Lin28a up‐regulation is associated with the formation of restenosis via promoting proliferation and migration of vascular smooth muscle cells

Abstract To explore the potential role of Lin28a in the development of restenosis after percutaneous transluminal angioplasty, double‐balloon injury surgery and mono‐balloon injury surgery were used to establish restenosis and atherosclerosis models, respectively, so as to better distinguish restenosis from atherosclerotic lesions. Immunohistochemical analysis revealed that significantly higher expression of Lin28a was observed in the iliac arteries of restenosis plaques than that of atherosclerosis plaques. Immunofluorescence studies showed the colocalization of Lin28a with α‐smooth muscle actin in restenosis plaques, rather than in atherosclerosis plaques, which suggested that Lin28a might be related to the unique behaviour of vascular smooth muscle cells (VSMCs) in restenosis. To further confirm above hypothesis, Lin28a expression was up‐regulated by transfection of Lenti‐Lin28a and inhibited by Lenti‐Lin28a‐shRNA transfection in cultured VSMCs, and then the proliferation and migration capability of VSMCs were detected by EdU and Transwell assays, respectively. Results showed that the proliferation and migration of VSMCs were significantly increased in accordance with the up‐regulation of Lin28a expression, while above behaviours of VSMCs were significantly suppressed after inhibiting the expression of Lin28a. In conclusion, the up‐regulation of Lin28a exerts its modulatory effect on VSMCs’ proliferation and migration, which may play a critical role in contributing to pathological formation of restenosis.


| INTRODUC TI ON
The incidence of type 2 diabetes mellitus (T2DM) is rapidly soaring at an alarming rate, imposing a considerable burden on public health. 1 Lower extremity atherosclerotic disease (LEAD), one of the frequent macrovascular complications of diabetes, accounts for one of the main factors causing foot ulceration and amputation, which leads to markedly reduced quality of life. 2 LEAD commonly implicates the tibial/peroneal artery and/or their branches. 3 Percutaneous transluminal angioplasty (PTA) is regarded as a primary approach in the treatment of LEAD. 4,5 However, restenosis which often occurs (incidence is 49.4% at 6 months after PTA and 52.5% at 1 year after PTA) in the dilated site of vessels after PTA restricts its long-term efficacy. 6 Treatment of LEAD is essential in preventing amputation, morbidity and mortality of diabetic patients. 7 Thus, the prevention and therapy of restenosis have been the keen spot in the process of LEAD treatment.
Our previous study has demonstrated that restenosis and atherosclerosis had distinct pathogenesis mechanisms. 8 Excessive migration and proliferation of vascular smooth muscle cells (VSMCs) were the dominant causes to develop the restenosis. 9 However, the particular mechanism involved in it remains unclear. It is necessary to further carry on the research on the exact mechanism of restenosis occurrence and interdicting this process is of great effect on the prevention and therapy of restenosis. 10 Therefore, we attempt to figure out the particular mechanisms of VSMCs' migration and proliferation in restenosis based on the previous study.
Lin28 was originally identified in Caenorhabditis elegans (C elegans) as a component of the heterochronic gene pathway that regulates developmental timing. 11 Lin28a, a homolog of Lin28, has been demonstrated to enhance tissue repair in some adult tissues in recent years. 12,13 It is reported that Lin28a reactivation can improve hair regrowth by promoting anagen in hair follicles and accelerate regrowth of cartilage, bone, and mesenchyme after ear and digit injury. 14 The process of restenosis plaque formation by further impairment of atherosclerotic arteries by PTA resembles the above tissue repair, 15 which speculated that Lin28a might be involved in the process of restenosis. However, the role of Lin28a in restenosis formation has not been elucidated yet.
In our research, restenosis and atherosclerosis rat models were established by using different methods, and the expression and the effect of Lin28a on the VSMCs' proliferation and migration were further explored.

| Animals and treatments
Male Sprague-Dawley (SD) rats (~120 g) were obtained from the Beijing Huafukang Bioscience Co. Inc. The rats were allowed to adapt to the new environment for at least 7 days, and then were fed with a high-fat diet during the whole experiment. The animal care and experiments were in conformity to the previous publication. 8 The protocol was authorized by the ethical committee of Qianfoshan Hospital Affiliated to Shandong University. All the rats were taken surgical procedures under anaesthesia with sodium pentobarbital.
High-fat diet was provided for all rats in the whole experiment.
Type 2 diabetes mellitus (T2DM) was induced by intraperitoneal injection of streptozocin (STZ, 27.5 mg/kg, Solarbio) to rats. After STZ injection for three days, the rat with fasting blood glucose (FBG) ≥11.1 mmol/L in three consecutive analyses was considered as a successful T2DM rat model. All eligible diabetic rats were assigned into three groups: restenosis, atherosclerosis and diabetic control group.
Non-diabetic rats were also included as the blank control group.
We developed models of restenosis and atherosclerosis in both diabetic rats. Surgical procedures for establishment of restenosis and atherosclerosis models were presented as followings.

| Restenosis models
After diabetes model was successfully established for one week, rats were anaesthetized with phenobarbital. An arteriotomy in the right femoral artery of diabetic rats was conducted with standard surgical procedures.
Firstly, a 1.5-mm, wire-guided balloon catheter (Medtronic, Inc.) was inserted into the iliac artery through the femoral artery. The balloon was blown up at 8, 10 and 12 atmospheric pressure (atm), respectively. And then, the inflated balloon was hauled from iliac artery to the femoral artery to induce endothelial denudation. After the operation, the balloon catheter was withdrawn, the vessel was ligated, and the surgical site was closed. In order to induce restenotisis plaques, in the fourth weeks after the first surgical procedure, additional PTA was performed if the iliac artery developed severe atherosclerotic damage, which was confirmed by ultrasound examination (Visualsonics). After anaesthesia, a midsagittal incision was executed in the dissected distal ends of the right femoral artery. A 1.5-mm, wire-guided balloon catheter was reinserted into the narrow injured iliac artery and the balloon was inflated to 12 atm three K E Y W O R D S Lin28a, migration, percutaneous transluminal angioplasty, proliferation, restenosis, vascular smooth muscle cells times for 5 seconds each time at the stenosis site. After the dilatation, catheter was removed and the femoral artery was ligated.

| Atherosclerosis models
Balloon-injured endothelial denudation of the normal iliac arteries in diabetic rats was performed to develop atherosclerotic plaques as the rats in restenosis group underwent the first balloon dilatation on the same day.

| EVG dye and Immunohistochemical staining
We chose the balloon-induced injury part of iliac artery and se-

| VSMCs isolation, purification and verification
Primary VSMCs were obtained by using explant culture method as described previously. 16 In brief, 6-week-old SD rats (sex unlimited) were anaesthetized with sodium pentobarbital (50 mg/kg ip), and the iliac artery was quickly excised and placed in cold Dulbecco's modified Eagle's medium (DMEM, with 5 mmol/L glucose, Gibco, ThermoFisher). The medial VSMCs layers were cut into 1 mm 3 tissues, and then were explanted in culture flask with DMEM medium containing 20% foetal bovine serum (FBS, Invitrogen, ThermoFisher), streptomycin (100 μg/mL) and penicillin (100 U/mL) and incubated in a humidified atmosphere with 5% CO 2 at 37°C. The medium was changed twice a week, and the cells should be passaged when grown to 80% confluence.
We purified VSMCs according to differential adherence of various cells. After the cell mixture was isolated, we digested the cell mixture by 0.25% tyrisin. And then the cell mixture was washed by serum-free M199 medium, and was resuspended by M199 complete medium. The culture flask containing cell mixture was standing for 40 to 50 minutes, and the fibroblast and endothelial cells were firstly adhered to the flask. Then, the culture flask was inverted, and the cell suspension was transferred into another culture flask. Repeating the above operation again and centrifuging the suspension. The cell pellet of VSMCs was retained after discarding the supernatant and resuspended into the culture flask.
In view of the specificity of SM22α expression in VSMCs, VSMCs were verified by immunofluorescence that all cells of SM22α and α-SMA were positively stained, which proved that the extracted primary cells were indeed VSMCs.

| Proliferation and migration assays
The proliferation of VSMCs in vitro was determined using a 5-ethynyl-2′-deoxeuridine (EdU) assay kit (RiboBio Co., Ltd). VSMCs Cell migration was evaluated by the Transwell chamber as described previously. 17 After transfected by Lenti-Lin28a, Lenti-Lin28a-shRNA and Lenti-Lin28a-shNC, VSMCs at 1.5 × 10 4 cells/100 µL were seeded in the upper chambers of Matrigel-coated 8-μm pore size transwell filters (Corning Life Sciences). In the lower chamber, 500 μL DMEM containing 10% foetal bovine serum was added, which used as a chemoattractant. After incubation for 24 hours, the cells that migrated to the lower surface of the filter were fixed in 4% paraformaldehyde for 15 minutes, and stained with crystal violet, and the nonmigrating cells in the upper surface of the filter were removed with a cotton-tipped swab. Then, the lower side of the filters was fixed in 4% paraformaldehyde for 15 minutes, and stained with crystal violet dyes. The images of migrated cells were taken at randomly selected fields using an OLYMPUS FSX100 imaging system (Olympus). The migration of VSMCs was evaluated as the mean number of cells in five randomly selected fields. The assay was repeated at least three times.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA levels were detected by qRT-PCR with One Step PrimeScriptmiRNA cDNA Synthesis Kit (Takara Bio Inc.). 18 The following conditions were used: 95°C for 30 seconds, 95°C for 5 seconds and 60°C for 20 seconds for 40 cycles. The relative expression level of Lin28a mRNA was calculated using the following formula: relative gene ex-  KGaA) overnight at 4°C. The next day, corresponding peroxidase-labelled goat anti-rabbit and anti-mouse secondary antibody (1:5000)

| Western blot analysis
were used as the second antibody, respectively. The peroxidase activity was detected using the FluorChem E enhanced chemiluminescent system (ProteinSimple). And ImageJ software (National Institutes of Health) was used to quantify the densitometer of the optical density of the bands.

| Colour doppler ultrasonogram
Ultrasonography was to periodically assess the vascular status. After diabetes model was established, the iliac vessels of rats in restenosis group were damaged and subsequently observed with ultrasonography in different time points. Figure 1B showed that atherosclerosis plaques were established at four weeks after balloon injury surgery.
Then the stenosed iliac arteries were recanalized after PTA surgery ( Figure 1C), and restenosis plaques were formed at four weeks ( Figure 1D).

| VSMCs were significantly richer in restenosis plaques than atherosclerosis plaques
Samples were collected from the iliac arteries of rats in distinct groups. CD31 was used to exhibit the position of endothelial cells ( Figure S1). The elastic membrane tissue was shown by elastic-van Gieson (EVG) staining, and the intima is located in the inside of the internal elastic lamina. Figure 2A showed the apparent intimal hyperplasia both in atherosclerosis and restenosis groups. α-SMA was the marker of VSMCs, and immunohistochemical analysis revealed that the expression of α-SMA was significantly higher in restenosis plaques when compared to that of atherosclerosis plaques (P < .05, Figure 2B) by calculating the percentage through calculating the area of positive staining in terms of percentage of the total area.

| Lin28a was differentially expressed in the plaques of restenosis and atherosclerosis
Since restenosis undergoes a process of tissue repair, 19 Lin28a was demonstrated to enhances tissue repair. 14 We then explored whether there existed differential expressions of Lin28a in restenosis and atherosclerosis plaques. Immunohistochemical results showed that Lin28a expression was significantly higher in restenosis plaques

| Lin28a was involved in the proliferation and migration of VSMCs
In vitro, primary VSMCs which were extracted from iliac arteries of rats ( Figure 5A) were verified by immunofluorescence staining for α-SMA ( Figure 5B) and SM22α ( Figure S3). Subsequently, Lin28a expression in the primary VSMCs was examined after regulation. qRT-PCR (P < .05, Figure 5D) and Western blot (P < .05, Figure 5C Figure 6C showed that the migrated cells labelled as the purple ones were significantly increased after overexpression of Lin28a. EdU and Transwell migration showed that the proliferation and migration of VSMCs were significantly increased with the up-regulated Lin28a expression, and were significantly decreased with the down-regulated expression of Lin28a (P < .05, Figure 6).

| D ISCUSS I ON
PTA is regarded as a vital treatment approach for patients with LEAD, especially for the patients with severe artery stenosis below popliteal artery. 20 However, nearly half of the arteries after PTA develop restenosis in the first year following operation. 21 To the best of our knowledge, there are few studies on the pathogenesis of restenosis after LEAD. Our previous studies found a unique mechanism of restenosis which was in consistence with others 22,23 and demonstrated that the excessive proliferation and migration of VSMCs from the media to the intima were the main cause of restenosis. 8 In the present study, the potential pathogenesis was further investigated and we found that Lin28a played a crucial role in regulating the proliferation and migration of VSMCs in restenosis.
Lin28, a highly conserved RNA-binding protein, was originally discovered in C elegans and it comprises a network of genes which can regulate the developmental timing. 11 Studies showed that mutations of Lin28 gene and other so-called 'heterochronic genes' perturbed developmental progression in the worm, and these characteristic events occur either earlier or later than normal. 24 Lin28 homologue involves Lin28a and Lin28b. 25 A large amount of data focus on the pro-neoplastic effect of Lin28b, 26,27 including colon tumours 26 and oral squamous cell carcinoma. 27 On the contrary, few studies focus on Lin28a. Lin28a is regarded as a principal element that participates in reprogramming mammalian somatic cells to pluripotent cells. 28 Differ from Lin28b which is closely linked with malignant proliferation, Lin28a weights towards initiating and maintaining cell proliferation. 29, 30 Hanna J et al previously indicated that the overexpression of Lin28a promoted cell division and led to the formation of induced pluripotent stem (iPS) cells more quickly. 31 Furthermore, transgenic mouse that overexpressed Lin28a was reported to have enlarged brain size and body weight as well as slightly increased thickness of F I G U R E 2 A was shown as overview of vessels. Immunohistochemistry staining for elastic-van Gieson (EVG), EVG-positive staining indicated the position of elastic tissue. The internal elastic lamina was labelled with red arrow and external elastic lamina was labelled with green arrow. AS, atherosclerosis group; RS, restenosis group. B, Immunohistochemistry staining for α-SMA in atherosclerosis (a) and restenosis (b) plaques. α-SMA was rarely observed in atherosclerosis plaque but filled in restenosis plaques. The red arrow indicated the positive staining for α-SMA. The left images of immunohistochemistry staining for α-SMA were taken at ×100 magnification and scale bar was 300 μm; the right part of picture A and B was magnified to ×200, respectively and scale bar was 75 μm. c, Quantification of positive staining for α-SMA in atherosclerosis and restenosis groups. α-SMA was significantly more in restenosis than atherosclerosis plaques. The values denote the positive area/ total area ± SEM. *Statistically significant difference (P < .05) the cerebral cortex. 29 Besides, the body size, crown-rump length and growth rate of Lin28a transgenic mice were also augmented without malignant transformation. 32 The above evidences presented the similarity to the process of restenosis plaques repair to some extent. Moreover, Lin28a expressed highly in the early stage of angiogenesis and it gradually decreased with the development of vascular maturation. 33 Based on the above evidences, we hypothesize that Lin28a might be involved in restenosis formation.

F I G U R E 3
Immunohistochemistry staining for Lin28a in atherosclerosis (A) and restenosis (B) plaques. Few Lin28a was observed in atherosclerosis plaques but more Lin28a was filled with restenosis plaques. The red arrow indicated the positive staining for Lin28a. The left images of immunohistochemistry staining for Lin28a were taken at ×100 magnification and scale bar was 300 μm; the right part of picture A and B was magnified to ×200, respectively and scale bar was 75 μm. C and D, Quantification of positive staining area and cells for Lin28a in atherosclerosis and restenosis groups. The values denote the positive area/ total area ± SEM. *Statistically significant difference (P < .05)

F I G U R E 4
Expression and colocalization of Lin28a (red) and α-SMA (green) in atherosclerosis (AS) and restenosis (RS) groups by immunofluorescence double staining. α-SMA is the specific marker of vascular smooth muscle cells (VSMCs), and the merged orange colour represented the Lin28a expression in VSMCs. The merged orange colour was enhanced in RS group. The image of immunofluorescence was taken at ×100 magnification and scale bar was 100 μm; a small part was magnified to ×400 and is shown on the top left corner of each picture. AS, atherosclerosis group; RS, restenosis group Our previous study suggested that the plaque formation of restenosis and atherosclerosis involved different pathophysiological mechanisms. In the development of atherosclerosis, hyperglycaemia played a critical role, but had little effect on the formation of restenosis after PTA. 8 Furthermore, we found that atorvastatin cannot arrest the occurrence of restenosis, which revealed blood lipids made rare difference on restenosis formation. 34 For exploring the specific mechanism of restenosis formation, two distinct rat models were established.

In our present research, abundant foam cells accumulation but few
VSMCs appeared in the atherosclerosis plaques. However, VSMCs were proved to be the dominant components of restenosis plaques, and the excessive proliferation and migration of VSMCs from the media to the intima were found in restenosis plaques, 8 which was in accordance with our present findings. Commonly, VSMCs mainly locate in the tunica media of the arteries, and are rarely observed in intima. Why do they migrate from tunica media to the intima after F I G U R E 5 The isolation and verification of primary vascular smooth muscle cells (VSMCs) and regulation of Lin28a in VSMCs. A, The schema graph of primary VSMCs isolation. B, The verification of primary VSMCs by immunofluorescence stain for α-SMA (red). The image of immunofluorescence was taken at ×200 magnification and scale bar was 75 μm. C, Lin28a protein expression was detected by western blot after regulated by Lenti-Lin28a, Lenti-Lin28a-shRNA and Lenti-Lin28a-shNC, respectively. D, Lin28a mRNA expression was detected by qRT-PCR after transfected by Lenti-Lin28a, Lenti-Lin28a-shRNA and Lenti-Lin28a-shNC, respectively. *Statistically significant difference (P < .05) PTA? The underlying mechanisms involved in it were investigated and we found that expression of Lin28a was significantly increased in restenosis plaques, while it was hardly to find in atherosclerosis plaques.
Combined with the function in controlling angiogenesis, 33 it is speculated that Lin28a played a key role in the formation and development of restenosis. As mentioned above, excessively migrated VSMCs are major elements of restenosis. Is Lin28a associated with VSMCs in restenosis? Our study showed that the Lin28a-positive cells were also α-SMA positive by double immunofluorescence staining, which suggested that enhanced Lin28a expression was closed related to the VSMCs in restenosis plaques. Subsequently, exact function of Lin28a on VSMCs was further examined in vitro.
Our studies indicated that the proliferation and migration of VSMCs were markedly decreased by Lin28a down-regulation. On the contrary, VSMCs showed a dramatic proliferation and migration trend with Lin28a up-regulation, which was consistent with the increased pathological manifestations of VSMCs in restenosis plaques, and this further presented Lin28a an underlying key regulator in the restenosis disease.
The limitation of our study is that the signalling mechanism of Lin28a in restenosis was not performed, which need further investigations in our future study.
In summary, our studies highlighted the fact that Lin28a is critically involved in the progression of restenosis. VSMCs contribute significantly to restenosis plaques, which is different from atherosclerosis. In contrast to atherosclerosis, Lin28a was highly expressed in VSMCs of restenosis. In addition, the strongly positive correlation between Lin28a expression and proliferation and migration of VSMCs was found, which raised the possibility that Lin28a could be served as a novel therapeutic target for restenosis. A better understanding of the relationship between Lin28a and cell proliferation of VSMCs will make us have an in-depth knowledge in the field of restenosis in LEAD.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S ' CO NTR I B UTI O N S
In this work, Jianjun Dong and Lin Liao conceived the study and de-