Biolegend, USA), anti‐MMP9 (1:100,
   ab38898, Abcam, USA), anti‐TEAD1 (1:100, ab133533, Abcam, USA),
   anti‐α‐SMA (1:200, ab7817, Abcam, USA), anti‐LUM (1:250, ab168348,
   Abcam, USA), anti‐GLI1 (1:100. sc‐515781, Santa Cruz, USA), anti‐CD41
   (1:100, sc‐373992, Santa Cruz, USA), anti‐CD68 (1:100, NB100‐683,
   Novus, USA). Normal isotype IgG (1:100, sc2027, Santa Cruz, USA) was
   used as a negative control. After three washes with PBS, cells or
   tissue sections were incubated with secondary antibodies Alexa Fluor
   647‐conjugated goat anti‐rabbit (1:200, A‐21244, Invitrogen, USA),
   Alexa Fluor 488‐conjugated goat anti‐rabbit (1:200, A‐11008,
   Invitrogen, USA), Alexa Fluor 594‐conjugated donkey anti‐rabbit (1:200,
   [189]R37119, Invitrogen, USA) or Alexa Fluor 594‐conjugated goat
   anti‐mouse (1:200, A‐11005, Invitrogen, USA) for 1 h at 37 °C in the
   dark. Nuclei were labeled with DAPI (Vector Laboratories), and cells
   were visualized using an LSM710 laser confocal microscope (Carl Zeiss,
   Germany). A close examination of each plane of the z‐stack was
   conducted using Zen 2009 Light Edition Software (Zeiss) to confirm that
   the presence of immunofluorescence staining coincided with a single
   DAPI nucleus. Image J was used to quantify the lesion area or the
   positive region.

Immunohistochemistry (IHC) Analysis

   For IHC, antigen retrieval was performed after paraffin sections were
   dewaxed and rehydrated. Then tissue sections were then blocked with 5%
   goat serum and incubated with primary antibodies targeting α‐SMA
   (1:200, ab7817, Abcam, USA), CD68 (1:100, NB100‐683, Novus, USA), and
   COL3A1 (1:100, sc‐8784, Santa Cruz, USA) at 37 °C for 1 h. After three
   washes with PBS, the sections were incubated with secondary antibodies
   at 37 °C for 1 h. The DAB Horseradish Peroxidase Color Development Kit
   (DA1015, Solarbio, China) was used to visualize the positive signals in
   the sections. Cell nuclei were counterstained with Mayer's hematoxylin
   solution. Image J was used to calculate the ratio of the positive area
   of α‐SMA, CD68, or COL3A1 relative to the total plaque area.

TUNEL Assay

   TUNEL assay was used to evaluate apoptotic cells according to the
   manufacturer's instruction (40307ES20, YEASEN, China). Image J was used
   to quantify TUNEL‐positive cells in each sample under 20 ×
   magnification.

Protein Extraction and Western Blotting (WB)

   Whole cell lysate was prepared by 1 × cell lysis buffer (9803, Cell
   Signaling Technologies, USA) with protease inhibitors (0 469 315 9001,
   Roche Molecular Biochemicals, USA) and phosphatase inhibitor (78 428,
   ThermoFisher Scientific, USA). Protein concentrations were determined
   by using a BCA protein assay kit. Proteins were separated by SDS–PAGE,
   transferred to polyvinylidene fluoride (PVDF) membranes, and incubated
   with antibodies diluted with a blocking buffer overnight at 4 °C,
   including anti‐FSP1 (1:1000, ab124805, Abcam, USA), anti‐FN1 (1:500,
   sc‐8422, Santa Cruz, USA), anti‐COL3A1 (1:500, sc‐514601, Santa Cruz,
   USA), anti‐TEAD1 (1:1000, ab133533, Abcam, USA), anti‐α‐SMA (1:1000,
   ab7817, Abcam, USA), anti‐β‐Catenin (1:1000, ab32572, Abcam, USA),
   anti‐Wnt4 (1:500, sc‐376279, Santa Cruz, USA), and anti‐GAPDH (1:5000,
   60004‐1‐Ig, Proteintech, USA). Then the PVDF membranes were incubated
   with HRP‐conjugated secondary antibodies for 1 h and bands were
   visualized using chemiluminescence (TANON, China) and viewed under an
   Amersham Imager 600 system (GE Health‐care, USA).

Real‐Time Quantitative RT‐PCR

   Total RNA was extracted using Trizol regent (Thermo Fisher, USA)
   according to the manufacturer's instructions. Purified RNA (1000 ng)
   was reverse‐transcribed using a Primer Script RT Reagent Kit (#rr820a,
   Takara, Japan). The quantitative RT‐PCR was then performed on 1 µg of
   cDNA product using FastStart Universal SYBR Green Master (QR0100,
   Roche, USA) on a Roche Light cycler. The RT‐PCR primers are listed in
   the Table [190]S2 (Supporting Information).

ScRNA‐Seq Data Processing

   The scRNA‐seq data for tdTomato^+ cells and zsGreen^+ cells from the
   aortas of HFD‐fed Myh11^Cre B6G/R mice ([191]GSE197073) were generated
   in the previous study.^[ [192]^17 ^] In addition, a publicly available
   scRNA‐seq dataset of the carotid artery ligation model ([193]GSE174098)
   was downloaded from the GEO database. The datasets were analyzed using
   Seurat v3.1.1 in R. The Myh11^CrePadi4^flox/flox part of scRNA‐seq were
   excluded from this data for further analysis. Quality control was
   performed to exclude cells with poor gene expression profiles,
   specifically those expressing fewer than 200 genes, or those with more
   than 10% mitochondrial genes among the total expressed genes. After
   normalization, Principal Component Analysis was performed and
   calculated the number of significant principal components using the
   Elbow Plot function. The Uniform manifold approximation and projection
   (UMAP) was performed for unsupervised clustering. The resolution was
   set to 0.6 because it allows for a better annotation of cell types. In
   addition, marker genes were defined based on the Find Marker function
   and literature.

SCENIC Analysis

   The R package SCENIC ([194]https://github.com/aertslab/SCENIC, version
   1.1.1‐10, RcisTarget 1.6.0, and AUCell 1.8.0) was used to analyze the
   enrichment of transcriptome factors in different cell subtypes.^[
   [195]^57 ^] The input matrices in SCENIC were the raw UMI counts
   obtained from Seurat. Following the standard SCENIC procedure, the
   GENIE3 method was used to identify potential transcription factor
   targets. In addition, the activity of each regulon in each cell was
   evaluated using AUCell, calculated as the area under the recovery
   curve, which integrated the expression ranks across all genes within a
   regulon.

Differential Expression and Pathway Enrichment Analysis

   DEGs of cell subtypes were identified using Find Markers in the Seurat
   package with the Wilcoxon test. |Log[2](FC)|>1 and adjusted P value
   < 0.05 were set as the cut‐off criteria. Pathway enrichment analysis
   was performed using QIAGEN‐Ingenuity Pathway Analysis (IPA) software
   ([196]www.ingenuity.com). The Fisher's Exact Test was used to calculate
   a statistical significance of the overlap of differentially expressed
   transcripts with canonical pathways, and pathways were selected based
   on statistical significance (P value<0.05) and enrichment (Z) score.
   All analyses were performed in R v4.3.3 statistical environment and the
   plots were generated with ggplot2 v3.5.1 library.

The Association between Biomarkers and Prognosis in Bulk RNA‐Seq Dataset

   The bulk RNA‐seq and prognostic data were downloaded from [197]GSE21545
   dataset. The Kaplan‐Meier curves were plotted based on the median
   expression levels of TNF‐α, FSP1, SPP1, and ACAT2 in R v4.3.3
   statistical environment.

Bulk RNA‐Seq and Bioinformatics Analysis

   Bulk RNA‐Seq dataset (PRJNA492803) for the aortic media from the TEAD1
   CKO group and control group mice was downloaded from NIH BioProject.
   The CKO group utilized an inducible smooth muscle–specific TEAD1 KO
   (knockout) mouse model. DEG analysis was performed using the limma R
   package. Genes with log[2]FC ≥1 and adjusted P value <0.05 were
   considered as significant DEGs. Subsequently, Gene Set Enrichment
   Analysis (GSEA) was conducted using the “GSEA” package to reveal
   signaling pathways associated with these significant DEGs.

CUT & Tag PCR

   With minor modifications, CUT & Tag was performed using the Hyperactive
   Universal CUT & Tag Assay Kit for Illumina (TD903, Vazyme, China). A
   total of 1 × 10^5 cells were gently washed twice with PBS. Then 10 µl
   of concanavalin A‐coated magnetic beads were added to each sample and
   incubated at room temperature for 10 min. After discarding the unbound
   cells, the bound cells were mixed with an anti‐Flag antibody (1:50,
   14 793, CST, USA) or rabbit normal IgG antibody (1:200, 2729, CST, USA)
   and incubated at 4 °C overnight with rotation. Next, cells were
   incubated in a secondary antibody, diluted in DIG wash buffer, at room
   temperature for 1 h, and washed three times with DIG wash buffer. The
   beads were then incubated with a pA/G‐Tnp adapter complex (0.04 µm) at
   room temperature for 1 h with rotation and washed three times with
   DIG‐300 buffer. DNA was purified using phenol‐chloroform isoamyl
   alcohol extraction and ethanol precipitation. To amplify the library,
   15 µl of purified DNA was mixed with 5 µl of a universal i5 and a
   uniquely barcoded i7 primer. A volume of 25 µl 2 × CUT & Tag
   amplification mix was added and mixed. The samples were placed in a PCR
   thermal cycler with a heated lid using the following program: 72 °C for
   3 min; 95 °C for 3 min; 18 cycles of 98 °C for 10 s and 60 °C for 5 s;
   final extension at 72 °C for 1 min and hold at 4 °C. PCR products were
   purified using VAHTS DNA Clean Beads (N411‐01, Vazyme, China).

Statistics

   Statistical analyses were performed using GraphPad Prism (Version 9.0).
   Results were displayed as mean ± standard error of the mean (SE).
   Student's t‐test or Mann‐Whitney U‐test was used to analyze the
   difference between standard and non‐normal variables. Differences
   across more than three groups were compared using one‐way ANOVA
   followed by a post hoc analysis with the Bonferroni test. Two‐sided and
   p < 0.05 was regarded as statistical significance.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   M.Z., Z.L., and Y.S. contributed equally to this work. W.P., M.W.F.,
   Q.Y., J.Z., and W.J. conceived and designed the study and experiments.
   M.Z., Z.L., and J.S. performed the experiments and data analyses. Y.S.
   and Q.Y. supervised the study. M.Z., Z.L., Y.Z., and S.G. interpreted
   the data and wrote the manuscript. All authors revised, critically
   discussed, and approved the final version of the manuscript.

Supporting information

   Supporting Information
   [198]ADVS-12-2407408-s003.docx^ (6.6MB, docx)

   Supplemental Table 1
   [199]ADVS-12-2407408-s002.xlsx^ (9KB, xlsx)

   Supplemental Table 2
   [200]ADVS-12-2407408-s001.xlsx^ (10.1KB, xlsx)

Acknowledgements