Mindin regulates vascular smooth muscle cell phenotype and prevents neointima formation.

Clinical Science (2015) 129, 129–145 doi: 10.1042/CS20140679

Mindin regulates vascular smooth muscle cell phenotype and prevents neointima formation Li-Hua Zhu*†1 , Ling Huang*†1 , Xiaojing Zhang‡1 , Peng Zhang*†, Shu-Min Zhang*†, Hongjing Guan*†, Yan Zhang†, Xue-Yong Zhu†, Song Tian†, Keqiong Deng*† and Hongliang Li*† *Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, China †Cardiovascular Research Institute of Wuhan University, Wuhan 430060, China ‡State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao 999078, China

Abstract Mindin/spondin 2, an extracellular matrix (ECM) component that belongs to the thrombospondin type 1 (TSR) class of molecules, plays prominent roles in the regulation of inflammatory responses, angiogenesis and metabolic disorders. Our most recent studies indicated that mindin is largely involved in the initiation and development of cardiac and cerebrovascular diseases [Zhu et al. (2014) J. Hepatol. 60, 1046–1054; Bian et al. (2012) J. Mol. Med. 90, 895–910; Wang et al. (2013) Exp. Neurol. 247, 506–516; Yan et al. (2011) Cardiovasc. Res. 92, 85–94]. However, the regulatory functions of mindin in neointima formation remain unclear. In the present study, mindin expression was significantly down-regulated in platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) and wire injury-stimulated vascular tissue. Using a gain-of-function approach, overexpression of mindin in VSMCs exhibited strong anti-proliferative and anti-migratory effects on VSMCs, whereas significant suppression of intimal hyperplasia was observed in transgenic (TG) mice expressing mindin specifically in smooth muscle cells (SMCs). These mice exhibited blunted VSMC proliferation, migration and phenotypic switching. Conversely, deletion of mindin dramatically exacerbated neointima formation in a wire-injury mouse model, which was further confirmed in a balloon injury-induced vascular lesion model using a novel mindin-KO (knockout) rat strain. From a mechanistic standpoint, the AKT (Protein Kinase B)−GSK3β (glycogen synthase kinase 3β)/mTOR (mammalian target of rapamycin)−FOXO3A (forkhead box O)–FOXO1 signalling axis is responsible for the regulation of mindin during intimal thickening. Interestingly, an AKT inhibitor largely reversed mindin-KO-induced aggravated hyperplasia, suggesting that mindin-mediated neointima formation is AKT-dependent. Taken together, our findings demonstrate that mindin protects against vascular hyperplasia by suppression of abnormal VSMC proliferation, migration and phenotypic switching in an AKT-dependent manner. Up-regulation of mindin might represent an effective therapy for vascular-remodelling-related diseases. Key words: mindin, neointima formation, vascular injury, vascular smooth muscle cell.

INTRODUCTION Mindin/spondin 2, along with F-spondin, is an evolutionarily conserved extracellular matrix (ECM) protein of the thrombospondin type 1 repeat (TSR) class molecules. Since it was initially identified in zebrafish as a component of the basal lamina, mindin has been shown to influence a broad range of biological functions in mammals [1]. Based on F-spondin domains 1 and 2 (FS1 and FS2) at the N-terminus and a TSR domain at the C-terminus

[2], which principally determine its binding partner, mindin can function as an integrin ligand, a pattern-recognition molecule and a regulator of various cellular functions, e.g. growth, differentiation, apoptosis and immune responses [3–5]. Given its multiple effects on these cellular processes, it is not surprising that mindin is implicated in immune diseases [6,7]. Additionally, mindin can alleviate hepatic steatosis, insulin resistance and obesity, which are indicative of its role in metabolic diseases [8,9]. As in the cardiovascular system, our previous

Abbreviations: AKTI, AKT inhibitor IV; BrdU, 5 -bromo-2 -deoxyuridine; ECM, extracellular matrix; ERK, extracellular-signal-regulated kinase; FOXO, forkhead box O; FS, F-spondin domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; H&E, haematoxylin and eosin; HASMC, human aortic smooth muscle cell; I/M, intima-to-media; JNK, c-Jun N-terminal kinase; KO, knockout; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; NTG, non-transgenic; PCNA, proliferating-cell nuclear antigen; PDGF, platelet-derived growth factor; RASMC, rat aortic smooth muscle cell; RIPA, radioimmunoprecipitation assay; SD, Sprague–Dawley; SMA, smooth muscle actin; SMC, smooth muscle cell; T7E1, T7 endonuclease 1; TALE, transcription activator-like effector; TG, transgenic; TS, thrombospondin; TSR, thrombospondin type 1 repeat; VSMC, vascular smooth muscle cell; WT, wild-type. 1 These authors contributed equally to this work. Correspondence: Dr Hongliang Li (email [email protected]).

C The Authors Journal compilation C 2015 Biochemical Society

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studies have demonstrated that mindin exerts important regulatory effects on cardiac remodelling and ischaemic stroke [10–12]. However, the role of mindin in neointima formation, a common vascular pathological condition involving vascular smooth muscle cell (VSMC) proliferation, migration and phenotypic switching, has remained poorly understood until recently. In many vascular conditions, including atherosclerosis, instent restenosis and vein bypass graft failure, as well as during their treatment, such as angioplasty, the intimal layer of blood vessel thickens, which primarily results from the proliferation and migration of VSMCs. Upon vascular injury, the interplay among activated inflammatory cells, platelets and smooth muscle cells (SMCs) cause the release of growth factors, especially platelet-derived growth factor (PDGF), thereby inducing remarkable phenotypic remodelling of VSMCs (i.e. a switch of VSMCs from a contractile phenotype to a synthetic phenotype) [13–15]. Compared with those with a contractile phenotype, VSMCs with a synthetic phenotype are more likely to proliferate and migrate [16]. Although numerous approaches have been utilized to explore the pathogenesis of vascular remodelling, the underlying mechanisms of intimal hyperplasia still require indepth investigation. Considering that ECM proteins are always involved in extensive and complex cellular and molecular events and that the ECM components thrombospondin (TS) and integrin, which are structurally related to mindin, are closely associated with VSMC function and vascular injury [17,18], we hypothesize that mindin has critically important roles in neointima formation. To evaluate our hypothesis, SMC-specific mindin transgenic (mindin-TG) and global mindin-knockout (mindin-KO) mice were used to investigate the effects of mindin on VSMC proliferation, differentiation and phenotypic switching as the results of vascular injury. Additionally, a novel rat strain, mindin-KO, was successfully generated to further confirm the role of mindin in intimal hyperplasia. Our results indicate that, based on the blockage of AKT signalling, mindin exhibits vascular-protective effects by modulating VSMC functions. Up-regulation of mindin might represent a novel therapeutic strategy for vascular disorders.

MATERIALS AND METHODS Animals and treatment The animal study protocols were approved by the Animal Care and Use Committee of Renmin Hospital at Wuhan University, China. The global mindin-deficient mice (mindin-KO, C57BL/6J background) were genotyped using the PCR method. The primers used for wild-type (WT) mice were 5 -CAGCCCTGACTGGTTTGTGGGC-3 and 5 -CCCTGGGACTCTGCTGTAGCCGCACG-3 and the primers used for mindinKO mice were 5 -CACCAGCAGCTTGAGACGTTCTGG-3 and 5 -CCTTCTACTGCCTTCTTGACGAG-3 . To generate SMCspecific mindin-TG mice, full-length mouse mindin cDNA was ligated into a vector with a mouse SM22α promoter, a 5 -HA (haemagglutinin)-tag and an SV40 (Simian vacuolating virus 40) polyA signal. The construct was then microinjected into fertilized mouse embryos (C57BL/6J

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background). The mindin-TG mice were identified using the primers 5 -ACGGCAGAGGGGTGACATCA-3 and 5 GGTTTGTCCAAACTCATCAATG-3 . Four independent TG lines were established. Western blot analyses were employed to confirm mindin overexpression in the mindin-TG mice. Mice were exposed to a 12-h light/12-h dark cycle with controlled temperature and humidity. Food and water were provided ad libitum. A total of seven to ten mice and five to seven rats per group were used for vascular injury. The littermates of mindin-KO mice or mindin-KO rats whose genotypes were mindin+/+ were used as WT. A suspension of AKT inhibitor IV (AKTI; CAS 68128188-9, Calbiochem 0.5 mg/kg/day) was freshly prepared and administered by intraperitoneal injection every 3 days. AKTI was administered 1 week before surgery and continued for 4 weeks after wire injury or sham operation. Mice in the control group received the same volume of DMSO.

Carotid artery wire injury model To establish a carotid artery injury mouse model, mice were first anaesthetized with sodium pentobarbital (80 mg/kg, intraperitoneally). Then the left carotid artery was carefully dissected under a dissecting microscope by a midline neck incision. The external carotid artery was ligated with an 8-0 suture immediately proximal to the bifurcation point. Vascular clamps were then applied to interrupt the blood flow of the internal and common carotid arteries. A transverse incision was made immediately proximal to the suture around the external carotid artery. A guidewire (0.38 mm in diameter; Cook) was then introduced into the arterial lumen towards the aortic arch and withdrawn five times with a rotating motion. After the guidewire was carefully removed, the vascular clamps were also removed and blood flow was restored. The skin incision was subsequently closed. The sham littermate control mice underwent the same procedures without the incision or injury. Animal tissues were collected at specific time points after surgery for use in morphological and biochemical assays.

Rat carotid artery balloon injury model Carotid artery balloon injury was induced in male SpragueDawley (SD) rats (300 g) as previously described [19]. In brief, after anaesthetization, the left common carotid artery of the rat was exposed through a midline cervical incision. A Fogarty 2F catheter was advanced from just below the proximal edge of the omohyoid muscle to the carotid bifurcation three times (Baxter). To achieve constant and equal degrees of vessel wall injury in all animals, we maintained a consistent balloon diameter and resistance during withdrawal. All of the procedures were performed by a single operator. The animals were subsequently processed for morphological and biochemical studies at specific time points after surgery.

Histological and morphometric analysis Mice were killed at 7, 14 or 28 days post-injury by an intraperitoneal injection of an overdose of sodium pentobarbital (150 mg/kg). Carotid arteries were collected after circulation perfusion and fixed with 4 % paraformaldehyde dissolved in PBS. The arteries were further fixed with formalin and embedded with paraffin. Serial cross-sections (3 μm) were obtained from the bifurcation

Mindin inhibits neointima formation

Table 1

Primary and secondary antibodies used in the Western blotting and immunofluorescence

Primary antibody

Secondary antibody

Name

Company

Mindin

Santa Cruz Biotechnology

Catalogue number sc-98927

Smoothelin

Santa Cruz Biotechnology

sc-28562

SMA

Abcam

ab7817

SM22α

Abcam

ab14106

PCNA

Cell Signaling Technology

#2586

Cyclin D1


#2978

MMP9


#2270

p-ERK1/2Thr202/Tyr204


#4370

ERK1/2


#4695

p-JNK1/2Thr183/Tyr185


#4668

JNK1/2


#9258

p-P38Tyr182


#4511

P38


#9212

p-FOXO1Ser256


#9461S

FOXO1


#2880S

p-FOXO3ASer318/321


#9465S

FOXO3A


#2497S

p-AKTThr308

Bioworld Technology

BS4009

AKT

Bioworld Technology

BS2987

p-GSK3β Ser9

Bioworld Technology

BS4084

GSK3β

Bioworld Technology

BS1402

p-mTORSer2448

Bioworld Technology

BS4706

mTOR

Bioworld Technology

BS3611

GAPDH

Bioworld Technology

MB001

Alexa Fluor 488-conjugated goat anti-mouse IgG

Invitrogen

A11001

Alexa Fluor 488-conjugated goat anti-rabbit IgG

Invitrogen

A11008

Alexa Fluor 568-conjugated goat anti-mouse IgG

Invitrogen

A11004

Alexa Fluor 568-conjugated goat anti-rabbit IgG

Invitrogen

A11011

site of the left carotid artery. For morphometric analysis, the sections were stained with haematoxylin and eosin (H&E) or VVG (Verhoeff ’s Van Gieson) after deparaffinization and rehydration. A single observer who was blinded to the treatment protocols determined the level of neointima formation based on the intima areas and intima-to-media (I/M) ratios using Image Pro Plus software (version 6.0, Media Cybernetics). A mean value was generated from five independent sections of each artery sample.

Immunofluorescence staining After a 5-min high-pressure antigen retrieval process (100× sodium citrate buffer, pH 6.0), the arterial sections were blocked in PBS with 10 % goat serum for 1 h and incubated overnight with primary antibodies at 4 ◦ C. Rat aortic SMCs (RASMCs) were seeded on glass coverslips placed in 24-well plates. Cells were fixed with freshly prepared 4 % paraformaldehyde for 15 min, followed by permeabilization with 0.2 % Triton X-100 in PBS for 5 min. Next, the slides were washed in PBS containing 10 % goat serum and incubated overnight with primary antibodies at 4 ◦ C. The sections were then washed with PBS and incubated with the appropriate secondary antibodies for 1 h at 37 ◦ C. The nuclei were stained with DAPI. The images were obtained using a confocal laser-scanning microscope (Fluoview 1000; Olympus) or a

fluorescence microscope (Olympus DX51) and DP2-BSW software (version 2.2). The integrated optical density (IOD) values or the ratios of PCNA (proliferating-cell nuclear antigen)-positive cells were obtained using Image Pro Plus software (version 6.0, Media Cybernetics). The primary and secondary antibodies are shown in Table 1.

Cell culture Human aortic SMCs (HASMCs) were acquired from the A.T.C.C. The primary VSMCs were enzymatically isolated from the thoracic aortas of male WT, mindin-TG and mindin-KO mice as well as SD rats through enzymatic digestion. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium with 10 % FBS (SV30087.02; HyClone) and 1 % penicillin/streptomycin in a 5 % CO2 /water-saturated incubator at 37 ◦ C. The cells used in the experiments were passaged three to five times.

Cell proliferation measurement using BrdU SMC DNA synthesis was evaluated based on the amount of 5 -bromo-2 -deoxyuridine (BrdU) incorporated. Primary mouse SMCs (5 × 103 /well) were growth-arrested in a 96-well microplate. After growing to 60 % confluence, the cells were serum-starved for 24 h and subsequently treated with 20 ng/ml


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PDGF-BB (ProSpec) for 48 h. BrdU was added for the last 2 h of treatment. BrdU incorporation was determined using a cell proliferation ELISA kit (Roche Diagnostics) according to the manufacturer’s protocol.

Migration assay SMC migration was determined using a modified Boyden chamber. In brief, murine aortic SMCs were trypsinized and washed. Once resuspended, approximately 5 × 104 cells were added to the top wells of Transwell-modified Boyden chambers in a 24-well Transwell dish (a 6.5-mm polycarbonate membrane containing 8 μm pores) and allowed to attach for 30 min. SMCs were exposed to medium with or without PDGF-BB (20 ng/ml) that was added to the lower chamber for 6 h. The cells that migrated to the bottom of the membranes were fixed and stained with 0.1 % Crystal Violet/20 % methanol and counted. Five randomly chosen high-power fields (×200) in three independent experiments were used to calculate the average number of migrated cells. Images were quantified using Image Pro Plus software.

Western blot analysis Cellular and mouse tissue proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (720 μl of RIPA, 20 μl of PMSF, 100 μl of Complete protease inhibitor cocktail, 100 μl of Phos-stop, 50 μl of NaF and 10 μl of Na3 VO4 in a final volume of 1 ml). After complete homogenization on ice, the samples were centrifuged. The supernatants were then separated on SDS/PAGE (10 % gel; Invitrogen) and transferred to an Immobilon-FL membrane (Millipore). After blocking with TBS containing 5 % non-fat dried milk, the membranes were probed with primary antibodies overnight at 4 ◦ C. The membrane was then incubated with a secondary IRDye® 800CW-conjugated antibody (Li-Cor Biosciences) and the Odyssey Imaging System (Li-Cor Biosciences) was used for signal detection. Protein expression levels were quantified and normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) loading control. The primary and secondary antibodies are shown in Table 1.

Generation of mindin-KO rats To create mindin-KO rats (SD background), we used the region surrounding exon 2 of mindin as a candidate transcription activator-like effector (TALE) target site. TALE DNA-binding domains were designed using the first version of TALEN targeter (https://tale-nt.cac.cornell.edu/node/add/talen-old) and assembled following ‘Unit Assembly’. The TALEN expression plasmids were linearized with PmeI (New England Biolabs, NEB) and used as templates for in vitro transcription using the mMessage mMachine T7 Ultra Kit (Ambion). Then, this mature mRNA was purified using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. TALEN mRNAs were diluted to 10 ng/μl with injection buffer [10 mM Tris/HCl and 0.1 mM EDTA (pH 7.4)]. Subsequently, 2 pl (picoliter) of this solution was microinjected into the cytoplasm of one-cell-stage embryos using a FemtoJet 5247 microinjection system under standard conditions. The surviving embryos were then surgically transferred to pseudo-pregnant recipient females and viable pups were obtained. F0 founder genotyping was performed

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by PCR amplification of a portion of the mindin gene spanning the TALEN target site, using the primers mindin checkF (5 -GGGCTGCAGTCCACGAGTTA-3 ) and mindin check-R (5 -TGCCCTGTAGCCTCCTGGAT-3 ). A 519-bp amplicon was denatured and re-annealed in NEB buffer 2. T7 endonuclease 1 (T7E1, 1 μl; NEB) was added to cleave the heteroduplex into approximately 295- and 225-bp nicked DNA fragments (45 min in a 37 ◦ C water bath). The surplus PCR products from the founders were TA-cloned into T vectors following the manufacturer’s instructions (TaKaRa) and sequenced using the M13-47 common primer. For F1 and F2 generation screening, the primers mindin198-F (5 -GGCGCCAACACTTCTCTCT-3 ) and mindin-198-R (5 -TGCTGTCTGGCTCCACTTG-3 ) was used to amplify the indel site. The PCR products were analysed via 2 % agarose gel electrophoresis in Tris/borate/EDTA (TBE) buffer. The WT allele yielded a 198-bp amplicon, whereas the mutant allele yielded a 176-bp amplicon.

Statistical analyses The data analysis was performed using SPSS software, version 17.0 (SPSS Inc.). The data are presented as the mean values + − S.D. The differences between the two groups were determined using the independent-samples Student’s t test. Differences among multiple groups were determined using a one-way ANOVA followed by the least significant difference (LSD) test or Tamhane’s T2 post-hoc test. A P-value less than 0.05 was considered to be statistically significant.

RESULTS Arterial injury inhibits mindin expression in VSMCs As the expression level of a protein can initially reflect its involvement in certain diseases, the change in mindin expression was first examined in primary RASMCs and HASMCs in vitro. PDGF-BB, classically considered a stimulant of VSMC remodelling [20,21], was used to stimulate RASMCs and HASMCs in the present study. As shown in Figures 1(A) and 1(B), when RASMCs and HASMCs were exposed to PDGF-BB (20 ng/ml) for 0, 6, 24 and 48 h, the expression of mindin was significantly decreased in a time-dependent manner. The in vivo expression profile of mindin was subsequently measured in a well-established wire injury-induced intimal hyperplasia model in C57BL/6 mice. At 7, 14 and 28 days after wire injury, the degree of neointimal formation, as reflected by increased intimal area and I/M ratio, was gradually elevated (Figure 1C). In situ immunofluorescence staining suggested the localization of mindin in VSMCs, which was indicated by the overlapping of mindin with the VSMC marker SMA (smooth muscle actin; Figure 1D). Consistent with in vitro investigations, the expression of mindin was dramatically reduced after vascular damage by wire injury, concomitant with a decrease in the level of SMA and an obvious increase in the level of the proliferative marker PCNA at 14 and 28 days post-injury (Figures 1D and 1E). Western blotting analysis further confirmed the varied expression profile of mindin during neointima formation (Figure 1F),


Figure 1

Mindin is down-regulated in VSMCs following stimulation (A and B) Mindin expression was examined in cultured RASMCs (A) and HASMCs (B) after treatment with PDGF-BB (20 ng/ml) by Western blotting using a specific mindin antibody. GAPDH served as a loading control. The relative expression of mindin was determined by densitometric analysis of immunoblots (*P < 0.05 compared with 0-h group; #P < 0.05 compared with the 6-h group and †P < 0.05 compared with 24-h group). The blots represent three independent experiments. (C) Representative histological sections of H&E-stained carotid arteries at 7, 14 and 28 days after injury. The insets in the upper panels were magnified and are presented in the bottom panels. The black arrows indicate inner elastic discs. The lesion was calculated using the intimal area and I/M ratio. Scale bar: 50 μm. (n = 10, *P < 0.05 compared with 7-day group; #P < 0.05 compared with 14-day group). (D and E) Immunofluorescence staining of mindin (red), SMA (green; D) and PCNA (red; E) are shown in representative sections of an injured artery at 14 and 28 days after wire injury or in representative sections of sham-operated artery. Intensities of SMA and mindin were quantified by scanning immunofluorescence and the level of PCNA-positive cells was evaluated (n = 3–5, *P < 0.05 compared with sham group;


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indicating that mindin might participate in VSMC dysfunction and vascular remodelling.

Mindin attenuates neointima formation To determine whether mindin is functionally involved in neointima formation, we created SMC-specific mindin-TG mice carrying full-length mouse mindin cDNA under the control of the SM22α promoter (Figure 2A) and four mindin-TG mouse lines were obtained. Western blotting showed that the expression of mindin was highest in line 4 with a 3.79-fold increase, therefore this line was used in subsequent experiments (Figure 2B). Under physiological conditions, the intimal area and I/M ratio in mindin-TG mice were similar to those in non-TG (NTG) controls; however, after carotid artery wire injury, the degree of neointimal formation in mindin-TG mice was strikingly lower compared with that in NTG mice, as evidenced by the dramatically reduced intimal area and I/M ratio in the injured vessels from mindin-TG mice (Figure 2C). To further confirm the inhibitory effects of mindin on neointima formation, WT and mindin-KO mice were subjected to wire injury and the intimal area and I/M ratio were calculated. Compared with WT mice, significantly higher increases in the lesion area and I/M ratio were observed in mindinKO mice at 14 and 28 days post-injury, indicating that mindin deficiency exacerbates intimal thickening (Figure 2D). Collectively, these results demonstrate that mindin attenuates neointima formation in response to injury.

the mindin-KO VSMCs were greatly enhanced compared with the control group (Figures 3C and 3D). Accordingly, we could reason that the protective effects of mindin on neointima formation are at least partially due to its anti-proliferative function in VSMCs.

Mindin suppresses VSMC migration In the formed neointima, VSMCs clearly show migratory properties [23]. To investigate the effect of mindin on the migration of VSMCs, primary VSMCs isolated from mindin-TG and mindin-KO mice were placed in a Boyden chamber in the presence or absence of PDGF-BB for 6 h. The migration of VSMCs in the NTG and WT groups was strongly induced by PDGFBB (20 ng/ml) treatment, which were dramatically inhibited by mindin overexpression and significantly aggravated by mindin deficiency (Figure 4A). Matrix metalloproteinases (MMPs), especially MMP9, are largely involved in and profoundly mediate the migration of VSMCs and the following neointima formation [24]. Therefore, we examined the mRNA and protein expression levels of MMP9 in wire injury-stimulated carotid arteries and PDGF-BB-stimulated primary VSMCs from mindin-TG and mindin-KO mice. PCR analysis and Western blotting revealed significantly elevated MMP9 expression post-stimulation, which was dramatically alleviated by mindin overexpression, whereas increased levels of MMP9 protein were observed with mindin deficiency (Figures 4B–4D).

Mindin inhibits VSMC proliferation

Mindin blunts phenotypic switching in response to arterial injury

Considering that VSMC proliferation is one of the most direct causes of neointima formation, the cellular proliferation markers PCNA and cyclin D1 [22] were detected and compared in injured carotid arteries from different groups. Similar to what was observed in the morphological investigation, compared with the controls, the PCNA-positive cell ratio was significantly lower in mindin-TG mice vessels, whereas this ratio was much higher in the mindin-KO group at 14 and 28 days after injury (Figure 3A). The suppression of mindin in VSMC proliferation was further validated using Western blot analysis. As shown in Figure 3(B), the expression levels of PCNA and cyclin D1 proteins were obviously decreased in the vessels of mindin-TG mice, whereas the opposite effect were observed in mindin-KO mice; however, interestingly, no significant difference in these proliferative markers was observed in the sham groups. Consistent with the in vivo results, our studies on primary aortic SMCs stimulated with PDGF-BB verified the antiproliferative capacity of mindin. After 48 h of PDGF-BB treatment, both the elevation in BrdU incorporation analysed by ELISA and the protein expression of PCNA and cyclin D1 measured by Western blotting were significantly attenuated in VSMCs from mindin-TG mice, whereas the proliferative properties of

We next evaluated the regulatory effect of mindin on the phenotypic switching of VSMCs upon lesion stimulation. In response to vascular injury, adventitial fibroblast and progenitor cells transform into myofibroblasts which express SMA and contribute to the neointima formation [25]. Thus, to exclude the potential effects of adventitial fibroblast and progenitor cells in the progression of neointima formation, we employed SMA, SM22α and Smoothelin to evaluate the phenotypic switching of VSMCs after vascular injury. Immunofluorescence staining revealed a negligible expression of SMA and SM22α in the injured vessels of NTG and WT mice, indicating strong phenotypic switching of VSMCs from the quiescent contractile phenotype to the active synthetic phenotype in response to arterial injury. However, in the mindin-TG group, the decrease in SMA and SM22α were much lower compared with those observed in the NTG controls, whereas the decreases in these genes in mindin-KO mice and VSMCs were more prominent than those observed in the WT group (Figure 5A). Consistent with the immunofluorescence analysis, Western blotting showed that, compared with NTG and WT controls after stimulation, the declines in the SMC-specific genes SMA, SM22α and Smoothelin were significantly ameliorated in the vessels and primary

#P < 0.05 compared with 14-day group). Scale bar: 50 μm. (F) Protein expression of mindin, PCNA and SM22α at 7, 14 and 28 days after wire injury was examined by Western blotting (n = 3 samples, three or four carotid arteries were collected as a sample; *P < 0.05 compared with sham group; #P < 0.05 compared with the 7-day post-injury group and †P < 0.05 compared with 14-day post-injury group). The blots represent three independent experiments. All data represent the means + − S.D.

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Figure 2

Mindin inhibits neointimal formation after vascular injury (A) Mouse mindin cDNA was cloned into a construct containing SM22α promoter to create TG mice specifically expressing mindin in SMC (mindin-TG). (B) Western blot showing the expression of mindin in the carotid arteries of NTG and mindin-TG mice. Each lane represents a different line. The blots represent three independent experiments. (C and D) EVG staining of the carotid arteries of NTG and mindin-TG mice (C) or WT and mindin-KO mice (D) at 14 and 28 days post-injury. The insets in the left panels were magnified and are presented in the right panels. The black arrows indicate inner elastic discs. Scale bar: 50 μm. Right panel: quantified intimal area and I/M ratio (n = 7–10, *P < 0.05, **P < 0.01, ***P < 0.001 compared with NTG or WT group). All data represent the means + − S.D.

VSMCs of mindin-TG mice, but greatly augmented in the vascular tissue and cells with mindin deficiency (Figures 5B and 5C). Taken together, our results demonstrated that the vascular injury-induced abnormal alteration of VSMC functions, i.e. proliferation, migration and phenotypic switching, during neointima formation could be efficiently prevented by the up-regulation of mindin.

Mindin down-regulated AKT–GSK3β signalling in VSMC The evidence that mindin ameliorates the VSMC disorder and neointima formation prompted us to elucidate the mechanism

underlying the regulatory effects of mindin. Several lines of evidence indicated a role of mitogen-activated protein kinases (MAPKs) in the control of intimal hyperplasia and the corresponding VSMC proliferation and migration [26,27]. In addition, given that mindin is a ligand of integrin which could mediate MAPK signalling [5,28], we investigated the potential involvement of MAPKs in mindin-mediated vascular protection. Compared with those of the non-injured sham controls, the levels of phosphorylated MAPKs were higher at 28 days after injury. Unexpectedly, the expressions of the phosphorylated c-Jun Nterminal kinase (JNK) 1/2, extracellular-signal-regulated kinase (ERK)1/2 and p38 proteins in the injured vessels of mindin-TG


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Figure 3

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Mindin inhibits VSMC proliferation (A) Immunofluorescence staining for PCNA (red) in the carotid arteries of NTG and mindin-TG mice or WT and mindin-KO mice at 14 and 28 days after wire injury. Quantification of PCNA-positive cell percentages in each group is shown in the right panel (n = 3, *P < 0.05 compared with NTG or WT group). Scale bar: 50 μm. (B) Expression levels of PCNA and cyclin D1 were determined using Western blotting and the bands were quantified by densitometric analysis (n = 3 samples, three or four carotid arteries were collected as one sample, *P < 0.05 compared with NTG or WT group). The blots represent three independent experiments. (C) Primary VSMCs were stimulated with 20 ng/ml PDGF-BB or control PBS for 48 h and the individual proliferation was reflected as the absorbance intensity of incorporated BrdU. (n = 8, *P < 0.05 compared with NTG or WT group). Results represent three independent experiments. (D) After stimulation with PDGF-BB, PCNA and cyclin D1 protein expression in VSMCs isolated from NTG and mindin-TG mice or WT and mindin-KO mice was analysed by Western blotting (*P < 0.05 compared with NTG or WT group). GAPDH was used for normalization. The blots represent three independent experiments. All data represent the means + − S.D.



Figure 4

Mindin blunts VSMC migration (A) A Transwell assay was used to test the migration ability of VSMCs and the representative microscopic observations of stained VSMCs is shown. Scale bar: 50 μm. The migrated cells were counted in five high-power fields (200×). Results represent three independent experiments. (B and C) Real-time PCR (B) and Western blotting (C) were performed to examine MMP9 mRNA and protein levels in vessels of indicated mice at 28 days after wire injury (n = 6 for real-time, n = 3 samples for Western blotting, three or four carotid arteries were collected as one sample, *P < 0.05 compared with NTG or WT group). (D) Western blotting assay was performed to examine MMP9 protein in cultured VSMCs isolated from NTG and mindin-TG mice or WT and mindin-KO mice treated with PDGF-BB (20 ng/ml) or control PBS for 24 and 48 h. GAPDH was used as an internal control (*P < 0.05 compared with NTG or WT group). The blots represent three independent experiments. All data represent the means + − S.D.

or -KO mice were comparable with those in the NTG or WT controls (Figure 6A). Since that AKT is crucial to VSMC remodelling and that our previous study demonstrated the ability of mindin to regulate cardiac hypertrophy and ischaemic stroke through the AKT–GSK3β (glycogen synthase kinase 3β) pathway [11,12,29], we hypothesized that AKT might be responsible for the vascular regulation of mindin. Therefore, phosphorylated AKT and its downstream molecules GSK3β, mTOR (mammalian target of rapamycin), FOXO3A (forkhead box O3A) and FOXO1 were examined in

the vascular injury models and control groups. After vascular injury, the proteins expressions of phosphorylated AKT, GSK3β, mTOR, FOXO3A and FOXO1 were significantly elevated in the NTG and WT groups; however, these increases were diminished by mindin-TG, whereas mindin deficiency enlarged these differences between the post-injury model group and the non-injury sham group (Figures 6B and 6C). Meanwhile, similar results were found in primary VSMCs isolated from gene-intervened mice in response to PDGF-BB (Figures 6D and 6E). Thus, the protective functions of mindin in intimal hyperplasia might be


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Figure 5

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Mindin attenuates VSMC phenotypic switching after injury (A) The expression levels of SMA and SM22α (green) in the carotid arteries of NTG and mindin-TG or WT and mindin-KO mice at 14 and 28 days after injury were examined using an immunofluorescence assay. The right panel shows the intensity of immunofluorescence. Scale bar: 50 μm. (n = 3, *P < 0.05 compared with NTG or WT group) (B) The protein levels of SMA, SM22α and Smoothelin in the carotid arteries of NTG and mindin-TG or WT and mindin-KO mice at 28 days after injury were detected by Western blotting and quantified in the bottom panel (n = 3 samples, three or four carotid arteries were collected as one sample, *P < 0.05 compared with NTG or WT group). (C) Cultured mouse VSMCs of NTG and mindin-TG or WT and mindin-KO mice were treated with PDGF-BB (20 ng/ml) or control PBS for 0, 24 and 48 h. SMA, SM22α and Smoothelin expression was assessed by Western blotting and intensities were quantified by scanning densitometry (*P < 0.05 compared with NTG or WT group). The blots represent three independent experiments. All data represent the means + − S.D.



Figure 6

Mindin down-regulates AKT signalling in response to vascular injury (A–C) Expression levels of MAPKs (A) and AKT signalling proteins in the carotid arteries of NTG and mindin-TG mice (B) or WT and mindin-KO (C) mice at 28 days after injury were assessed by Western blotting (n = 3 samples, three or four carotid arteries were collected as one sample, *P < 0.05 compared with NTG or WT group). (D and E) Cultured primary VSMCs of NTG and mindin-TG (D) or WT and mindin-KO mice (E) were treated with PDGF-BB (20 ng/ml) or control PBS. Phosphorylated levels of AKT and its downstream proteins, GSK3β, FOXO3A, FOXO1 and mTOR, were detected by Western blotting and quantified by scanning densitometry (*P < 0.05 compared with NTG or WT group). The blots represent three independent experiments. All data represent the means + − S.D.


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Figure 7

The regulatory effects of mindin on neointimal formation are AKT-dependent (A) Mice were treated with AKTI or control PBS after the wire-injury operation and their carotid arteries were collected at 28 days post-injury. Phosphorylated levels of AKT and its downstream protein GSK3β were examined at by Western blotting and quantified by scanning densitometry (*P < 0.05 compared with PBS group post-injury). (B) Representative H&E-stained carotid artery sections from indicated groups obtained 28 days after wire injury. The insets in the upper panels were magnified and are presented in the bottom panels. The black arrows indicate inner elastic discs. Intimal area and the I/M ratio were measured (n = 6–8, *P < 0.05 compared with WT group; #P < 0.05 compared with mindin-KO group). Scale bar: 50 μm. (C) Expression levels of PCNA, SMA and SM22α in injured vessels were examined by immunofluorescence assay. Quantification of PCNA-positive cells (left), expression of cyclin D1 (middle) and SMA (right) are shown in the bottom panel (n = 3–4, *P < 0.05 compared with WT group; #P < 0.05 compared with mindin-KO group). All data represent the means + − S.D.

regulated, at least in part, through the inhibition of the AKT signalling pathway.

The regulatory effects of mindin on neointimal formation are AKT-dependent Considering that variation in AKT signalling occurred during mindin-regulated neointima formation, we further determined whether the regulatory effects of mindin are dependent on AKT. AKTI or DMSO (control) was administered 1 week before surgery and continued for 4 weeks after wire injury or sham opera-

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tion and the intimal hyperplasia was examined at 28 days. Western blotting revealed that the phosphorylation of AKT was lower in the AKTI treatment group than in DMSO controls post-injury (Figure 7A). Morphological staining showed that AKTI treatment efficiently alleviated the intimal area and I/M ratio after wire injury compared with WT mice treated with DMSO (Figure 7B). More importantly, the administration of AKTI almost completely abolished the promotional effect of mindin deficiency on neointima formation in response to carotid artery wire injury (Figure 7B).


Next, we evaluated whether the influence of AKTI on neointima formation results from its regulation in VSMC functions. Immunofluorescence examination of carotid arteries from AKTIand DMSO-treated mice indicated that, in WT and mindin-KO groups respectively, AKTI greatly inhibited and largely abolished vascular injury-induced VSMC dysfunction, as evidenced by the dramatically decreased PCNA-positive cells and increased expression of SMC markers (Figure 7C). Hence, the present study provides credible evidence that the regulatory effects of mindin on neointima formation are AKT-dependent.

Mindin KO promotes neointimal formation in a balloon-induced vascular injury rat model Although the beneficial effects of mindin on wire injury-induced vascular neointima formation have been validated in mice, the pathological intimal hyperplasia may vary significantly depending on the specific animal models. With this in mind, a novel rat strain, mindin-KO, was successfully generated and the carotid balloon injury rat model was utilized in subsequent experiment to further confirm the function of mindin. The mindin-KO rat strain was created using TALEN technology. The region surrounding exon 2 was selected as the target site and undergone genetic disruption (Figure 8A). A total of ten live-born offspring were generated, six of which were identified as cleaved products using T7E1 assay (Figure 8B). The precise locations of the indel mutation were determined through TA cloning and sequencing (Figure 8C). The found number 4-1(a1) was chosen for the further establishment of mindin22/+ and mindin22/22 rat strains. Mindin deficiency was verified through PCR analysis and Western blotting (Figures 8D and 8E). A classic carotid balloon injuryinduced neointimal formation was then established in the SD and mindin-KO rats, suggested by the obviously increased intima area and I/M ratio. Consistent with the results obtained in mice with wire injury-induced intimal hyperplasia, the increase in these two phenotypic indexes, i.e. intima area and I/M ratio, were significantly promoted by mindin-KO in rats (Figure 8F). Collectively, the deleterious effects of mindin deficiency in a rat model of balloon injury-induced neointima formation provide additional evidence supporting the critical roles of mindin in vascular injury.

DISCUSSION The pathological growth, migration and phenotypic switching of VSMCs often occur after vascular diseases and their treatments, leading to intimal thickening and restenosis in vessels [22,23,30,31]. The mechanisms underlying neointima formation have remained hitherto elusive. In the present study, we provide the first evidence that mindin acts as a crucial mediator of neointima formation based on its essential regulatory functions in VSMC proliferation, migration and phenotypic switching. Notably, in addition to identifying the protective role of mindin in primary VSMCs and mouse models of intimal hyperplasia, a novel mindin-KO rat strain further validated the detrimental effects of mindin deficiency on intimal thickening. Additionally,

our investigations highlighted a role for the AKT signalling pathway and determined that the beneficial effects of mindin on neointima formation are AKT-dependent. ECM proteins have extensive roles in histological architecture and biological information transduction and influence both physiological and pathological conditions through their regulations of cell survival, differentiation, mobility, adhesion and migration [32,33]. In the vascular system, ECM proteins provide a supportive medium for blood vessels. Through receptors on the cellular surface, these proteins interact with different sets of vascular cells and thereby regulate the development and/or remodelling of vessels [33,34]. Several ECM proteins, e.g. laminins and fibronectin, are intimately involved in endothelial functions [35,36]. Additionally, integrin and TSR have regulatory effects on VSMC phenotypic switching [37,38]. Mindin is one of the ligands of integrin and is structurally similar to TS. Previous studies on mindin have primarily focused on its roles in the immune, nervous and metabolic systems [8,9,11,39]; however, the role of mindin in neointima formation, a common vascular disorder, is unknown. In the present study, we employed gain- and loss-of-function approaches to demonstrate that mindin regulates VSMC functions and ameliorates intimal thickening. Overexpression of mindin dramatically reduced wire injury-triggered or balloon injury-induced neointima formation and PDGF-BB-stimulated VSMC proliferation, migration and phenotypic switching, whereas mindin deficiency accelerated and exacerbated this pathological condition. Therefore, mindin can be considered as a novel regulator of neointimal formation, indicating a crucial involvement of this protein in vascular diseases. Previous studies indicated that, dependent on its FS1/FS2 and TSR domain, mindin exerts extensively modulatory effects on the cellular behaviours [39]. For instance, mindin can directly bind to its TSR receptor on macrophages and function as an opsonin for the phagocytosis of bacteria [4]. Besides, in neuronal cells, mindin accelerates the outgrowth of various cell populations, including sensory, hippocampal and commissural neurons [39]. In our previous studies, we found that mindin can interact with cardiomyocytes and suppress cell hypertrophy [12]. However, the role of mindin on VSMCs remains unclear, although its analogues, TS and F-spondin, can be activated by PDGFBB and can promote the proliferation and migration of VSMCs [38,40,41]. In the present study, we demonstrated that mindin is down-regulated upon exposure to PDGF-BB and blocks the proliferation, migration and phenotypic switching of VSMC. This result suggest that mindin acts as a direct regulator of VSMCs. The apparently contradictory effects of mindin and its analogues (TS and F-spondin) on VSMC function might be explained by the difference in their structures and the corresponding signalling pathways that they affect. Numerous studies have been performed to explore the mechanisms that underlie the biological functions of mindin and several of these studies identified the role of mindin as an integrin ligand, thus regulating integrin signalling complexes and downstream pathways [3,5]. As one part of the classic downstream pathway, AKT signalling has been demonstrated to primarily participate in the initiation and augmentation of intimal hyperplasia [42]. In response to PDGF-BB stimulation or vessel


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Figure 8

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Generation of mindin-KO rats via TALEN technology and the detrimental effect of mindin deficiency on balloon injury-induced neointimal hyperplasia in rats (A) Schematic representation showing TALEN target site in exon 2 of rat mindin gene. (B) Agarose gel photograph of the T7E1 digestion assay showing the digestion products of the predicted size from WT SD (519 bp) and mutant rats (295 and 225 bp). The arrows indicated the size of the bands. (C) DNA sequences of cloned PCR products from WT and created founder rats. (D) F2 rats were generated by mating F1 heterozygotes (HZ) and were genotyped via PCR and agarose gel electrophoresis. The 198-bp band represents the WT allele and the 176-bp band represents the mutant allele. (E) Western blotting results showing the expression of mindin in WT and mindin-KO rats. (F) Representative histological sections of H&E-stained rat carotid arteries at 7 and 14 days after balloon injury. The insets in the upper panels were magnified and are presented in the bottom panels. The black arrows indicate inner elastic discs. Intimal area and the I/M ratio were measured. Scale bar: 50 μm. (n = 7, *P < 0.05 compared with control group). All data represent the means + − S.D.



injury, AKT can be significantly activated and the up-regulated AKT subsequently promotes the proliferation, migration and dedifferentiation of VSMC by activating phosphorylated GSK3β and mTOR, thereby exacerbating neointima formation [43–46]. Consistent with our previous studies, overexpression of mindin greatly inhibited the activation of AKT signalling [11,12]. As expected, treatment with a pharmacological AKT inhibitor almost completely abolished the promotional effect of mindin-KO on neointimal formation through inhibiting VSMC proliferation, migration and phenotypic switching, suggesting an AKTdependent effect of mindin on neointima formation. However, the molecular mechanisms that link mindin to AKT merit further investigation. In the last few decades, the TG mouse has become the principal mammalian model organism for biomedical research [47]. However, compared with mice, rats exhibit greater similarities to human pathophysiological conditions; thus rats have been consistently chosen for current research on clinical diseases [48]. In studies of neointima formation, the balloon injury-induced vascular injury model in rats is suitable for laboratory research [49–51]. However, the gene-disruption mindin rat strain was unavailable until recently. In our present study, a mindin-KO rat line was successfully generated using TALEN technology, a powerful tool for creating specific genetically engineered animals via genome editing and for introducing targeted double-strand breaks (DSBs) [52]. In the present study, this rat strain was utilized to further confirm the beneficial effects of mindin on intimal hyperplasia. More importantly, the created mindin-KO rat strain greatly facilitates in-depth investigations of the biological functions of mindin and its underlying molecular events. In conclusion, in the present study, using both in vitro VSMC dysfunction model and in vivo mouse and rat intimal thickening models, an ECM protein, mindin, was identified as a novel bona fide suppressor of neointima formation based on its mediatory effects on VSMC proliferation, migration and phenotypic switching. The ability of mindin to alleviate neointima formation is dependent on its inhibition of AKT signalling. The present study proposes that targeting mindin may be a promising strategy for the treatment of intimal hyperplasia-related vascular diseases.

plies that up-regulation of mindin might serve as an effective therapy for neointima formation related vascular diseases.

AUTHOR CONTRIBUTION

Li-Hua Zhu, Ling Huang and Xiaojing Zhang designed and performed the experiments, analysed the data and wrote the manuscript. Peng Zhang and Shu-Min Zhang analysed the data and wrote the manuscript. Hongjing Guan, Yan Zhang, XueYong Zhu, Song Tian and Keqiong Deng performed the experiments. Hongliang Li designed the experiments and wrote the manuscript.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers NO. 81170086, NO. 81370209 and NO. 81370365]; the National Science and Technology Support Project [grant numbers NO. 2011BAI15B02, NO. 2012BAI39B05, NO. 2013YQ030923-05, 2014BAI02B01 and 2015BAI08B01]; the Key Project of the National Natural Science Foundation [grant number NO. 81330005]; the National Basic Research Program China [grant number NO. 2011CB503902]; and The National Science Fund for Distinguished Young Scholars [grant number NO. 81425005].

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Received 27 October 2014/16 February 2015; accepted 9 March 2015 Published as Immediate Publication 9 March 2015, doi: 10.1042/CS20140679


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PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Syndecan-1 regulates vascular smooth muscle cell phenotype.

LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity.

Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.

Interferon regulatory factor 7 protects against vascular smooth muscle cell proliferation and neointima formation.

16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP.

Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis.

Genistein suppresses leptin-induced proliferation and migration of vascular smooth muscle cells and neointima formation.

Myocardin regulates vascular smooth muscle cell inflammatory activation and disease.

Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing.

Chronic kidney disease alters vascular smooth muscle cell phenotype.

Anthocyanidins, novel FAK inhibitors, attenuate PDGF-BB-induced aortic smooth muscle cell migration and neointima formation.

New endoplasmic reticulum stress regulator, Gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.

Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury.

Rab5a‑mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells.

Oxidative stress induces early-onset apoptosis of vascular smooth muscle cells and neointima formation in response to injury.

Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1.

Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.

Mebendazole reduces vascular smooth muscle cell proliferation and neointimal formation following vascular injury in mice.

The flavo-oxidase QSOX1 supports vascular smooth muscle cell migration and proliferation: Evidence for a role in neointima growth.

Caveolin-3 Promotes a Vascular Smooth Muscle Contractile Phenotype.

The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

Enhanced caveolin-1 expression in smooth muscle cells: Possible prelude to neointima formation.

L-arginine-dependent vascular smooth muscle relaxation and cGMP formation.