PCSK9 promotes vascular neointimal hyperplasia through non-lipid regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi:10.21203/rs.3.rs-4278817/v1

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PCSK9 promotes vascular neointimal hyperplasia through non-lipid regulation of vascular smooth muscle cell proliferation, migration, and autophagy

https://doi.org/10.21203/rs.3.rs-4278817/v1

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We aim to explore the impact of Proprotein convertase subtilisin-kexin type 9 (PCSK9) and its inhibitor evolocumab on neointimal hyperplasia. Wild type and PCSK9 knockout (PCSK9-/-) mice were subjected to ligation of the common carotid artery, with or without subcutaneous injection of PCSK9i Evolocumab. Mouse aortic vascular smooth muscle (MOVAS) cells were pretreated with evolocumab or under siRNA-mediated suppression of PCSK9, and then exposed to platelet-derived growth factor type BB(PDGF-BB), a major promoter of MOVAS transformation to a proliferative phenotype. PCSK9 was upregulated in ligated carotid arteries and PDGF-BB-treated MOVAS cells. PCSK9-/- mice showed decreased intimal area and intimal/ media area ratio, downregulation of proliferation and autophagy, which was coincidence with wild-type mice treated with evolocumab. In MOVAS cells fortified with evolocumab or silencing of PCSK9, PCNA, Beclin1, p62, LC3 were downregulated, additionally, EdU-positive cells decreased, cell viability reduced, migration ability was weakened, and the number of autophagosomes and autolysosomes decreased after the treatment. We also identified the PI3K/AKT/mTOR signaling molecules as potential PCSK9 targets mediating proliferative effect in MOVAS cells. To sum up, our results suggest that PCSK9 has intrinsic properties to promote proliferation, migration and autophagy in VSMCs independent of its lipid- regulating role. The proliferative effects of PCSK9 may be mediated by the PI3K/AKT/mTOR signaling pathway. These data provide additional evidence for PCSK9i in cardiovascular disease beyond the low-density lipoprotein (LDL)-lowering benefit.

Biological sciences/Cell biology/Autophagy

Biological sciences/Cell biology/Cell growth

Biological sciences/Cell biology/Cell migration

Biological sciences/Physiology/Cardiovascular biology

VSMC

PCSK9

evolocumab

proliferation

autophagy

neointimal hyperplasia

At present, cardiovascular disease (CVD) remains a major public burden threatening human health[1]. Exploration for the diagnosis and treatment of cardiovascular diseases has been ongoing, it is clear that neointimal hyperplasia is a key pathological process[2]. As an important component of the vascular wall, vascular smooth muscle cells (VSMCs) can be categorized into two phenotypes: contractile phenotype and secretory phenotyped (including proliferative and migratory phenotypes), excessive proliferation and migration of VSMCs along with abnormal autophagy are the primary stimulus for intimal hyperplasia in blood vessels[3-5]. An increasing number of interventions have been reported to target neointima hyperplasia, however, there is still a lack of strong experimental and clinical evidence. A better understanding of the mechanisms underlying the onset and development of neointimal hyperplasia is a matter of urgency.

Autophagy is an intracellular process that degrades cytoplasmic proteins and damaged organelles in response to harsh environments to adjust cell function and maintain homeostasis[6]. In VSMCs, it is activated by many factors including hypoxia, starvation, reactive oxygen species (ROS), metabolic stress, growth factors, cytokines and certain drugs[7]. Accumulating evidence suggests that activation of autophagy in VSMCs is a considerable regulator of neointima formation[8, 9, 4]. Moderate autophagy prevents abnormal proliferation of VSMCs by timely clearance of damaged organelles and misfolded proteins, while under the stimulation of pathological factors, excessive autophagy provides the material basis and energy for the proliferation of VSMCs by decomposing macromolecular substances, thereby promoting their proliferation[7, 4, 3, 10]. The effect of autophagy on the proliferation of vascular smooth muscle cells requires more in-depth studies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is mainly synthesized by the liver, intestine and kidney also have a small amount of synthesis. It is an essential molecule that mainly promotes the degradation of the low-density lipoprotein (LDL) receptor, resulting in restricted LDL cholesterol clearance, thus increasing serum low-density lipoprotein cholesterol (LDL-C) [11]and emerging as a risk predictor and biotarget in atherosclerotic cardiovascular disease[12, 13]. PCSK9 is also expressed in endothelial cells, macrophages and VSMCs[14, 15]. Higher PCSK9 expression has been reported in the aortic bifurcation, iliac bifurcation of low-shear stress mouse aorta[16], and also in the “shoulder” regions of vulnerable atherosclerotic plaques[17], and is involved in the process of atherosclerotic disease independently of its LDL cholesterol regulation[18]. PCSK9 aggravates hypoxia-induced endothelial cell pyroptosis in critical limb ischemia[19]. PCSK9 also regulates mitophagy, which contributes to reperfusion injury after myocardial infarction via the BNIP3 pathway[20]. As for VSMCs, it is controversial whether PCSK9 inhibits or promotes VSMCs proliferation[21, 17, 22]. The effect of PCSK9 on VSMC autophagy is still unclear.

PCSK9 inhibitors (PCSK9i), including evolocumab, alirocumab and small interfering RNA inclisiran, are now becoming widely used in the clinic and have shown potent lipid-lowering effects[23, 24] and beneficial effects on plaque stabilization and regression[25]. While a recent real-world study showed that the risk of in-stent restenosis after PCI was significantly reduced by evolocumab, this was mainly based on non-lipid effect, and the mechanism underlying such an observation is unclear[26]. Increasing evidence showed that PCSK9i display possible beneficial effects independent of LDL cholesterol levels. Administration of evolocumab within 24 hours of admission to hospital in patients with myocardial infarction can significantly improve post-infarction myocarditis[27]. Evolocumab reduces infarct size via suppressing autophagy in cardiac tissue[28]. PCSK9i possess intrinsic properties for anti-inflammatory, anti-autophagic, and antioxidant in endothelial cells[29]. However, it is unknown whether PCSK9i are able to counteract with the autophagy and proliferation in VSMC.

This study aims to explore the potential non-lipid effect of PCSK9 and its inhibitor in modulating neointimal hyperplasia by regulating VSMC proliferation, migration, and autophagy. We used PCSK9 knockout (PCSK9^-/-) mice to identify a potential novel lipid-independent target of PCSK9: PCSK9 deficiency inhibited VSMC autophagy activation, reduced excessive proliferation and migration, and slowed down the pace of neointimal hyperplasia. Our results provided further evidence for PCSK9 to promote the proliferation of VSMCs and confirmed for the first time that PCSK9 involved into regulating VSMCs autophagy process. We also demonstrated that PCSK9i evolocumab could also inhibit the progression of the above process, providing further factual evidence to support the in-depth exploration of PCSK9 as a therapeutic target for vascular neointimal hyperplasia and for the clinical application of PCSK9i.

PCSK9 upregulated in carotid ligation in vivo model and platelet derived growth factor‐BB (PDGF-BB) ‐ treated MOVAS cells in vitro model.

To explore whether PCSK9 is related to intimal hyperplasia, we ligated the left common carotid artery of mice to acquire vascular intimal hyperplasia model. After 14 d, 21 d, the common carotid arteries were harvested, Western blot analysis showed that the expression of PCNA (Fig. 1A and C), a marker of proliferation, was gradually elevated as the ligation time prolongs, indicating the successful establishment of the model. Autophagy markers such as Beclin1(Fig. 1A and D), p62(Fig. 1A and E), LC3(Fig. 1A and F) were upregulated consistent with PCNA, showing that autophagy was activated after ligation. PCSK9 was markedly elevated in vessels after ligation (Fig. 1A-B), suggesting that PCSK9 may be involved in intimal hyperplasia and the regulation of autophagy process after ligation.

Since the excessive VSMC proliferation and impaired autophagy play an important role in the development of vascular restenosis and atherosclerosis, we induced MOVAS cells proliferation and autophagy using PDGF-BB[30, 31]. Western blot was performed and the results showed that PDGF-BB significantly increased autophagy markers Beclin1, p62, LC3 consistent with proliferation marker PCNA expression, and PCSK9 level was increased in MOVAS cells after PDGF-BB stimulation (Fig. 1G-H), suggesting that PCSK9 may be involved in the modulation of proliferation and autophagy in MOVAS cells.

Evolocumab and knockout of PCSK9 inhibited ligation‐induced intimal hyperplasia

To detect the effect of PCSK9i on intimal hyperplasia, wild-type mice were injected subcutaneously with evolocumab immediately after the procedure, then again two weeks later, and to determine the effect of knockout of PCSK9 on intimal hyperplasia, PCSK9^-/- mice were subjected to the same procedure without using evolocumab. Arteries were harvested after 14, 21 and 28 days after ligation to observe the effect of both on the process of intimal hyperplasia (Fig. 2A). Hematoxylin and eosin staining was performed (Fig. 2B), and the morphological parameters lumen area, intima area, media area and intima/media ratio were determined (Fig. 2C). Our results suggested that intima area and intima/media ratio were significantly increased in ligated arteries compared to the sham group, however, lumen area was significantly reduced and media area was not significantly different, indicating the successful establishment of the model. When using evolocumab or knockout of PCSK9, intima area and intima/media ratio reduced, while lumen area increased conversely. The results mentioned above showed the same trend at different time points. All of above suggest that in wild type mice, the ligation of carotid arteries resulted in significant neointima formation in the left carotid artery compared with mice only threaded without ligation, while evolocumab and knockout of PCSK9 can inhibit the progression of intimal hyperplasia.

To evaluate the expression level of relevant protein markers, immunohistochemistry (IHC) staining was performed (Fig. 2D, Fig. S1A and Fig. S1C) and the average optical density (AOD) of IHC (Fig. 2E, Fig. S1B and Fig. S1D) was calculated to represent the expression level of protein markers. Our results showed that proliferation marker PCNA, and autophagy markers Beclin1, p62, LC3 were upregulated in ligated arteries compared with sham group, when using evolocumab or knockout of PCSK9, the expression level reduced. The results also showed the same trend at different time points.

The results suggest that PCSK9 possibly hasten the proliferation and autophagy in the intimal hyperplasia process, while evolocumab and knockout of PCSK9 can alleviate this pathological process.

Evolocumab inhibits MOVAS cells proliferation, migration and autophagy levels

PCSK9 was upregulated in MOVAS cells treated with PDGF-BB, therefore we hypothesized that inhibition of PCSK9 function may alleviate VSMC proliferation, migration and autophagy.

Evolocumab was added to the serum-starved MOVAS cells together with PDGF-BB stimulation, and the effect of evolocumab on MOVAS cells proliferation was assessed by Western blot and Edu assay. Western blot results displayed lower expression of PCNA in the PDGF-BB + evolocumab group than that in the PDGF group (Fig. 3A-B). Similarly, evolocumab apparently decreased the percentage of Edu‐positive cells in PDGF-BB‐treated MOVAS cells as assessed by the EdU assay (Fig. 3C-D). Additionally, the CCK‐8 assay showed that evolocumab decreased the cell viability after 24 and 48 hours of treatment (Fig. 3E). Moreover, the migration assay showed that evolocumab inhibited the migration of MOVAS cells (Fig. 3F-G). These results suggest that inhibition PCSK9 function alleviates proliferation and migration of PDGF-BB-stimulated MOVAS cells.

The effect of evolocumab on autophagy of MOVAS cells was assessed by Western blot, the results revealed lower expression of Beclin1, p62 and LC3 in the PDGF-BB + evolocumab group than that in the PDGF-BB group (Fig. 3A-B). And autophagic flow assay was conducted to observe the effect of evolocumab on the autophagic process in MOVAS cells (Fig. 3H), in the merged image, the number of autophagosomes (yellow dots) and autolysosomes (red dots) were counted and used to evaluate the level of autophagy, the results revealed that evolocumab decreased the number of autophagosomes and autolysosomes (Fig. 3I). These findings demonstrate that evolocumab inhibits VSMC autophagy.

Silencing of PCSK9 inhibits MOVAS cells proliferation, migration and autophagy levels

To further verify the role of PCSK9 on proliferation, migration and autophagy of MOVAS cells, we utilized siRNA to knock down PCSK9 and ensured the transfection efficiency (Fig S1).

The effect of PCSK9 knockdown on MOVAS cells proliferation was assessed by Western blot and Edu assay. Western blot results displayed lower PCNA expression in the PDGF-BB + si-PCSK9 group than that in the PDGF-BB + si-NC group (Fig. 4A-B). Similarly, knockdown of PCSK9 apparently decreased the percentage of Edu‐positive cells in PDGF-BB‐treated MOVAS cells as assessed by the EdU assay (Fig. 4C-D). Additionally, the CCK‐8 assay showed that silencing of PCSK9 decreased the cell viability of MOVAS cells after 24 and 48 hours of treatment (Fig. 4E). Moreover, the migration assay showed that silencing of PCSK9 inhibited the migration of MOVAS cells (Fig. 4F-G). These results suggest that silencing of PCSK9 may alleviate proliferation and migration of PDGF-BB-stimulated MOVAS cells.

The effect of silencing of PCSK9 on autophagy of MOVAS cells was assessed by Western blot, the results revealed lower expression of Beclin1, p62 and LC3 in the PDGF-BB + si-PCSK9 group than that in the PDGF-BB + si-NC group (Fig. 4A-B). And autophagic flow assay was conducted to observe the effect of PCSK9 silencing on the autophagic process in MOVAS cells (Fig. 4H), in the merged image, the number of autophagosomes (yellow dots) and autolysosomes (red dots) were counted and used to evaluate the level of autophagy, the results revealed that silencing of PCSK9 decreased the number of autophagosomes and autolysosomes (Fig. 4I). These findings demonstrate that silencing PCSK9 inhibits MOVAS cells autophagy. Results above further supported the hypothesis that PCSK9 aggravated proliferation, migration and autophagy in VSMC.

Silencing of PCSK9 inhibits MOVAS cells proliferation by suppressing the PI3K/AKT/mTOR signaling pathway

The PI3K/AKT/mTOR signaling pathway is one of the important pathways for regulating cell proliferation and growth, which is also involved in the progression of cardiovascular diseases[32], so we speculated that PCSK9 silencing might suppress MOVAS cell proliferation through PI3K/AKT/m-TOR signaling pathway. To verify this hypothesis, we examined the changes in total protein (t-PI3K, t-AKT and t-mTOR) and phosphorylated protein (p-PI3K, p-AKT and p-mTOR) expression of the pathway by Western blot (Fig. 5A), the ratio of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were calculated by quantification analysis. The results showed that, silencing of PCSK9 significantly reduced the ratio of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR in PDGF-BB-treated MOVAS cells (Fig. 5B-D). These results indicate that silencing of PCSK9 can suppress activation of the PI3K/Akt/mTOR signaling pathway in PDGF-BB-treated MOVAS cells. However, these effects were reversed by co-treatment with 740 Y-P, a PI3K agonist. Meanwhile, the depression of PCNA expression in MOVAS cells induced by silencing of PCSK9 was significantly enhanced by activating the PI3K/Akt/mTOR signaling pathway (Fig. 5E). Collectively, these data implied that silencing of PCSK9 suppressed MOVAS cells proliferation by suppressing the PI3K/AKT/mTOR signaling pathway.

Vascular intimal hyperplasia is the major pathological process in multiple vascular diseases, including atherosclerosis and restenosis after stent implantation. Excessive VSMC proliferation and migration, along with impaired autophagy contribute to intimal hyperplasia, although the molecular mechanisms are still not fully understood[4]. The discovery of new targets that inhibit VSMC proliferation and migration may provide viable ideas for the treatment of endometrial proliferative diseases. In the current study, we identified the role of PCSK9 in vascular intimal hyperplasia by using C57BL/6 wild type and PCSK9^-/- mice. Consistent with previous reports[21, 17, 22], our results show that PCSK9 is upregulated in the intimal hyperplasia model, and the expression levels of PCSK9 and proliferation coming along with autophagy markers rise in a proportional ratio as ligation duration increased, indicating that PCSK9 is involved in the regulation of intimal hyperplasia, while hyperplasia was suppressed when using PCSK9 inhibitor evolocumab or knock-out PCSK9 in vivo throughout the hyperplastic process(Fig. 2B). Consequently, PCSK9 aggravates neointimal hyperplasia through by activating of VSMC proliferation, migration and autophagy.

The effect of autophagy on vascular smooth muscle proliferation is difficult to determine, some studies have reported that autophagy inhibits vascular smooth muscle cell proliferation, while others have confirmed that autophagy promotes the proliferation process[3, 7, 10, 33]. High levels of autophagy have been observed in vivo by partial ligation of the left mouse carotid artery and by balloon injury of the rat carotid artery, when autophagy activation was inhibited, VSMC phenotype switching and neointima proliferation were prevented[34, 35]. Furthermore, knockdown of JMJD3[34] or Meox1[35] could suppress autophagic activation resulting in attenuation of injury-induced neointima formation in a rat carotid artery balloon injury model. Our research results showed that autophagy and proliferation were co-directed changes in both the carotid artery ligation model and the PDGF-BB-induced MOVAS cells model. We believe that, in our experimental models, autophagy degrades useless organelles and proteins in cells, providing more raw materials and energy to accelerate proliferation, and PCSK9 promotes this process.

Additionally, in our study, the expression levels of Beclin1, p62, and LC3 were used to evaluate the degree of autophagy, the trend of p62 and LC3 was consistent, which was different from previous reports[31]. Classically, as a representative of a selective substrate for autophagy, the more p62 is consumed, the higher the level of autophagy, the content of p62 is opposite to the level of autophagy[36, 37]. On the other hand, p62 is also an initiating factor of the autophagy process[38, 39], at this point, p62 is not only an autophagy marker to evaluate the level of autophagy. Autophagic flux assay showed that the process of converting autophagosomes into autolysosomes was not affected, indicating that the autophagic process was unobstructed in our models. In our opinion, the upregulation of p62 may have promoted the progression of autophagy in our models. Although our findings confirmed that deletion or knockdown of PCSK9 could suppress autophagy, the mechanism by which PCSK9 regulates autophagy remains to be elucidated.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a role in the development of central nervous system development, atherosclerosis, immune response, tumorigenesis and other biological functions[40]. As a classical target for lipid-lowering therapy, PCSK9’s atherogenic effects were initially attributed to its role in lipid metabolism, while emerging evidence shows that PCSK9 may potentially affect the cardiovascular system independently of its cholesterol-regulating effect. Circulating PCSK9 induces macrophage activation via LDLR-independent mechanisms in vein graft lesion development[41]. PCSK9 promotes the endothelial cell apoptosis via the JNK/p38 MAPK pathway in atherosclerosis[42]. The effect of PCSK9 on VSMC proliferation is still controversial, as mentioned above, overexpression of PCSK9 inhibits proliferation but induces polyploidization, senescence and apoptosis of cultured human aortic SMCs. In aged mice, SMC-specific PCSK9 knockout exacerbates neointima formation and ameliorates arterial stiffening[17]. While another study showed that PCSK9 knockout appears to maintain the contractile phenotype of SMCs by reducing cell proliferation, exogenous expression of PCSK9 in PCSK9 knockout SMCs induces cell proliferation[21]. In addition, PCSK9 knockout significantly reduced vascular stenosis and the intimal hyperplasia area in graft vascular disease[22]. Our results showed that PCSK9 was upregulated in the PDGF-BB-induced MOVAS cells model, silencing of PCSK9 suppressed MOVAS cells proliferation, and intimal hyperplasia was slowed in PCSK9 knockout mice, indicating that PCSK9 has a positive effect on VSMC proliferation.

Evolocumab is a widely used lipid-lowering agent in clinical practice for reducing cardiovascular risk[43]. In addition, it can inhibit inflammation and autophagy of cardiomyocytes after myocardial infarction[20, 27], alleviate vascular inflammation in the process of atherosclerosis[18] and reduce the risk of in-stent restenosis after PCI based on non-lipid effect[26]. In vivo, Evolocumab exerts its effect by binding to circulating PCSK9, thereby inhibiting the binding of PCSK9 to LDLR in the liver[44]. As for vascular smooth muscle cells in vitro, we speculate that evolocumab enters the cells through endocytosis and binds to intracellular PCSK9 to exert its effects (Fig.6). Our study showed that evolocumab had similar effects on proliferation and autophagy in hypertrophic vascular intima of mice to those of PCSK9 knockout, evolocumab exhibited a strong inhibitory effect on neointimal hyperplasia, although evolocumab probably alleviated the proliferation of vascular intima dependent on its lipid-lowering effect in vivo, evolocumab still demonstrated its inhibitory effects on MOVAS cells proliferation and migration of MOVAS cells in vitro experiments. We therefore concluded that the inhibitory effect of evolocumab on intimal hyperplasia was not solely dependent on its lipid-lowering effect, which would explain the in-stent restenosis outcome. To our knowledge, this was the first study to show that evolocumab depressed VSMC autophagy, and future investigations are needed to illustrate its underlying mechanism.

The PI3K/AKT/mTOR signaling pathway is a classic signaling pathway in the eukaryotic signaling network that regulate a variety of cellular functions, especially cell proliferation and metabolism[45]. Studies have confirmed that the PI3K/AKT/mTOR pathway is an important growth factor or cytokine signaling pathway involved in VSMC proliferation and migration[32, 46]. Here we provid direct evidence that PCSK9 enhances proliferation via the PI3K/AKT/mTOR signaling pathway in VSMC. PCSK9-siRNA markedly decreased the phosphorylation of PI3K, AKT, mTOR and proliferation marker PCNA, which can be reversed by the addition of 740Y-P (a PI3K agonist). The results of our study are consistent with the prediction that the PI3K/AKT/mTOR-dependent pathway may be involved in PCSK9-mediated vascular smooth muscle proliferation. Our results appear to contradict previous research on the causal relationship between PCSK9 and mTOR signal levels, which reported that increased mTORC1 activity led to reduced activity of HNF1α, resulting in decreased PCSK9 expression in the liver[47]. In our opinion, mTOR is located at the center of a complex cellular signaling network and participates in multiple biological processes. Moreover, the main biological functions exhibited by PCSK9 may vary in different tissues. Therefore, there may be corresponding differences in different models.

In conclusion, our findings suggest that in endovascular proliferative disease, PCSK9 is upregulated in the hypertrophic vascular intima and promotes vascular smooth muscle proliferation via the PI3K/AKT/mTOR signaling pathway. Together with PCSK9-mediated excessive autophagy, hypertrophic vascular intima worsens over time. Direct inhibition of PCSK9 may be an effective therapeutic approach for the prevention of vascular intimal hyperplasia diseases independent its lipid regulatory effect (Fig. 6).

Ethics statement

Animal experiments were approved by the First Hospital of Hebei Medical University’s Ethics Committee and conducted in the light of the ARRIVE guidelines.

Animals

Eight-week-old C57BL/6 wild type male mice were purchased from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, Hebei, China). Eight-week-old C57BL/6 pure-line PCSK9 gene knockout (PCSK9^-/-) male mice were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China). The vascular intimal hyperplasia model was established by ligating the common carotid artery of mice as described previously[48]. Briefly, the animals were anaesthetized through inhalation of 2% isoflurane and ligation was performed on the left common carotid artery nearly before the bifurcation. The wild-type mice were randomly divided into sham, ligation, ligation + evolocumab groups, PCSK9^-/- mice were all included in the ligation of PCSK9^-/-micegroup (Fig. 2A). Mice in the sham group were only threaded without ligation. Mice in the ligation + evolocumab group were injected subcutaneously with evolocumab (25 mg/kg, immediately after the procedure, then again two weeks later) which was diluted with water for injection. The remaining mice underwent left carotid artery ligation only. All the mice were housed in a pathogen-free laboratory with a 12-hour light/dark cycle at 22 ℃, and had free access to food and water. After 14, 21 and 28 days of the procedure in each group, the mice were euthanized by an overdose of isoflurane inhalation, and the common carotid arteries were harvested for subsequent histological observations or detection of protein expression levels.

Hematoxylin and eosin staining

Vascular tissues were fixed with 4% paraformaldehyde for 16 hours and embedded in paraffin. Each artery was cut to 4 μm thickness. Sections were dewaxed and rehydrated by immersion in xylene and graded ethanol series. The sections were then stained with hematoxylin for 5 minutes and eosin for 15 seconds for hematoxylin and eosin (H&E) staining, washed in 1% hydrochloric acid alcohol, dehydrated, and fixed. All images were obtained using a microscope (LEICA DM 2000, USA), the cross-sectional lumen area, intimal area and medial area were measured by Image J image analysis software, following with calculation of the intima/medial area (I/M) ratio, which represents the formation or degree of neointima.

Immunohistochemistry staining

Briefly, sections prepared as described above were dewaxed and rehydrated with xylene and graded ethanol series followed by antigen retrieval in an oven (60°C for 2 h). Sections were blocked with 5% BSA (room temperature for 30 min) and incubated with primary antibodies against PCNA (1:1,000, Proteintech, 10205-2-AP), Beclin1 (1:50, Proteintech, 11306-1-AP), p62 (1:200, abcam, ab91526) and LC3 (1:200, Proteintech, 14600-1-AP) (4°C overnight). Sections were then incubated with secondary antibodies in a water bath (37℃ for 20 min). Subsequently, sections were visualized with diaminobenzidine (MedChemExpress, HY-W025920) and finally counterstained with hematoxylin. All the sections were observed under the light microscope. Image J software was used to calculate the average optical density (AOD) for evaluating expression levels of protein markers.

Cell culture and treatment

Mouse aortic vascular smooth muscle (MOVAS) cells (ATCC, Shanghai, China) were cultured in low glucose DMEM (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were placed in a humidified atmosphere containing 5% CO2 at 37 ℃.

Platelet‐derived growth factor (PDGF)‐BB was incubated with MOVAS cells for 24 hours to induce phenotypic transformation[30]. Small interfering RNA (siRNA) targeting PCSK9 (si-PCSK9) and its negative control(si-NC) were transfected into MOVAS cells. GP-transfect-Mate (GenePharma, Shanghai, China) was used for RNA transfection in accordance with the manufacturer’s instructions. The siRNAs used in this study were purchased from Ruibo Biotechnology Co., LTD (Guangzhou, China): PCSK9(5’-GAACCTACATTGTGGTGCT-3’).

MOVAS cells were seeded in culture flasks for 12 hours before serum starvation for 12 hours, and then divided into the following groups for further treatment for 24 hours: (1) control (CTRL) group; (2) PDGF‐BB (25 ng/ml) group; (3) PDGF-BB + evolocumab (200 ug/ml) group; (4) PDGF-BB + si‐NC (100 nM) group; (5) PDGF-BB + si‐PCSK9 (100 nM) group; (6) PDGF-BB + si‐PCSK9 (100 nM) + 740 Y-P (20 uM) group.

Western blot analysis

Total protein from MOVAS cells and mouse carotid artery tissue was extracted with RIPA lysis buffer (Seven Biotech, SW104, Beijing, China) plus PMSF (Seven Biotech, SW106), and when phosphorylation levels of proteins was required, phosphatase inhibitors (MedChemExpress, HY-K0021 and HY-K0022) were enriched additionally. Total protein concentrations were evaluated and quantified using BCA protein assay kits (Seven Biotech, SW101-02). Equal amounts of protein from each sample were separated by SDS-PAGE, the separated proteins were transferred to polyvinylidene fluoride (PVDF) blotting membranes, and blocked with 5% skimmed milk. The membranes were incubated with primary antibodies against PCSK9 (1:1,000, abcam,ab31762), PCNA (1:10,000,Proteintech,10205-2-AP), and Beclin-1 (1:1,000,Proteintech,11306-1-AP), p62 (1:1,000,abcam,ab91526), LC3Ⅰ/Ⅱ (1:1,000,Proteintech,14600-1-AP), β-actin (1:1,000, Proteintech,20536-1-AP)，p-PI3K p85(Tyr458)/p55(Tyr199) (1:1,000, cell signaling technology,#4228), PI3K (1:1,000, abcam,ab191606), p-AKT (1:2,000, cell signaling technology,#4060)，AKT (1:1000,cohesion biosciences,CPA1036), p-mTOR (1:2,000, Proteintech,67778-1-lg)，and mTOR (1:5,000, Proteintech,66888-1-lg) overnight at 4℃. Afterward, the membrane was rinsed and incubated with HRP-conjugated anti-mouse (1:10,000, Proteintech, SA00001-1) or anti-rabbit (1:10,000, Proteintech, SA00001-2) secondary antibodies at 37 ℃ for 1 hour at room temperature. β-actin was used as internal reference. At last, images were captured using a chemiluminescence imaging system (Bio-Rad, USA), and Image J software was used to calculate the greyscale values of the protein bands.

EdU incorporation assay

The 5-ethynyl-2’ -deoxyuridine (EdU) incorporation assay was performed using the EdU Cell Proliferation Kit with Alexa Fluor 555 (Meilunbio, Dalian, China) according to the manufacturer’s instructions. Briefly, MOVAS cells were seeded in a 6‐well plate, after different treatments, the cells were incubated in DMEM medium supplemented with 10 μM EdU solution for 2 h, fixed with 4% paraformaldehyde, incubated with 0.3% TritonX-100, stained with Click Additive Solution, and the nuclei were stained with 1 × Hoechst 33342, images were photographed using a fluorescence microscope (OLYMPUS IX71). EdU‐positive cells were counted using Image J software.

Cell viability assay

The cell viability assay was performed using the Cell Counting Kit-8 (CCK-8, MedChemExpress) according to the manufacturer’s instructions. Briefly, MOVAS cells were seeded in a 96‐well plate, after different treatments, the cells were incubated with a 10% CCK-8 solution for 3 h, and the absorbance was measured at a wavelength of 450 nm using a microplate reader.

Cell migration analysis

Migration levels were measured using a wound healing assay. Briefly, MOVAS cells were seeded in a 6‐well plate, when the cells reached 90% confluence, a wound was created using a pipette tip with a volume of 200 μL, resulting in a linear blank area, subsequently cells were subjected to different treatments. Images were photographed at the appropriate time points using a Leica DMi1 inverted microscope. The percentage of the closed area was measured using the ImageJ software.

Autophagic flux assay

MOVAS cells were seeded in a 6‐well plate with prepared cell sheets, after 12 hours of incubation, adenovirus encoding LC3 with dual fluorescence labeling (HBAD-mRFP‐GFP-LC3, HANBIO, China) was infected into the cells for 6 hours, and then the cells were divided into groups for different treatment, 24 hours later, the cells were washed with PBS, fixed with 4% paraformaldehyde, and the cell sheets were placed on the glass slide, followed by nuclear staining with DAPI at room temperature for 15 min and neutral gum sealing piece. Images were obtained by a Confocal Laser Scanning Microscope System (Leica). In the merged images, the autophagosomes (yellow dots) and autolysosomes (red dots) were counted using the ImageJ software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. Quantitative data derived from at least three independent experiments are presented as mean ± standard error of mean (SEM). For comparisons between paired groups, the paired t-test was used for normally distributed data. For unpaired groups, the unpaired t-test was used for the normal/homoscedastic data, and the Mann-Whitney U test was conducted for the non-normal/non-homoscedastic data. For multiple groups with a single factor, the one-way ANOVA followed by Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test was conducted for normal/homoscedastic data. For multiple groups with two factors, the two-way ANOVA followed by Sidak’s multiple comparisons test was performed for normally distributed data. Value of p < 0.05 was considered statistically significant difference.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Author contributions

Qian Zhang: Conceptualization, Investigation, Methodology, Validation, Writing Original Draft. Mengdan Miao: Investigation, Methodology, Data Curation, Software. Shanhu Cao: Methodology, Validation, Investigation. Lixia Chen: Visualization, Software. Da Liu: Methodology, Investigation. Mei Wei: Visualization, Investigation, Methodology. Shaoguang Sun: Methodology, Supervision, Resources. Sheng Jin: Methodology, Supervision. Lihua Dong: Visualization, Software. Yajuan Yin: Methodology, Validation, Investigation. Gang Liu: Project Administration, Supervision. Mingqi Zheng: Resources, Funding Acquisition, Supervision. All authors participated in and approved the final version of the paper.

Conflict of interest

The authors declare they do not have any conflict of interests.

Financial support

This Study was supported by National Natural Science Foundation of China (No.82204011); China Hebei International Joint Research Center for Structural Heart Disease; S&T Program of Hebei (22377719D, 203777117D); Natural Science Foundation of Hebei Province (H2021206399); Hebei Health Care Commission (20231904); Hebei Province Finance Department Project (LS202101).

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PCSK9 promotes vascular neointimal hyperplasia through non-lipid regulation of vascular smooth muscle cell proliferation, migration, and autophagy

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