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Introduction

Atherosclerosis is a chronic inflammatory disease of the large and medium-sized arteries, and is one of the most prominent causes of mortality worldwide (1-3). Atherosclerosis has been identified as the most common pathological process underlying diseases of the coronary, carotid and peripheral arteries (4). Several types of cells play crucial roles during the development of atherosclerosis, including endothelial cells, intimal smooth muscle cells and leukocytes (5,6). In normal vascular tissue, the normal morphology and functional integrity of endothelial cells permits them to engulf bacteria, as well as senescent and necrotic tissue. However, the lack of morphological integrity and dysfunction of vascular endothelial cells promotes inflammation and arterial thrombosis (7). In addition, damaged endothelial cells may undergo phenotypic changes in a process known as endothelial-to-mesenchymal transition (EMT), which allows endothelial cells to transform into mesenchymal- or myofibroblast-like cells (8). EMT is a complicated process characterized by the loss of specific endothelial markers, such as platelet endothelial cell adhesion molecule-1 and vascular epithelial calcitonin, and the acquisition of mesenchymal markers (9). Accumulating evidence has demonstrated that EMT is closely associated with the pathological processes of atherosclerosis and increases plaque calcification and plaque instability, leading to vascular stenosis (narrowing of the lumen) and ischemia in atherosclerotic tissues (10,11).

Semaphorins are a large class of secreted or transmembrane proteins, which were originally identified as regulators of neuronal growth (12). Previous studies have revealed that semaphorins serve important roles in several physiological processes, including angiogenesis, vascular development and immune responses (13,14). Semaphorin 7A (Sema7A) is a class 7 semaphorin that has a glycophosphatidylinositol-anchored domain (15). Numerous studies have shown that Sema7A and its membrane receptor, β1 integrin, exerted regulatory effects on neuronal growth, numerous cancer types and the inflammatory response (16-18). Furthermore, Sema7A and β1 integrin have been reported to be associated with atherosclerosis progression in previous studies (19,20). However, to the best of our knowledge, research on the biological role of Sema7A in endothelial cell injury and EMT-associated atherosclerosis is currently limited.

The present study was undertaken to investigate the expression of Sema7A and β1 integrin and their biological functions in atherosclerosis, and examine the effects of Sema7A/β1 integrin on the biological functions of oxidized low-density lipoprotein (ox-LDL)-treated HUVECs. The aim was to determine whether Sema7A can regulate cellular injury, apoptosis and EMT in ox-LDL-treated HUVECs by binding to β1 integrin, and whether the Sema7A/β1 integrin axis may serve as a novel target for the treatment of endothelial cell injury and dysfunction in atherosclerosis.

Materials and methods

Cell culture and treatment

HUVECs were obtained from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% streptomycin/penicillin, and maintained with 5% CO2 at 37˚C. To induce inflammatory injury and EMT, HUVECs were pretreated with 50 µg/ml ox-LDL (Peking Union-Biology Co. Ltd) for 24 h at 37˚.

Plasmid construction and cell transfection

To knock down Sema7A expression, specific short hairpin (sh)RNA targeting Sema7A (shRNA-Sema7A-1/2) and corresponding negative control (NC) shRNA (shRNA-NC) were synthesized by Shanghai Integrated Biotech Solutions Co., Ltd. To overexpress β1 integrin, a pcDNA3.1 expression vector containing the entire length of β1 integrin (Ov-β1 integrin) and empty vector (Ov-NC) were constructed by Shanghai GenePharma Co., Ltd. These recombinants were transfected into HUVECs at 37˚, using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After transfection for 48 h, cells were harvested for subsequent experiments. In addition, transfected HUVECs were treated with 50 µg/ml ox-LDL for 24 h at 37˚, and harvested as aforementioned.

Cell Counting Kit-8 (CCK-8) assay

The effects of Sema7A and β1 integrin on HUVEC viability were detected using a CCK-8 assay. Briefly, HUVECs were seeded into 96-well plates (5x103 cells/well) and transfected with shRNA-Sema7A with or without Ov-β1 integrin and corresponding NCs for 48 h prior to treatment with 50 µg/ml ox-LDL for 24 h. Following the incubation, 10 µl CCK-8 reagent (MedChemExpress) was added to each well and incubated for a further 2 h at 37˚C. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA was extracted from HUVECs from different treatment groups using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a HiScriptQ RT SuperMix for qPCR (Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol. qPCR was subsequently performed using a SYBR Premix ExTaq kit (Takara Bio, Inc.) on an ABI Prism 7900 Real-Time PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR thermocycling conditions were as follows: 95˚C for 3 min, followed by 35 cycles of denaturation at 95˚C for 30 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 1 min. A final extension step at 72˚C for 7 min was performed for each assay. Each treatment group was ran five times. GAPDH was used as the endogenous control. The relative expression of the target genes was quantified using the 2-ΔΔCq method (21). The primer sequences used are as follows: Sema7A: Forward, 5'-TTCAGCCCGGACGAGAACT-3' and reverse, 5'-GAACCGAGGGATCTTCCCAT-3'; β1 integrin: Forward, 5'-AGGAATGTTACACGGCTGCT-3' and reverse, 5'-ACCAAGTTTCCCATCTCCAG-3'; and GAPDH: Forward, 5'-GAAAGCCTGCCGGTGACTAA-3' and reverse, 5'-GCATCACCCGGAGGAGAAAT-3'.

ELISA

HUVECs were transfected with shRNA-Sema7A or shRNA-NC and Ov-β1 integrin or Ov-NC, followed by treatment with ox-LDL for 24 h at 37˚C. Then, the levels of inflammatory factors, including IL-1β, IL-6 and C-C motif chemokine ligand 2 (CCL2), in the culture supernatant were measured using ELISA kits (cat. no. H002, H007-1-1 and H318-1, respectively; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

Western blotting

Total protein was extracted from ox-LDL-induced HUVECs transfected with shRNA-Sema7A with or without Ov-β1 integrin and corresponding NCs using RIPA lysis buffer (CoWin Biosciences). Total protein was quantified using a Detergent Compatible Bradford Protein assay kit (Beyotime Institute of Biotechnology) and the proteins were separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% non-fat milk for 1 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4˚C: Anti-Sema7A (1:1,000; cat. no. ab255602), anti-β1 integrin (1:1,000; cat. no. ab52971), anti-intracellular adhesion molecule (ICAM)-1 (1:1,000; cat. no. ab282575), anti-vascular cell adhesion molecule (VCAM)-1 (1:2,000; cat. no. ab134047), anti-Bax (1:2,000; cat. no. ab182733), anti-cleaved caspase-3 (1:500; cat. no. ab32042), anti-caspase-3 (1:5,000; cat. no. ab32351), anti-CD31 (1:1,000; cat. no. ab9498), anti-von Willebrand factor (vWF) (1:1,000; cat. no. ab154193), anti-α-smooth muscle actin (α-SMA) (1:1,000; cat. no. ab7817), anti-Snail family transcriptional repressor 2 (Slug) (1:1,000; cat. no. ab51772), anti-zinc finger E-box binding homeobox 1 (ZEB1) (1:500, ab203829) and anti-GAPDH (1:1,000; cat. no. ab8245; all from Abcam). Following primary antibody incubation, the membranes were washed four times with 0.05% PBS-Tween-20 and incubated with secondary antibodies [goat anti-rabbit IgG, HRP, cat. no. 7074; or goat anti-mouse IgG, HRP, cat. no. 7076; Cell Signaling Technology (1:2,000)] for 1 h at room temperature. Protein bands were visualized using an ECL detection system (Beyotime Institute of Biotechnology) and semi-quantified using ImageJ software (version 1.49; National Institutes of Health).

Flow cytometry

The effects of the knockdown of Sema7A and Ov-β1 integrin on cell apoptosis were evaluated using flow cytometry. Briefly, HUVECs were transfected with shRNA-Sema7A with or without Ov-β1 integrin for 48 h, then induced for 24 h at 37˚C with ox-LDL. The cells were harvested, washed and resuspended in binding buffer, then incubated with 5 µl Annexin V-FITC for 15 min and 10 µl PI (10 mg/ml) for 5 min in the dark. Apoptotic cells were detected by a flow cytometry (Accuri C6; BD Biosciences) and analyzed using FlowJo software (version 10.2; FlowJo LLC).

Immunofluorescence staining

Transfected or control HUVECs were plated into six-well plates with coverslips at a density of 4x105 cells/well and allowed to reach 70-80% confluence. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.1% Triton X-100 for permeabilization for 7 min, both at room temperature. The slides were then blocked with 5% skimmed milk diluted in 0.01 M PBS for 1 h at room temperature, and incubated with an anti-α-SMA primary antibody (1:100; cat. no. ab184675; Abcam) overnight at 4˚C. Following incubation, the slides were washed four times with PBS and incubated with a goat anti-mouse IgG H&L secondary antibody (1:200; cat. no. ab150113; Abcam) for 2 h at room temperature. Nuclei were counterstained with DAPI (Vector Laboratories, Inc.) for 5 min at room temperature. Finally, the slides were observed using a fluorescence microscope (magnification, x200; Olympus Corporation).

Statistical analysis

Statistical analysis was performed using SPSS version 19.0 software (IBM Corp.) and GraphPad Prism version 5.0 (GraphPad Software, Inc.) was used to draw the graphs. Data are presented as the mean ± SD from at least three independent experiments. Statistical differences between multiple groups were determined using one-way ANOVA followed by Tukey's multiple comparisons test, while statistical differences between two groups were determined using an unpaired, two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Sema7A and β1 integrin expression is upregulated in ox-LDL-induced HUVECs

ox-LDL plays a key role in endothelial cell injury during the progression of atherosclerosis (22). To simulate lipid accumulation-induced dysfunction of the endothelium in atherosclerosis, HUVECs were treated with 50 µg/ml ox-LDL for 24 h. As shown in Fig. 1A, the mRNA expression levels of Sema7A and β1 integrin were significantly in HUVECs upregulated following ox-LDL treatment. Similarly, ox-LDL upregulated the protein expression levels of Sema7A and β1 integrin in HUVECs compared with the control group (Fig. 1B). These data suggested that the aberrant expression of Sema7A and β1 integrin in HUVECs was associated with ox-LDL stimulation.

Figure 1 Sema7A and β1 integrin expression is upregulated in HUVECs in response to ox-LDL. (A) Sema7A and β1 integrin mRNA expression in untreated or ox-LDL-induced HUVECs was detected by reverse transcription-quantitative PCR analysis. (B) Western blot assay was performed to measure the protein levels of Sema7A and β1 integrin in untreated or ox-LDL-induced HUVECs. Data are expressed as mean ± SD. ***P<0.001 vs. control. Sema7A, semaphorin 7A; ox-LDL, oxidized low-density lipoprotein.

Sema7A knockdown and Ov-β1 integrin ameliorate ox-LDL-induced impaired HUVEC viability

To determine the role of Sema7A and β1 integrin in atherosclerosis, the effects of Sema7A and β1 integrin on the activities of HUVECs were investigated. As presented in Fig. 2A and B, Sema7A-knockdown vectors were transfected into HUVECs and the transfection efficiency was evaluated using RT-qPCR analysis and western blotting. According to the results, shRNA-Sema7A-1 exhibited the best interference efficiency, and was thus selected for the following experiments. Subsequently, the effect of Sema7A knockdown on β1 integrin expression was determined. The results revealed that ox-LDL treatment notably upregulated the mRNA and protein expression level of β1 integrin compared with the control group, while transfection with shRNA-Sema7A-1 inhibited the ox-LDL-induced upregulated expression of β1 integrin (Fig. 2C and D). Furthermore, β1 integrin overexpression vectors were transfected into Sema7A-knockdown cells and the transfection efficiency was investigated (Fig. 2E-G). The results of the CCK-8 assay revealed that cell viability was markedly repressed following ox-LDL induction, while the knockdown of Sema7A alleviated the suppressive effects of ox-LDL on cell viability. However, the overexpression of β1 integrin reversed the Sema7A knockdown-induced increase in HUVEC viability (Fig. 2H).

Figure 2 Effects of Sema7A and β1 integrin on HUVEC viability. (A) Western blot assay was performed to measure the protein expression of Sema7A in ox-LDL-induced HUVECs transfected with shRNA-Sema7A. (B) RT-qPCR analysis was performed to measure the mRNA expression of Sema7A in ox-LDL induced HUVECs transfected with shRNA-Sema7A. (C) Western blot assay was performed to measure the protein levels of β1 integrin in ox-LDL induced HUVECs transfected with shRNA-Sema7A. (D) RT-qPCR analysis was performed to measure the mRNA expression of β1 integrin in ox-LDL-induced HUVECs transfected with shRNA-Sema7A. (E) RT-qPCR analysis was performed to measure the mRNA expression of β1 integrin in HUVECs transfected with Ov-β1 integrin. (F) Western blot assay was performed to measure the protein level of β1 integrin in Sema7A-silenced HUVECs transfected with Ov-β1 integrin. (G) RT-qPCR analysis was performed to measure the mRNA expression of β1 integrin in Sema7A-silenced HUVECs transfected with Ov-β1 integrin. (H) HUVEC viability was measured using a Cell Counting Kit-8 assay. Data are expressed as mean ± SD. ***P<0.001, ##P<0.01 and ###P<0.001; $P<0.05. Sema7A, semaphorin 7A; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative PCR; shRNA, short hairpin RNA; Ov-, overexpression vector; NC, negative control.

Sema7A knockdown and Ov-β1 integrin inhibit the ox-LDL-induced release of inflammatory cytokines and adhesion factors in HUVECs

Inflammation and adhesion factors are closely associated with the loss of morphological and functional integrity of vascular endothelial cells in atherosclerosis (10). As shown in Fig. 3A, the production of IL-1β, IL-6 and CCL2 was significantly increased in HUVECs induced with ox-LDL compared with control cells. The knockdown of Sema7A abrogated the secretion of IL-1β, IL-6 and CCL2, while the overexpression of β1 integrin enhanced the Sema7A knockdown-mediated decrease in the levels of IL-1β, IL-6 and CCL2. Moreover, the results from RT-qPCR and western blot analyses demonstrated that the stimulation with ox-LDL markedly upregulated the expression levels of ICAM-1 and VCAM-1 in HUVECs. The knockdown of Sema7A repressed the production of these adhesion factors, while these effects were blocked by Ov-β1 integrin (Fig. 3B and C).

Figure 3 Effects of Sema7A and β1 integrin on the levels of inflammatory cytokines and adhesion factors in HUVECs. (A) IL-1β, IL-6 and CCL2 levels in ox-LDL induced HUVECs were examined after transfection with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. Western blot assay and reverse transcription-quantitative PCR were carried out to detect the (B) protein levels and (C) mRNA expressions of ICAM-1 and VCAM-1 in ox-LDL induced HUVECs transfected with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. Data are expressed as mean ± SD. ***P<0.001 vs. control; ###P<0.001 vs. shRNA-NC; $P<0.05, $$P<0.01 and $$$P<0.001 vs. shRNA-Sema7A + Ov-NC. Sema7A, semaphorin 7A; ox-LDL, oxidized low-density lipoprotein; CCL2, C-C motif chemokine ligand 2; shRNA, short hairpin RNA; Ov-, overexpression vector; NC, negative control; ICAM-1, intracellular adhesion molecule; VCAM, vascular cell adhesion molecule-1.

Sema7A knockdown and Ov-β1 integrin prevent the apoptosis of HUVECs in response to ox-LDL

Next, the effects of Sema7A knockdown and Ov-β1 integrin transfection on the apoptosis of HUVECs were analyzed. As illustrated in Fig. 4A and B, the apoptotic rate of HUVECs induced with ox-LDL was markedly increased compared with the control cells. The knockdown of Sema7A reduced ox-LDL-induced cell apoptosis, while the overexpression of β1 integrin reversed the suppressive effect of Sema7A knockdown on the apoptosis of HUVECs treated with ox-LDL. Furthermore, the incubation of HUVECs with ox-LDL downregulated Bcl-2 expression levels and upregulated the expression levels of Bax and cleaved caspase-3. The knockdown of Sema7A reversed the effects of ox-LDL, while transfection with Ov-β1 integrin reversed the Sema7A knockdown-induced upregulation of Bcl-2 protein expression levels and downregulation of Bax and cleaved caspase-3 expression levels (Fig. 4C).

Figure 4 Effects of Sema7A and β1 integrin on the apoptosis in ox-LDL-induced HUVECs. (A and B) Cell apoptosis was evaluated by flow cytometry in HUVECs transfected with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. (C) Bcl-2, Bax, caspase-3 and cleaved caspase-3 protein levels in HUVECs were determined using western blot assay after transfection with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. Data are expressed as mean ± SD. ***P<0.001 vs. control; ###P<0.001 vs. shRNA-NC; $P<0.05, $$P<0.01, $$$P<0.001 vs. shRNA-Sema7A + Ov-NC. Sema7A, semaphorin 7A; ox-LDL, oxidized low-density lipoprotein; shRNA, short hairpin RNA; Ov-, overexpression vector; NC, negative control.

Sema7A knockdown and Ov-β1 integrin attenuate ox-LDL-induced EMT in HUVECs

To further determine the functional role of Sema7A and β1 integrin in the EMT of HUVECs treated with ox-LDL, the protein and mRNA expression levels of EMT-related proteins were analyzed using western blotting and RT-qPCR, respectively. As shown in Fig. 5A and B, the expression levels of α-SMA, Slug and ZEB1 were upregulated, while the expression levels of CD31 and vWF were downregulated in HUVECs following induction with ox-LDL, and these effects were reversed by the knockdown of Sema7A and overexpression of β1 integrin. In addition, the results of the immunofluorescence assay revealed that α-SMA expression levels were significantly upregulated after HUVECs were induced with ox-LDL, whereas they were downregulated by the knockdown of Sema7A expression. However, the overexpression of β1 integrin inhibited the effects of Sema7A knockdown on ox-LDL-induced EMT in HUVECs (Fig. 5C).

Figure 5 Effects of Sema7A and β1 integrin on ox-LDL-induced epithelial-to-mesenchymal transition in HUVECs. (A) Western blot assay was used to determine the protein levels of α-SMA, Slug, ZEB1, CD31 and vWF in ox-LDL-induced HUVECs transfected with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. (B) Reverse transcription-quantitative PCR analysis was used to measure mRNA expression of α-SMA, Slug, ZEB1, CD31 and vWF were estimated by in ox-LDL-induced HUVECs transfected with shRNA-Sema7A in the presence and absence of Ov-β1 integrin. (C) Immunofluorescence assay was performed to assess α-SMA protein levels in ox-LDL-induced HUVECs transfected with shRNA-Sema7A in the presence or absence of Ov-β1 integrin. Data are expressed as mean ± SD. ***P<0.001 vs. control; ###P<0.001 vs. shRNA-NC group; $P<0.05, $$P<0.01, $$$P<0.001 vs. shRNA-Sema7A + Ov-NC group. Sema7A, semaphorin 7A; ox-LDL, oxidized low-density lipoprotein; α-SMA, α-smooth muscle actin; vWF, von Willebrand factor; Slug, Snail family transcriptional repressor 2; ZEB1, zinc finger E-box binding homeobox 1; shRNA, short hairpin RNA; Ov-, overexpression vector; NC, negative control.

Discussion

Atherosclerosis is the main cause of morbidity and mortality in cardiovascular diseases, and is responsible for more lives than all cancer types combined (23). The results of the present study revealed that the expression of Sema7A and β1 integrin was dysregulated ox-LDL-induced HUVECs. In addition, Sema7A and β1 integrin were serve key roles in ox-LDL-induced HUVECs by regulating cell viability, inflammatory responses, apoptosis and EMT. The knockdown of Sema7A expression increased cell viability, inhibited the release of inflammatory factors and adhesion molecules, suppressed cell apoptosis and inhibited the EMT process, whereas the overexpression of β1 integrin reversed the effects of Sema7A silencing in HUVECs induced with ox-LDL.

Endothelial cells exist in the walls of blood and lymphatic vessels and have unique functions in vascular biology (24). Vascular endothelial cell injury is a common pathological basis of various cardiovascular diseases and represents the early stage of atherosclerosis (25). Hyperlipidemia, a cardiovascular risk factor, promotes the accumulation of plasma LDL in the arterial wall, which drives the formation of atherosclerotic plaques, and induces endothelial cell injury and apoptosis during the occurrence and development of atherosclerosis (26,27). ox-LDL is a causal risk factor for atherosclerosis and binds to the scavenger receptor in endothelial cells to promote the secretion of inflammatory cytokines, inducing apoptosis and further promoting EMT (28,29).

Vascular endothelial cell inflammation plays a central role in all stages of atherosclerosis, from lesion initiation to progression and destabilization (30). A previous study reported that ox-LDL induced a local inflammatory response, which inflamed the endothelium and upregulated the expression of adhesion molecules, leading to the adhesion of circulating leukocytes to the endothelium (31). Thus, in the present study, ox-LDL was used to stimulate HUVECs to mimic hyperlipidemia-induced endothelial cell injury. The results demonstrated that ox-LDL treatment significantly reduced HUVEC viability and induced the production of inflammatory factors, including IL-1β, IL-6 and CCL2. In addition, the expression levels of ICAM-1 and VCAM-1 were markedly upregulated after HUVECs were exposed to 50 µg/ml ox-LDL for 24 h. The present study also revealed that the knockdown of Sema7A abrogated the generation of ox-LDL-induced inflammatory factors, IL-1β, IL-6 and CCL2, while the overexpression of β1 integrin reversed these effects of Sema7A-knockdown. As expected, the upregulated expression levels of ox-LDL-induced ICAM-1 and VCAM-1 were downregulated by the knockdown of Sema7A and recovered by Ov-β1 integrin transfection. These data indicated that silencing of Sema7A may protect against ox-LDL-induced inflammatory injury in endothelial cells by regulating β1 integrin.

Inflammatory responses induced by injury stress signals, such as ox-LDL, mediate further interactions between monocytes and endothelial cells, resulting in increased apoptosis of endothelial cells, which may initiate atherosclerosis (32-34). Wu et al (35) found that ox-LDL significantly induced HUVEC apoptosis, while the knockdown of proprotein convertase subtilisin/kexin type 9 suppressed apoptosis via the Bcl/Bax/caspase-9/caspase-3 signaling pathway. In addition, Zhong et al (36) demonstrated that myocardial infarction-associated transcript promoted cell proliferation and inhibited apoptosis in ox-LDL-induced atherosclerosis cell models by modulating the microRNA-181b/STAT3 signaling axis. Thus, the suppression of vascular endothelial cell apoptosis may promote pathological remission of atherosclerosis (37). In the present study, the apoptotic rate was increased in HUVECs exposed to ox-LDL, which was accompanied with downregulated expression levels of Bcl-2 and upregulated expression levels of Bax and cleaved caspase-3. However, these changes were mitigated by the knockdown of Sema7A, while β1 integrin-overexpression abolished the inhibitory effects of Sema7A silencing, which is consistent with previous reports.

EMT serves key roles in plaque instability and atherosclerosis progression (38,39). Previous studies have reported that the formation and instability of atherosclerotic plaques are associated with endothelial cells acquiring morphological and phenotypic properties of mesenchymal cells, such as increased proliferation and migration, and the secretion of leukocyte adhesion molecules, extracellular matrix and matrix metalloproteinases (40). An accumulating number of studies have found that Sema7A and its receptor, β1 integrin, contributed to vascular endothelial cell injury and the pathophysiology of atherosclerosis (19,20,41). In the present study, the expression levels of α-SMA, Slug and ZEB1 were found to be upregulated, while the expression levels of CD31 and vWF were downregulated under the induction of ox-LDL. The knockdown of Sema7A rescued the expression levels of EMT-related proteins, while β1 integrin-overexpression counteracted the effects of Sema7A silencing on EMT in HUVECs. These findings indicated that the knockdown of Sema7A or overexpression of β1 integrin may protect HUVECs against ox-LDL-induced EMT. In addition, the present study focused on the regulatory role of Sema7A silencing in ox-LDL HUVECs through modulating β1 integrin expression, but did not explore whether Sema7A is involved in the regulation of β1 integrin expression in ox-LDL HUVECs. Furthermore, the results of the present study were not verified in animal experiments. Thus, the relationship between Sema7A and β1 integrin and the underlying mechanisms will be further investigated in vitro and in vivo in future studies.

In conclusion, the findings of the present study suggested that the knockdown of Sema7A/β1 integrin may protect HUVECs from ox-LDL-induced injury by inhibiting the stimulation of inflammatory responses, cell apoptosis and EMT. Therefore, Sema7A and β1 integrin may represent novel targets for the treatment of endothelial cell injury and dysfunction in atherosclerosis. Collectively, the findings of the present study may provide potential targets and therapeutic strategies against vascular endothelial cell inflammation and EMT to promote the pathological remission of atherosclerosis.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Medical Guidance (TCM) Science and Technology Support Project of Science and Technology Commission of Shanghai Municipality, China (grant no. 17401933700), the Scientific Research Project of Shanghai Municipal Commission of Health and Family Planning, China (grant no. 201640039) and the Peak Plateau Subject of Shanghai University of Traditional Chinese Medicine (Special Project for Clinical Talents), China (grant no. 171319).

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XS and DL designed and supervised the experiments. XS, JM, GY, HW and HL performed the experiments. HW analyzed the data and HL searched the literature. XS, JM and DL wrote and revised the manuscript. XS and DL have seen and can confirm the authenticity of all the raw data. All the authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References