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Bves maintains vascular smooth muscle cell contractile phenotype and protects against transplant vasculopathy via Dusp1-dependent p38MAPK and ERK1/2 signaling

  • Jin-Xin Liu
    Affiliations
    Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Tong Huang
    Affiliations
    The Eight Affiliated Hospital, Sun Yat-sen University, Shenzhen, China
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  • Dawei Xie
    Affiliations
    State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Qihong Yu
    Correspondence
    Corresponding author. Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Wuhan 430030, China.
    Affiliations
    Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Clinical Medical Research Center of Hepatic Surgery at Hubei Province, Wuhan, China
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Open AccessPublished:August 14, 2022DOI:https://doi.org/10.1016/j.atherosclerosis.2022.08.010

      Highlights

      • Bves is downregulated in the medial VSMCs in aortic allograft and in cultured VSMCs stimulated by PDGF.
      • Bves expression is essential for maintaining the VSMC contractile phenotype.
      • Genetic knockdown of Bves inhibits Dusp1 expression and promotes p38MAPK and ERK1/2 phosphorylation.
      • VSMC-specific Bves overexpression in aortic allografts delays neointimal formation after transplantation.

      Abstract

      Background and aims

      Vascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear.

      Methods

      We used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development.

      Results

      Here, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation.

      Conclusions

      Bves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

      Graphical abstract

      Keywords

      1. Introduction

      Vascular smooth muscle cells (VSMCs) are the major cellular components in the tunica media of the arterial wall. In healthy vessels, VSMCs retain a highly differentiated contractile phenotype and display remarkable plasticity [
      • Owens G.K.
      • Kumar M.S.
      • Wamhoff B.R.
      Molecular regulation of vascular smooth muscle cell differentiation in development and disease.
      ]. Compelling evidence has demonstrated that fully differentiated VSMCs counteract the progression of a broad spectrum of cardiovascular diseases, including atherosclerosis, restenosis, aneurysm, transplant vasculopathy and pulmonary hypertension [
      • Bennett M.R.
      • Sinha S.
      • Owens G.K.
      Vascular smooth muscle cells in atherosclerosis.
      ,
      • Inoue T.
      • Node K.
      Molecular basis of restenosis and novel issues of drug-eluting stents.
      ,
      • Yang K.
      • Ren J.
      • Li X.
      • Wang Z.
      • Xue L.
      • et al.
      Prevention of aortic dissection and aneurysm via an ALDH2-mediated switch in vascular smooth muscle cell phenotype.
      ,
      • Xiao Y.
      • Christou H.
      • Liu L.
      • Visner G.
      • Mitsialis S.A.
      • et al.
      Endothelial indoleamine 2,3-dioxygenase protects against development of pulmonary hypertension.
      ,
      • Abrahimi P.
      • Liu R.
      • Pober J.S.
      Blood vessels in allotransplantation.
      ]. These disorders depend on the phenotypic transition of VSMCs from a contractile phenotype to a synthetic phenotype in response to stressed or pathological conditions, characterized by the reduced expression of contractile proteins and excessive VSMC migration and proliferation that underscores vascular remodeling [
      • Owens G.K.
      • Kumar M.S.
      • Wamhoff B.R.
      Molecular regulation of vascular smooth muscle cell differentiation in development and disease.
      ]. Recently, the certain roles of several growth factors, cytokines, and transcription and epigenetic factors in driving the VSMC phenotypic transition have been reported [
      • Owens G.K.
      • Kumar M.S.
      • Wamhoff B.R.
      Molecular regulation of vascular smooth muscle cell differentiation in development and disease.
      ,
      • Alexander M.R.
      • Owens G.K.
      Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease.
      ]. However, there is still a limited understanding of the regulation of VSMC contractile phenotype in the pathogenesis of these vascular diseases.
      Blood vessel epicardial substance (Bves, also known as Popdc1) is a prototypical member of the Popdc gene family that encodes an evolutionarily conserved transmembrane protein principally localized to the plasma membrane [
      • Froese A.
      • Breher S.S.
      • Waldeyer C.
      • Schindler R.F.
      • Nikolaev V.O.
      • et al.
      Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.
      ]. The Bves protein consists of one extracellular N-terminus, three transmembrane domains and one intracellular Popeye domain and is widely expressed in various tissue types in embryos and adults [
      • Reese D.E.
      • Zavaljevski M.
      • Streiff N.L.
      • Bader D.
      bves: a novel gene expressed during coronary blood vessel development.
      ,
      • Reese D.E.
      • Bader D.M.
      Cloning and expression of hbves, a novel and highly conserved mRNA expressed in the developing and adult heart and skeletal muscle in the human.
      ]. Popdc proteins have multiple modes to maintain organ homeostasis and mediate cellular signaling in both development and diseases [
      • Amunjela J.N.
      • Swan A.H.
      • Brand T.
      The role of the Popeye domain containing gene family in organ homeostasis.
      ]. First discovered in developing cardiac tissue, Bves performs known functions in regulating cardiac morphogenesis and exerts beneficial effects on smooth muscle cell differentiation during intracardiac vasculogenesis [
      • Reese D.E.
      • Zavaljevski M.
      • Streiff N.L.
      • Bader D.
      bves: a novel gene expressed during coronary blood vessel development.
      ]. The important roles of Bves in skeletal muscle regeneration, heart rhythm control, stress and inflammatory signaling have also been well established [
      • Froese A.
      • Breher S.S.
      • Waldeyer C.
      • Schindler R.F.
      • Nikolaev V.O.
      • et al.
      Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.
      ,
      • Amunjela J.N.
      • Swan A.H.
      • Brand T.
      The role of the Popeye domain containing gene family in organ homeostasis.
      ]. Studies involving mutant mice have identified Bves as a novel caveolae-associated protein or cyclic 3′,5′-adenosine monophosphate (cAMP)-binding protein that plays an important protective role in ischemia/reperfusion injury, oxidative stress and environmental stress [
      • Froese A.
      • Breher S.S.
      • Waldeyer C.
      • Schindler R.F.
      • Nikolaev V.O.
      • et al.
      Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.
      ,
      • Shetty M.S.
      • Ris L.
      • Schindler R.F.R.
      • Mizuno K.
      • Fedele L.
      • et al.
      Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity.
      ,
      • Alcalay Y.
      • Hochhauser E.
      • Kliminski V.
      • Dick J.
      • Zahalka M.A.
      • et al.
      Popeye domain containing 1 (Popdc1/Bves) is a caveolae-associated protein involved in ischemia tolerance.
      ], as well plays a positive role in reducing neointimal lesions in several models of vascular injury [
      • Sassi Y.
      • Lipskaia L.
      • Vandecasteele G.
      • Nikolaev V.O.
      • Hatem S.N.
      • et al.
      Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation.
      ,
      • Indolfi C.
      • Di Lorenzo E.
      • Rapacciuolo A.
      • Stingone A.M.
      • Stabile E.
      • et al.
      8-chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima formation induced by balloon injury in vivo.
      ]. Popdc proteins have been proposed to be a part of the protein network linked to cAMP signaling, and aberrant Bves signaling is involved in abnormal cell behaviors and diseases. In vitro studies of Bves loss in tumor cells have shown a prominent impact on enhancing proliferation, migration and the epithelial-to-mesenchymal transition (EMT) [
      • Williams C.S.
      • Zhang B.
      • Smith J.J.
      • Jayagopal A.
      • Barrett C.W.
      • et al.
      BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma.
      ,
      • Amunjela J.N.
      • Tucker S.J.
      Dysregulation of POPDC1 promotes breast cancer cell migration and proliferation.
      ]. Manipulating Bves expression in NIH 3T3 cells resulted in changes in cell shape and negatively regulated cell survival and movement [
      • Smith T.K.
      • Hager H.A.
      • Francis R.
      • Kilkenny D.M.
      • Lo C.W.
      • et al.
      Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity.
      ]. In most adult organisms, Bves-expressing cells have a similar phenotype or function in nature. Although Bves expression is essential for the acquisition of VSMC contractile properties, to date, the functional importance of Bves in VSMC phenotypes and related vascular diseases has not been determined.
      In this study, we identified the VSMC phenotype related gene Bves in aortic grafts and in vitro VSMCs by using RNA sequencing, and found that maintaining the VSMC contractile phenotype was the most noticeable effect of Bves. Specifically, Bves is required for the transcriptional expression of dual-specificity protein phosphatase 1 (Dusp1) and its dependent dephosphorylation and inactivation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in vivo and in vitro. Furthermore, we found an inverse correlation between VSMC-specific Bves expression and the development of neointimal lesions in a rat model of graft arteriosclerosis. These findings provide mechanistic insight into potential treatment and prevention strategies for vascular proliferative diseases.

      2. Materials and methods

      Expanded materials and methods are provided in the Supplementary Material. All supporting materials are available from the corresponding author upon reasonable request.

      2.1 Animals

      Male Brown Norway (BN) and Lewis rats about 4 weeks old were obtained from HFK Bioscience Co. (Beijing, China), and housed in a specific pathogen-free environment. All procedures involving animal subjects were approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; IACUC approval no. [2021] 2582) and conducted according to the guidelines of National Institutes of Health (NIH) for the Care and Use of Laboratory Animals.

      2.2 Aortic transplantation model and tissue preparation

      Rat model of abdominal aortic transplantation was established using a modified technique as our previous study [
      • Yu Q.
      • Li W.
      • Xie D.
      • Zheng X.
      • Huang T.
      • et al.
      PI3Kgamma promotes vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis via a SOX9-dependent mechanism.
      ,
      • Zheng X.
      • Yu Q.
      • Shang D.
      • Yin C.
      • Xie D.
      • et al.
      TAK1 accelerates transplant arteriosclerosis in rat aortic allografts by inducing autophagy in vascular smooth muscle cells.
      ]. Briefly, male Lewis rats were used as donors, syngeneic recipients and allogeneic recipients. Male BN rats served as donors. After anesthesia with an inhaled anesthesia mixture of isoflurane (3%) and oxygen (1 L/min), a segment of abdominal aorta approximately 1 cm in length was isolated from the donors and gently perfused 3–5 times with pre-cooling DMEM. The clean aortic grafts were soaked into opti-MEM medium containing lentiviruses expressing Bves with a specific Tagln promoter (GeneChem, Shanghai, China) or the corresponding negative control (Vector) (2 × 107 TU/ml) in the presence of polybrene (5 μg/ml; Sigma-Aldrich, St. Louis, USA) for 60 min at room temperature. Subsequently, the aortic graft was transplanted orthotopically into the position below the renal artery and above the iliac bifurcation. A proximal end-to-end microsurgical anastomosis was performed with 11–0 single interrupted nylon suture. The ischemic time was approximately 30 min. Metamizole (50 mg/100 ml water) was used for painkilling for 3 days after transplantation.
      The recipient rats were euthanized with an overdose of pentobarbital sodium (180 mg/kg) 2 and 8 weeks after transplantation. For transcriptome microarray and PCR analyses at 2 weeks, after in situ cardiac perfusion with ice-cold sterile saline, the adventitia of aortic grafts was removed and the endothelium was destroyed by advancing an angioplasty guide wire into aortic lumen and pulling back 2–3 times, and medial VSMCs were collected. For histological and immunostaining analyses, rats were situ perfused with ice-cold sterile saline followed by perfusion fixation with 4% paraformaldehyde, aortic grafts were then harvested and fixed with 4% paraformaldehyde overnight at room temperature.

      2.3 RNA sequencing (RNA-seq) analysis

      Total RNA was extracted from the medial VSMCs in aortic grafts, PDGF-treated VSMCs and siBves-transfected VSMCs using TRIzol Reagent (Invitrogen) following the manufacturer's instructions. Differentially expressed genes between two groups were evaluated using DESeq2 with adjusted p value < 0.05 and |log2 (fold change) | ≥ 1 were considered to be significantly different expressed genes. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using Goatools and KOBAS. All raw data have been uploaded to the Sequence Read Archive (SRA, NCBI) database under the BioProject accession number PRJNA862185.

      2.4 Histology and morphometry

      The arterial segments were dehydrated in ethanol, infiltrated in xylene and embedded in paraffin. Serial cross-sections (5 μm thick) were cut and 5 cross-sections (250 μm apart) were selected and stained with hematoxylin-eosin (H&E) and elastic tissue fibers-Verhoeff's Van Gieson (EVG). Morphometry analyses of digital images of stained sections were performed by two independent investigators blinded to the experimental design using Image-Pro Plus software (Media Cybernetics, Inc. Silver Spring, MD, USA). The lumen area, intimal area, medial area, and total vessel area were measured at each level as previously described [
      • Yu Q.
      • Li W.
      • Xie D.
      • Zheng X.
      • Huang T.
      • et al.
      PI3Kgamma promotes vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis via a SOX9-dependent mechanism.
      ]. The mean media and intima area, intima/media ratio, and lumen stenosis ratio was calculated.

      2.5 Immunostaining

      Immunohistochemistry and immunofluorescence staining were performed on paraffin-embedded sections using specific primary antibodies, positive cell area and counting were conducted as previously reported [
      • Yu Q.
      • Li W.
      • Jin R.
      • Yu S.
      • Xie D.
      • et al.
      PI3Kgamma (phosphoinositide 3-kinase gamma) regulates vascular smooth muscle cell phenotypic modulation and neointimal formation through CREB (cyclic AMP-response element binding protein)/YAP (Yes-Associated protein) signaling.
      ].

      2.6 Cell culture and treatment

      Primary VSMCs were isolated from thoracic aorta of BN rats using an enzymatic digestion method as described by Geisterfer AA et al. [
      • Geisterfer A.A.
      • Peach M.J.
      • Owens G.K.
      Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells.
      ]. VSMCs were cultured with DMEM/F12 (Life Technology, Grand Island, USA) containing 10% FBS (Life Technology, Grand Island, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Life Technology, Grand Island, USA) at 37 °C and 5% CO2. All in vitro experiments were performed on cells passaged for 3 to 6 generations.
      VSMCs were transfected with small interfering RNA targeting Bves (siBves; RiBo biotechnology, Guangzhou, China) and Dusp1 (siDusp1; RiBo biotechnology, Guangzhou, China) using transfection reagent Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
      For Bves and Dusp1 overexpression, cultured vSMCs were transfected with plasmid pcDNA3.1 encoding rat Bves gene (GeneChem, Shanghai, China) and Dusp1 gene (GeneChem, Shanghai, China), respectively, by using transfection reagent Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

      2.7 Western blotting

      Western blotting was performed as previously described [
      • Yu Q.
      • Li W.
      • Jin R.
      • Yu S.
      • Xie D.
      • et al.
      PI3Kgamma (phosphoinositide 3-kinase gamma) regulates vascular smooth muscle cell phenotypic modulation and neointimal formation through CREB (cyclic AMP-response element binding protein)/YAP (Yes-Associated protein) signaling.
      ], using corresponding primary antibodies. Protein blots were imaged using the ChemiDoc imaging system (Bio-Rad, California, USA) and quantified using the software AlphaEase FC (Alpha Innotech, California, USA).

      2.8 Quantitative RT-PCR

      Total RNA was isolated from rat aortic grafts or cultured VSMCs using TRIzol Reagent (TaKaRa Biotechnology, Dalian, China), according to the manufacturer's guidelines. The amount of target mRNA was calculated by the 2-۵۵Ct relative quantification method. Glyceraldehydec-3-phosphate dehydrogenase (Gapdh) was used for normalization.

      2.9 Cell proliferation assay

      Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and EdU incorporation assay as previously reported [
      • Zheng X.
      • Yu Q.
      • Shang D.
      • Yin C.
      • Xie D.
      • et al.
      TAK1 accelerates transplant arteriosclerosis in rat aortic allografts by inducing autophagy in vascular smooth muscle cells.
      ].

      2.10 Cell migration assay

      VSMC migration was evaluated by Transwell migration assay as previously described [
      • Yu Q.
      • Li W.
      • Jin R.
      • Yu S.
      • Xie D.
      • et al.
      PI3Kgamma (phosphoinositide 3-kinase gamma) regulates vascular smooth muscle cell phenotypic modulation and neointimal formation through CREB (cyclic AMP-response element binding protein)/YAP (Yes-Associated protein) signaling.
      ]. Migration rate was estimated by the number of cells quantified in five different fields.

      2.11 Statistical analysis

      Statistical analyses were undertaken using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA) software. All values are presented as the mean ± SEM. Data distribution and equal variance analysis were determined using Shapiro-Wilk test and Levene test, respectively. When normal distribution was confirmed, unpaired two-tailed Student t-test was performed for statistical differences between two independent groups, or one-way ANOVA with Bonferroni post hoc test was conducted for multiple group comparisons. p < 0.05 was considered statistically significant.

      3. Results

      3.1 Bves expression is downregulated in aortic allografts and PDGF-treated VSMCs

      A change in the VSMC phenotype is a major feature of the pathogenesis of neointimal formation during the development of transplant vasculopathy [
      • Abrahimi P.
      • Liu R.
      • Pober J.S.
      Blood vessels in allotransplantation.
      ,
      • Bojakowski K.
      • Religa P.
      • Bojakowska M.
      • Hedin U.
      • Gaciong Z.
      • et al.
      Arteriosclerosis in rat aortic allografts: early changes in endothelial integrity and smooth muscle phenotype.
      ]. To visualize novel aortic gene expression responsible for modulating VSMC phenotype plasticity, we established a rat model of graft arteriosclerosis and applied RNA sequencing to analyze medial VSMCs in rat aortic grafts after aortic transplantation and in cultured VSMCs treated with PDGF, a well-characterized inducer leading to a change in the VSMC phenotypic identity [
      • Kaplan-Albuquerque N.
      • Van Putten V.
      • Weiser-Evans M.C.
      • Nemenoff R.A.
      Depletion of serum response factor by RNA interference mimics the mitogenic effects of platelet derived growth factor-BB in vascular smooth muscle cells.
      ]. After rigorous quality control and filtering, 110 upregulated and 86 downregulated genes were commonly identified (|log2 (fold change) | > 1; p adj. < 0.01) in the aortic allografts and PDGF-treated VSMCs. A Gene Ontology (GO) analysis of these downregulated genes combined with a literature search revealed the top 10 enriched terms linked to vascular function and homeostasis. More specifically, we noted that the Bves gene was enriched in the terms “smooth muscle cell differentiation” and “smooth muscle cell development” (Fig. 1A), indicating a potential link between Bves and the VSMC identity. Interestingly, almost all well-known VSMC contractile phenotype markers, such as myocardin (Myocd), Acta2, Calponin1 (Cnn1) and Tagln, were also identified in the list of these downregulated genes (Fig. 1B). A subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the decrease in Bves mRNA expression in rat aortic allografts compared with that in the isograft controls following transplantation (Fig. 1C). Consistently, the immunofluorescence staining of the aortic grafts showed that Bves expression was restricted to the medial layer of the aortic grafts, and the staining intensity of Bves in the aortic allografts was markedly lower than that in the isografts at 2 weeks (Fig. 1D). In parallel, the in vitro studies revealed that PDGF treatment dramatically inhibited Bves expression in cultured VSMCs at both the mRNA (Fig. 1E) and protein (Fig. 1F) levels. Specifically, a reduction in Bves was mainly observed in the cell membrane of VSMCs treated with PDGF, as measured by immunofluorescent staining (Fig. 1G). Additionally, the expression profile analyses screened a positive correlation between Bves and the expression of VSMC contractile phenotype markers, including Myocd (Fig. 1H), Acta2 (Fig. 1I), Cnn1 (Fig. 1J) and Tagln (Fig. 1K), in both aortic allografts and isograft controls, suggesting that Bves may serve as a novel housekeeping gene maintaining the contractile phenotype of VSMCs.
      Fig. 1
      Fig. 1Bves expression is downregulated in aortic allografts and PDGF-treated VSMCs.
      (A) Gene Ontology analysis of downregulated and overlapping genes (|log2FC (allograft/isograft)| > 1; p adjust <0.01) in medial cells of isografts (n = 10 rats) and allografts (n = 9 rats) 2 weeks after transplantation and PDGF-BB-treated VSMCs, as determined by RNA sequencing. The top 10 terms and corresponding 35 genes are shown. (B) Heatmap shows these 35 differentially downregulated and overlapping genes. (C) qRT-PCR analysis of Bves mRNA expression within medial cells of aortic grafts 2 weeks after transplantation (n = 10 rats per group). (D) Representative cross sections of aortic grafts immunostained for Bves (green) 2 weeks after transplantation. Cell nuclei were stained with Hoechst (blue). Scale bar: 100 μm. A, adventitial; L, lumen. The staining intensity was quantified as the percentage of Bves positive area in the media of aortic grafts (n = 15 rats per group). (E) qRT-PCR analysis of Bves mRNA in PDGF (20 ng/ml)-treated VSMCs for 24 h (n = 6). (F) Western blotting and densitometric analysis of Bves protein in VSMCs exposed to PDGF (20 ng/ml) for 24 h (n = 6). (G) Immunofluorescence staining of Bves (red) in VSMCs stimulated with PDGF (20 ng/ml) for 24 h. Cell nuclei were stained with Hoechst (blue). Scale bar: 50 μm. Bves expression positively correlated with Mycod (H), Acta2 (I), Cnn1 (G) and Tagln (K) expression in medial cells of aortic allografts (red) and isografts (blue) 2 weeks after transplantation. **p < 0.01 and ***p < 0.001.

      3.2 Bves maintains the contractile phenotype of VSMCs

      We further dissected whether Bves downregulation was associated with VSMC dedifferentiation. siRNA-mediated Bves knockdown in VSMCs resulted in significantly reduced protein levels of VSMC contractile phenotype markers (Fig. 2A). VSMCs with Bves knockdown exhibited greater proliferative activity than control VSMCs, as indicated by a CCK-8 assay (Fig. 2B) and an EdU incorporation assay (Fig. 2C). Furthermore, cell migration was markedly suppressed by the Bves knockdown, as determined by a Transwell assay (Fig. 2D). The PDGF treatment in vitro was sufficient to recapitulate the changes in gene expression associated with VSMC dedifferentiation [
      • Kaplan-Albuquerque N.
      • Van Putten V.
      • Weiser-Evans M.C.
      • Nemenoff R.A.
      Depletion of serum response factor by RNA interference mimics the mitogenic effects of platelet derived growth factor-BB in vascular smooth muscle cells.
      ]. We found that Bves overexpression promoted the VSMC contractile phenotype, as determined by an enhanced basal expression of VSMC contractile phenotype proteins, specifically, the mitigation of the PDGF-induced repression of VSMC contractile phenotype proteins (Fig. 2E), and the amelioration of aberrant VSMC proliferation (Fig. 2F and G) and migration (Fig. 2H) induced by PDGF. To verify the functional contribution of Bves to VSMC differentiation in vitro, we generated smooth muscle specific, Bves-overexpressing lentivirus driven by the smooth muscle-specific Tagln promoter and infected donor allografts. Bves-overexpressing or control arteries from donors were transplanted into recipients. The qRT-PCR results demonstrated that endogenous Bves expression was dramatically downregulated in the aortic allografts relative to that in the isograft controls, while the local delivery of the Bves-overexpressing lentivirus markedly restored Bves expression in the allografts and mitigated the alloimmune injury-induced downregulation of VSMC contractile phenotype genes 2 weeks after transplantation (Fig. 2I), confirming the crucial role of Bves in controlling VSMC differentiation in vivo. Subsequently, immunofluorescence staining of the proliferation markers ki67 and Cyclin D1 was performed to evaluate cell proliferation in the medial layers of the aortic grafts. We found more ki67-and Cyclin D1-positive medial VSMCs in the allografts than in the isograft controls, whereas the number of positive cells was diminished in the Bves-overexpressing aortic allografts at 2 weeks posttransplantation (Fig. 2J). Taken together, these results emphasize that Bves is critical for maintaining the VSMC contractile phenotype in vivo and in vitro.
      Fig. 2
      Fig. 2Bves maintains the contractile phenotype of VSMCs.
      (A) Western blotting and densitometric analysis of Bves, Myocd, Acta2, Cnn1 and Tagln proteins expression in VSMCs transfected with siBves or negative siControl (n = 6). (B) CCK-8 assay was performed on VSMCs transfected with siBves for indicated time. (C) VSMC proliferation was evaluated by EdU (red) incorporation in siBves-transfected VSMCs for 24 h. Scale bar: 50 μm. Quantification of percentage of EdU positive cells is shown (n = 6). (D) Transwell assay was performed in VSMCs transfected with siBves. Representative images and quantification of number of migrated VSMCs on the bottom of transwell membrane (n = 6). (E) Western blotting and densitometric analysis of Bves, Myocd, Acta2, Cnn1 and Tagln proteins expression in VSMCs transfected with plasmids overexpressing Bves or its control Vector together with PDGF (20 ng/ml) stimulation (n = 6). VSMCs were transfected with plasmids overexpressing Bves or its control vector followed by PDGF (20 ng/ml) stimulation for 24 h, (F) CCK-8 assay was performed to measured cell viability; (G) EdU incorporation assay was performed to detect cell proliferation as presented as the percentage of EdU-positive cells; (H) Transwell assay was performed to assess cell migration, respectively (n = 6). (I) qRT-PCR analysis of Bves, Myocd, Acta2, Cnn1 and Tagln mRNA within the media of aortic grafts transfected with recombinant lentiviral vectors carrying Bves gene and a specific Tagln promoter (n = 10 rats per group). (J) Representative cross sections of aortic grafts immunostained for ki67 and Cyclin D1 2 weeks after transplantation. Cell nuclei were stained with Hoechst. Scale bar: 100 μm. Quantification of the number of ki67-and Cyclin D1-positive cells is shown (n = 15 rats per group). *p < 0.05, **p < 0.01 and ***p < 0.001.

      3.3 VSMC-specific Bves overexpression attenuates neointimal formation in aortic allografts

      Because Bves is repressed in aortic allografts and plays a functional role in the VSMC phenotypic transition, we subsequently investigated the impact of VSMC-derived Bves on the development of graft arteriosclerosis. A VSMC-specific Bves-overexpressing lentivirus with a Tagln promoter was used to infect donor allografts. We found that the local delivery of the Bves-overexpressing lentivirus markedly restored the downregulation of Bves expression induced by alloimmune injury in the donor allografts 8 weeks after transplantation, as measured by qRT-PCR (not shown). Our previous study confirmed an increase in neointimal formation in aortic allografts compared with that in isograft controls [
      • Yu Q.
      • Li W.
      • Xie D.
      • Zheng X.
      • Huang T.
      • et al.
      PI3Kgamma promotes vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis via a SOX9-dependent mechanism.
      ]. H&E and EVG staining showed that the formation of neointimal lesions in the Bves-overexpressing donor allografts was markedly decreased relative to that in the control allografts (Fig. 3A). The intimal area of donor allografts infected with the Bves-overexpressing lentivirus was thinner than that of the control allografts (Fig. 3B). Bves overexpression significantly reduced the ratio of the intimal-to medial-area (Fig. 3C), leading to a substantial decline in the lumen stenosis ratio (Fig. 3D). Notably, no statistical difference was observed in the medial area between the Bves-overexpressing allografts and control allografts (Fig. 3E). Further immunohistochemical staining of the donor grafts revealed that Bves overexpression in VSMCs generally increased the Acta2 signals in the media of the aortic allografts (Fig. 3F). Furthermore, the Ki67 staining in the graft sections was markedly reduced by the Bves overexpression (Fig. 3F), indicating the negative role of Bves in controlling VSMC proliferation in vivo. These data suggest that Bves ameliorates neointima formation by inhibiting VSMC proliferation and accumulation in the neointima of aortic allografts.
      Fig. 3
      Fig. 3VSMC-specific Bves overexpression attenuates neointimal formation in aortic allografts.
      (A) Representative images of H&E staining and EVG staining in cross sections of aortic allografts transfected with recombinant lentiviral vectors carrying Bves gene and a specific Tagln promoter 8 weeks after transplantation. Scale bar: 100 μm. Black boxes indicate the enlarged sections of aortic grafts. Quantification of intima area (B), intima/media ratio (C), lumen stenosis ratio (D) and media area (E) (n = 15 rats per group). (F) Representative cross sections of Bves gene transfected aortic allografts immunostained for Acta2 and ki67 8 weeks after transplantation. Scale bar: 100 μm. The staining intensity was quantified as the percentage of Acta2 positive area in the media of aortic allografts and the quantification of the number of ki67 positive cells is shown (n = 15 rats per group). *p < 0.05 and **p < 0.01.

      3.4 Dusp1 is a downstream target of Bves in VSMCs

      To further examine the mechanism by which Bves regulates VSMC phenotypic plasticity, we knocked down Bves in cultured VSMCs using siRNA transfection. The RNA sequencing of these cells identified 648 differentially expressed genes after Bves knockdown, including 191 downregulated and 457 upregulated genes (|log2 (fold change) | >1; p adj. < 0.01) (Fig. 4A). Of all 191 downregulated genes, 20 genes overlapped within the list of downregulated genes between aortic allografts and PDGF-treated VSMCs and, thus, were likely putative targets of Bves. Importantly, Bves and VSMC contractile phenotype genes (Myocd, Acta2, Cnn1 and Tagln) were identified among these downregulated genes (Fig. 4B). An unbiased inspection of the potential physiological functions of these 20 common genes by a KEGG pathway analysis revealed significant enrichment in the MAPK signaling pathway (Fig. 4C), which was previously reported to be functionally linked to vessel biological functions, including VSMC proliferation and migration [
      • Gennaro G.
      • Menard C.
      • Michaud S.E.
      • Deblois D.
      • Rivard A.
      Inhibition of vascular smooth muscle cell proliferation and neointimal formation in injured arteries by a novel, oral mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor.
      ,
      • Proctor B.M.
      • Jin X.
      • Lupu T.S.
      • Muglia L.J.
      • Semenkovich C.F.
      • et al.
      Requirement for p38 mitogen-activated protein kinase activity in neointima formation after vascular injury.
      ]. Among these two candidate targets, dual-specificity protein phosphatase 1 (Dusp1) was preferentially selected for subsequent validation. We found that Bves knockdown significantly inhibited Dusp1 expression at both the mRNA (Fig. 4D) and protein (Fig. 4E) levels in VSMCs compared to that in the control VSMCs. Consistent with the RNA sequencing results, PDGF treatment decreased Dusp1 expression, and Bves overexpression was able to upregulate the baseline Dusp1 levels and significantly reversed the PDGF-induced inhibition of Dusp1 (Fig. 4F and G). Moreover, a similar repressive effect on Dusp1 expression was observed in the medial layer of the donor allografts, while the restoration of Bves largely alleviated alloimmune injury-induced repression of Dusp1 transcription (Fig. 4H) and immunostaining signals (Fig. 4I) in the donor allografts at 2 weeks after transplantation, highlighting the critical role of Bves in Dusp1 expression in VSMCs.
      Fig. 4
      Fig. 4Dusp1 is a downstream target of Bves in VSMCs.
      (A) Volcano plot shows genes regulated by Bves knockdown in VSMCs as measured by RNA sequencing (n = 4). (B) Venn diagram shows overlapping genes in aortic grafts 2 weeks after transplantation, PDGF-treated VSMCs for 24 h and siBves-transfected VSMCs based on RNA sequencing data. Heatmap shows the 20 overlapping genes. (C) KEGG enrichment analysis shows top 5 terms of biological process of the 20 overlapping genes. Size of circles represents the number of overlapping genes. (D) qRT-PCR analysis of Dusp1 mRNA in VSMCs transfected with siBves (n = 6). (E) Western blotting analysis of Dusp1 protein in VSMCs transfected with siBves, representative blots (Ei) and densitometric analysis (Eii) are shown (n = 6). VSMCs were transfected with plasmids overexpressing Bves or its control vector followed by PDGF (20 ng/ml) stimulation, (F) Dusp1 mRNA expression was measured by qRT-PCR (n = 6); (G) Dusp1 protein was detected by Western blotting, representative blots and densitometric analysis are shown (n = 6). (H) qRT-PCR analysis of Dusp1 mRNA within the media of aortic grafts transfected with recombinant lentiviral vectors carrying Bves gene and a specific Tagln promoter (n = 10 rats per group). (I) Representative cross sections of Bves gene transfected aortic allografts immunostained for Dusp1 at 2 weeks. Scale bar: 100 μm. The staining intensity was quantified as the percentage of Dusp1 positive area in the media of aortic allografts (n = 15 rats per group). *p < 0.05 and **p < 0.01.

      3.5 Bves-driven Dusp1 expression is required for the VSMC contractile phenotype

      Next, we interrogated the relevance of Dusp1 in the VSMC contractile phenotype controlled by Bves in vitro. Consistent with the repressive effect of Bves knockdown on maintaining VSMC differentiation, Dusp1 knockdown significantly diminished the basal expression of VSMC contractile phenotype markers in cultured VSMCs (Fig. 5A). Additionally, Dusp1 overexpression circumvented the PDGF-induced repression of the protein expression of these markers (Fig. 5B). The combination of Bves knockdown and Dusp1 overexpression in PDGF-stimulated VSMCs showed that Dusp1 overexpression drastically reversed the Bves knockdown-induced downregulation of VSMC contractile protein expression (Fig. 5B). Furthermore, Dusp1 overexpression attenuated the functional contribution of Bves knockdown to VSMC proliferation and migration, as indicated by an EdU incorporation assay (Fig. 5C) and a Transwell assay (Fig. 5D), respectively. In vivo, the local delivery of Dusp1 in medial VSMCs in aortic allografts by lentivirus-mediated gene transfection recapitulated the effect of Bves overexpression on the histological features of aortic allografts 8 weeks after allograft transplantation (Fig. 5E), as indicated by the decrease in the intima area (Fig. 5F) and the declined ratio of the intima to media (Fig. 5G) that led to the reduction in lumen stenosis in aortic allografts (Fig. 5H). Altogether, these findings demonstrate that Bves maintains the VSMC contractile phenotype by targeting Dusp1 in VSMCs.
      Fig. 5
      Fig. 5Bves-driven Dusp1 expression is required for the VSMC contractile phenotype.
      (A) Western blotting and densitometric analysis of Dusp1, Myocd, Acta2, Cnn1 and Tagln protein expression in VSMCs transfected with siDusp1 or corresponding negative siControl (n = 6). (B) VSMCs were cotransfected with Bves siRNA and Dusp1 overexpressing plasmid together with PDGF (20 ng/ml) stimulation for 24 h, protein expression of Dusp1, Myocd, Acta2, Cnn1 and Tagln were determined by Western blotting, densitometric analysis of these proteins is shown (n = 6). (C) EdU incorporation assay was performed on VSMCs cotransfected with Bves siRNA and Dusp1 overexpressing plasmid. Cell nuclei were stained with Hoechst (blue). Scale bar: 50 μm. Quantification of the number of EdU positive cells is shown (n = 6). (D) VSMCs were cotransfected with Bves siRNA and Dusp1 overexpressing plasmid, cell migration was detected by Transwell assay and quantification of the migrated cell number is shown (n = 6). (E) Representative images of H&E staining and EVG staining in cross sections of aortic allografts transfected with recombinant lentiviral vectors carrying Dusp1 gene and a specific Tagln promoter 8 weeks after transplantation. Scale bar: 100 μm. Quantification of intimal area (F), intima/media ratio (G) and lumen stenosis ratio (H) (n = 10 rats per group). *p < 0.05, **p < 0.01 and ***p < 0.001.

      3.6 Effects of Bves on the phosphorylation of p38MAPK and ERK1/2

      Dusp1 is a known inhibitor of MAPK kinase signaling that probably inactivates p38MAPk, ERK1/2 and c-Jun N-terminal kinase (JNK) via dephosphorylation. Given the regulation of Bves in Dusp1, we examined the impacts of Bves on the potent activation of the MAPK signaling pathway. As shown in Fig. 6A, the levels of phosphorylated p38MAPK and ERK1/2 were significantly reduced in VSMCs after Bves and Dusp1 knockdown, but no obvious difference was observed in JNK phosphorylation between the two groups. In line with the in vitro findings, the immunofluorescence staining showed significant decreases in either p38MAPK or ERK1/2 phosphorylation in the media of the aortic allografts after the VSMC-specific overexpression of Bves and Dusp1 in vivo (Fig. 6B), demonstrating that Bves mediated the Dusp1-dependent dephosphorylation of p38MAPK and ERK1/2. Further validation of the effects of p38MAPK and ERK1/2 activation on the VSMC phenotype was performed in Bves knockdown VSMCs. We found that treatment with either the p38MAPK-specific inhibitor SB203580 or the ERK1/2-specific inhibitor PD98059 markedly abrogated the downregulation of VSMC contractile phenotype proteins induced by Bves knockdown in vitro VSMCs (Fig. 6C). Taken together, these findings demonstrate that Dusp1-dependent p38MAPK and ERK1/2 dephosphorylation and inactivation are likely the predominant causal factors by which Bves maintains the VSMC contractile phenotype.
      Fig. 6
      Fig. 6Effects of Bves on the phosphorylation of p38MAPK and ERK1/2.
      (A) Western blotting and densitometric analysis of p-p38MAPK, p38MAPK, p-ERK1/2, ERK1/2, p-JNK and JNK expression in VSMCs transfected with siBves or siDusp1 (n = 6). (B) Representative cross sections of Bves or Dusp1 gene transfected aortic allografts immunostained for p-p38MAPK and p-ERK1/2 2 weeks after transplantation. Scale bar: 100 μm. The staining intensity was quantified as the percentage of p-ERK1/2 and p-p38MAPK positive area in the media of aortic allografts (n = 15 rats per group). (C) VSMCs transfected with siBves were treated with p38MAPK-specific inhibitor SB203580 or ERK1/2-specific inhibitor PD98059, representative blots and densitometric analysis of Myocd, Acta2, Cnn1 and Tagln proteins are shown (n = 6). ns, no significance. *p < 0.05 and **p < 0.01.

      4. Discussion

      Understanding the potential molecular mechanisms controlling the VSMC contractile phenotype plays a pivotal role in exploring the endogenous protective factors against transplant vasculopathy. In this study, we used an RNA sequencing approach to identify novel and reduced Bves expression in the vascular walls of rat allograft arteriosclerosis models and VSMCs undergoing phenotypic modulation in response to PDGF stimulation in vitro. The comprehensive analyses of the functional changes in Bves in vitro VSMCs revealed that Bves expression was necessary for maintaining the VSMC contractile phenotype by mediating Dusp1 expression and subsequent p38MAPK and ERK1/2 inactivation. Furthermore, we report that VSMC-specific Bves and Dusp1 overexpression in aortic allografts prevents the changes in VSMC contractile phenotype and restrains the formation of neointimal lesions and lumen stenosis in aortic allografts, which may provide new mechanistic insight into therapeutic strategies for transplant vasculopathy.
      Bves was initially identified as a novel biomarker of the development of coronary blood vessels and was reported to be highly expressed in arterial vascular channels during intracardiac vasculogenesis [
      • Reese D.E.
      • Zavaljevski M.
      • Streiff N.L.
      • Bader D.
      bves: a novel gene expressed during coronary blood vessel development.
      ]. The accumulation of Bves in VSMCs in differentiated intracardiac arteries prior to the expression of VSMC-specific contractile proteins reveals an association between Bves and the cellular regulation of smooth muscle differentiation [
      • Reese D.E.
      • Zavaljevski M.
      • Streiff N.L.
      • Bader D.
      bves: a novel gene expressed during coronary blood vessel development.
      ]. Bves has a vasculoprotective effect against ischemia/reperfusion injury, oxidative stress and environmental stress [
      • Froese A.
      • Breher S.S.
      • Waldeyer C.
      • Schindler R.F.
      • Nikolaev V.O.
      • et al.
      Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.
      ,
      • Amunjela J.N.
      • Swan A.H.
      • Brand T.
      The role of the Popeye domain containing gene family in organ homeostasis.
      ,
      • Alcalay Y.
      • Hochhauser E.
      • Kliminski V.
      • Dick J.
      • Zahalka M.A.
      • et al.
      Popeye domain containing 1 (Popdc1/Bves) is a caveolae-associated protein involved in ischemia tolerance.
      ]. These notions are supported by our findings in this study showing that Bves gene expression was reduced in the vascular wall of rat allograft arteriosclerosis models and VSMCs undergoing PDGF stimulation in vitro, and changes in Bves gene expression were associated with a decreased expression of VSMC contractile genes in aortic allografts 2 weeks after transplantation. PDGF is a critical mitogen and chemoattractant correlated with the development of cardiac allograft vasculopathy [
      • Lemstrom K.B.
      • Koskinen P.K.
      Expression and localization of platelet-derived growth factor ligand and receptor protein during acute and chronic rejection of rat cardiac allografts.
      ]. Evidence suggests that PDGF and PDGF receptor β (PDGFRβ) are potent regulators of the VSMC differentiation program and the formation of neointimal lesions [
      • Dong L.H.
      • Wen J.K.
      • Miao S.B.
      • Jia Z.
      • Hu H.J.
      • et al.
      Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFRbeta-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia.
      ]. Studies in animal models of graft arteriosclerosis demonstrate that the phenotypic transition in VSMCs towards a dedifferentiated phenotype may occur as early as one week after transplantation and seems to be closely related to the pathogenesis of transplant arteriosclerosis [
      • Alexander M.R.
      • Owens G.K.
      Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease.
      ,
      • Bojakowski K.
      • Religa P.
      • Bojakowska M.
      • Hedin U.
      • Gaciong Z.
      • et al.
      Arteriosclerosis in rat aortic allografts: early changes in endothelial integrity and smooth muscle phenotype.
      ]. The results of the gain- and loss-of-function assays suggest that Bves alone is sufficient and essential for maintaining the VSMC contractile phenotype in vitro, specifically, Bves plays a critical role in repressing VSMC proliferation and migration, which are key cellular processes during the development of transplant arteriosclerosis [
      • Alexander M.R.
      • Owens G.K.
      Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease.
      ,
      • Yu Q.
      • Li W.
      • Xie D.
      • Zheng X.
      • Huang T.
      • et al.
      PI3Kgamma promotes vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis via a SOX9-dependent mechanism.
      ].
      The data in the present study further show that Bves overexpression in medial VSMCs in aortic allografts was associated with decreased Acta2-positive VSMCs, reduced cell proliferation within the lesions and decreased neointimal formation, thereby providing in vivo evidence that restoring VSMC-specific Bves expression delays alloimmune injury-induced formation of neointimal lesions in aortic allografts. These findings are supported by previous studies showing that highly proliferative and migratory VSMCs contribute to the neointimal formation [
      • Alexander M.R.
      • Owens G.K.
      Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease.
      ]. To date, there is no direct evidence regarding the role of Bves in VSMC behavior and vascular homeostasis. Interestingly, although Bves is widely expressed, Bves appears to have a common function and behavior in a variety of cell types because of its same membrane labelling pattern [
      • Wada A.M.
      • Reese D.E.
      • Bader D.M.
      Bves: prototype of a new class of cell adhesion molecules expressed during coronary artery development.
      ]. Bves interacts with guanine nucleotide exchange factor T (GEFT) protein, which has previously been identified as a negative regulator of GEFT activity [
      • Smith T.K.
      • Hager H.A.
      • Francis R.
      • Kilkenny D.M.
      • Lo C.W.
      • et al.
      Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity.
      ], and promotes actin cytoskeletal arrangement [
      • Smith T.K.
      • Hager H.A.
      • Francis R.
      • Kilkenny D.M.
      • Lo C.W.
      • et al.
      Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity.
      ], alterations in cell morphology, neurite outgrowth [
      • Bryan B.A.
      • Cai Y.
      • Liu M.
      The Rho-family guanine nucleotide exchange factor GEFT enhances retinoic acid- and cAMP-induced neurite outgrowth.
      ], cell differentiation and skeletal muscle regeneration [
      • Bryan B.A.
      • Mitchell D.C.
      • Zhao L.
      • Ma W.
      • Stafford L.J.
      • et al.
      Modulation of muscle regeneration, myogenesis, and adipogenesis by the Rho family guanine nucleotide exchange factor GEFT.
      ], and inhibits cell motility and cell proliferation [
      • Guo X.
      • Stafford L.J.
      • Bryan B.
      • Xia C.
      • Ma W.
      • et al.
      A Rac/Cdc42-specific exchange factor, GEFT, induces cell proliferation, transformation, and migration.
      ]. Indeed, alterations in the cell shape and actin cytoskeleton integrity could potentially affect cell proliferation in adhesion-dependent cells [
      • Huang S.
      • Chen C.S.
      • Ingber D.E.
      Control of cyclin D1, p27(Kip1), and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension.
      ]. In vitro studies suggest an important function of Bves in promoting epicardial cell directional movement that is essential for tissue morphogenesis and wound healing [
      • Benesh E.C.
      • Miller P.M.
      • Pfaltzgraff E.R.
      • Grega-Larson N.E.
      • Hager H.A.
      • et al.
      Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition.
      ,
      • Ripley A.N.
      • Chang M.S.
      • Bader D.M.
      Bves is expressed in the epithelial components of the retina, lens, and cornea.
      ,
      • Ripley A.N.
      • Osler M.E.
      • Wright C.V.
      • Bader D.
      Xbves is a regulator of epithelial movement during early Xenopus laevis development.
      ]. Moreover, studies have identified Bves as an effector protein of the second messenger cyclic 3′,5′-adenosine monophosphate (cAMP) [
      • Shetty M.S.
      • Ris L.
      • Schindler R.F.R.
      • Mizuno K.
      • Fedele L.
      • et al.
      Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity.
      ], which seems important for maintaining VSMC quiescence in healthy vessels and preventing VSMC proliferation, migration and neointimal formation following vascular injury [
      • Sassi Y.
      • Lipskaia L.
      • Vandecasteele G.
      • Nikolaev V.O.
      • Hatem S.N.
      • et al.
      Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation.
      ,
      • Indolfi C.
      • Di Lorenzo E.
      • Rapacciuolo A.
      • Stingone A.M.
      • Stabile E.
      • et al.
      8-chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima formation induced by balloon injury in vivo.
      ], highlighting the importance of Bves in neointimal formation and vascular remodeling in aortic allografts.
      The downstream events of Bves signaling activation remain poorly understood. Notably, our transcriptomic data show that Bves affects the VSMC contractile phenotype and neointimal formation by directly modulating Dusp1-dependent MAPK signaling. Dusp1 is a threonine/tyrosine dual-specificity phosphatase that belongs to the Dusp family, which is implicated in inflammation, immune regulation and responses to oxidative stress [
      • Lawan A.
      • Shi H.
      • Gatzke F.
      • Bennett A.M.
      Diversity and specificity of the mitogen-activated protein kinase phosphatase-1 functions.
      ,
      • Jeffrey K.L.
      • Camps M.
      • Rommel C.
      • Mackay C.R.
      Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses.
      ]. Dusp1 can dephosphorylate and inactivate MAPKs, including p38MAPK, ERK1/2 and JNK, with a high degree of specificity depending on the cell type, genetic context and extracellular stimulation [
      • Lawan A.
      • Shi H.
      • Gatzke F.
      • Bennett A.M.
      Diversity and specificity of the mitogen-activated protein kinase phosphatase-1 functions.
      ,
      • Zhao Q.
      • Wang X.
      • Nelin L.D.
      • Yao Y.
      • Matta R.
      • et al.
      MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock.
      ]. The same results were observed in vitro VSMCs, and we showed that Dusp1 knockdown preferentially phosphorylated p38MAPK and ERK1/2 but had no effect on JNK phosphorylation. Additionally, we found that Bves knockdown in VSMCs transcriptionally repressed Dusp1 expression and phenocopied the same effects as Dusp1 knockdown on MAPK activation, suggesting that Bves promotes the VSMC contractile phenotype via Dusp1-dependent p38MAPK and ERK1/2 activation. A recent study showed that mitogen activation of MAPK signaling was essential for the binding of ternary complex factors (TCFs) to their cofactor serum response factor (SRF), which regulates the transcription of target genes involved in proliferation [
      • Wozniak M.A.
      • Cheng C.Q.
      • Shen C.J.
      • Gao L.
      • Olarerin-George A.O.
      • et al.
      Adhesion regulates MAP kinase/ternary complex factor exchange to control a proliferative transcriptional switch.
      ,
      • Gualdrini F.
      • Esnault C.
      • Horswell S.
      • Stewart A.
      • Matthews N.
      • et al.
      SRF Co-factors control the balance between cell proliferation and contractility.
      ]. It is currently well established that TCFs directly compete with the myocardin family, including myocardin and myocardin-related transcription factors (MRTFs), for access to SRF, resulting in a decreased expression of VSMC contractile genes and elevated proliferation [
      • Mack C.P.
      Signaling mechanisms that regulate smooth muscle cell differentiation.
      ]. This evidence may explain our findings that blocking p38MAPK and ERK1/2 by using SB203580 and PD98059, respectively, could markedly restore Bves knockdown-induced downregulation of VSMC contractile proteins, offering a possible mechanism for the regulation of Bves in VSMC differentiation.
      More specifically, endogenous Dusp1 expression may be necessary for the inhibition of VSMC growth, proliferation and migration by inactivating p38MAPK and/or ERK1/2 in response to various environmental cues [
      • Begum N.
      • Ragolia L.
      High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation.
      ,
      • Li C.
      • Hu Y.
      • Mayr M.
      • Xu Q.
      Cyclic strain stress-induced mitogen-activated protein kinase (MAPK) phosphatase 1 expression in vascular smooth muscle cells is regulated by Ras/Rac-MAPK pathways.
      ]. It is plausible that the ectopic expression of Dusp1 is capable of blocking Bves knockdown-induced VSMC proliferation and migration. Studies involving in a carotid injury model showed that SMC-specific p38αMAPK expression promoted p38 MAPK activation and neointima formation after vascular injury [
      • Proctor B.M.
      • Jin X.
      • Lupu T.S.
      • Muglia L.J.
      • Semenkovich C.F.
      • et al.
      Requirement for p38 mitogen-activated protein kinase activity in neointima formation after vascular injury.
      ]. Systemic treatment with an ERK1/2 pharmacological inhibitor in rats may result in a decrease in VSMC proliferation that protects against neointimal lesion development induced by arterial balloon injury [
      • Gennaro G.
      • Menard C.
      • Michaud S.E.
      • Deblois D.
      • Rivard A.
      Inhibition of vascular smooth muscle cell proliferation and neointimal formation in injured arteries by a novel, oral mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor.
      ]. Accordingly, Dusp1, which is an upstream signaling molecule that induces specific overexpression in VSMCs is sufficient to rescue alloimmune injury-induced neointimal formation in vivo.
      Unlike other cAMP effector proteins, such as protein kinase A (PKA) and exchange factors directly activated by cAMP, the transmembrane protein Bves binds cAMP with a high affinity and exerts a unique effect on cAMP-dependent signaling due to its specific cAMP-binding domains [
      • Shetty M.S.
      • Ris L.
      • Schindler R.F.R.
      • Mizuno K.
      • Fedele L.
      • et al.
      Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity.
      ,
      • Brand T.
      • Schindler R.
      New kids on the block: the Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.
      ]. This notion is supported by a study in which Bves mediated cAMP signaling and regulated the biological activity of interacting proteins [
      • Brand T.
      • Simrick S.L.
      • Poon K.L.
      • Schindler R.F.
      The cAMP-binding Popdc proteins have a redundant function in the heart.
      ]. Of interest, in breast cancer cells, cAMP interacts with Bves to upregulate Bves expression and promote cell proliferation and migration [
      • Amunjela J.N.
      • Tucker S.J.
      Dysregulation of POPDC1 promotes breast cancer cell migration and proliferation.
      ]. Based on a previous study, the cAMP-driven transcriptional expression of Dusp1 has been identified as a beneficial inducer responsible for the anti-inflammatory effects of airway SMCs [
      • Patel B.S.
      • Prabhala P.
      • Oliver B.G.
      • Ammit A.J.
      Inhibitors of phosphodiesterase 4, but not phosphodiesterase 3, increase beta2-agonist-induced expression of antiinflammatory mitogen-activated protein kinase phosphatase 1 in airway smooth muscle cells.
      ]. A new question raised by our study is whether and how Bves controls Dusp1 transcriptional expression via cAMP signaling in VSMCs, and the potential mechanism should be further explored. Additionally, previous studies have illustrated that Acta2-expressing cells in neointimal lesions are likely derived from resident medial VSMCs, adventitial progenitor cells and circulating progenitors [
      • Atkinson C.
      • Horsley J.
      • Rhind-Tutt S.
      • Charman S.
      • Phillpotts C.J.
      • et al.
      Neointimal smooth muscle cells in human cardiac allograft coronary artery vasculopathy are of donor origin.
      ,
      • Kramann R.
      • Goettsch C.
      • Wongboonsin J.
      • Iwata H.
      • Schneider R.K.
      • et al.
      Adventitial MSC-like cells are progenitors of vascular smooth muscle cells and drive vascular calcification in chronic kidney disease.
      ,
      • Wan M.
      • Li C.
      • Zhen G.
      • Jiao K.
      • He W.
      • et al.
      Injury-activated transforming growth factor beta controls mobilization of mesenchymal stem cells for tissue remodeling.
      ]. Although the generation of VSMC-specific Bves overexpression in aortic allografts provides direct evidence of the functional contribution of Bves to the medial VSMC phenotype and neointimal formation in vivo, the extent of the role of Bves in other cell types in this process remains fully unknown, and thus needs to be further investigated.
      In summary, our work identified the critical role of VSMC-enriched Bves in the maintenance of VSMC differentiation by controlling Dusp1-dependent ERK 1/2 and p38MAPK inactivation. Restoring Bves expression in medial VSMCs in aortic allografts reduced VSMC proliferation and accumulation, which led to a reduction in neointimal lesions, suggesting a novel therapeutic target for proliferative cardiovascular diseases.

      Financial support

      This study was supported by the grants from the National Natural Science Foundation of China to Qihong Yu (No. 82000391 ) and Jin-Xin Liu (No. 82101199 ). This study was also supported by the grant from China Postdoctoral Science Foundation to Qihong Yu (No. 2021M691142 ).

      CRediT authorship contribution statement

      Jin-Xin Liu: Methodology, Data curation, Visualization, Writing – original draft, Funding acquisition. Tong Huang: Investigation, Methodology, Resources, Supervision. Dawei Xie: Investigation, Validation, Software, Resources. Qihong Yu: Conceptualization, Methodology, Data curation, Validation, Writing – original draft, Funding acquisition, Writing – review & editing.

      Declaration of interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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