GRAPHICAL ABSTRACT

Highlights

• E2F5 expression was elevated in diabetic atherosclerosis (DAS).

• E2F5 downregulation alleviated the symptoms of DAS in mice.

• E2F5 knockdown inhibited the phenotypic transformation of VSMCs.

• E2F5 knockdown inhibited the Wnt/β-catenin pathway in DAS.

INTRODUCTION

Diabetes is one of the fastest growing diseases, by 2040 the global diabetes people will rise to 629 million [1]. The complications related to diabetes especially vascular complications have caused serious social health burden [2]. Diabetic cardiovascular disease complications are the main cause of death in patients with diabetes, affect about 32.2% of all people with diabetes [3]. Among them, diabetic atherosclerosis (DAS) is the most common cardiovascular complication of diabetes [4]. Atherosclerosis is a chronic disease that mainly involves endothelial-cell dysfunction, inflammatory response, and lipid deposition and calcification in the arterial vessel wall [5]. Importantly, atherosclerosis is considered to be a critical pathologic basis for the development of various cardiovascular and cerebrovascular diseases [6]. This highlights the importance and urgency of researching the mechanisms of DAS and exploring therapeutic options.

E2F transcription factors (E2Fs) are a family of members that play critical roles in modulating the balance of cell cycle through the transcriptional axis [7,8], which have been identified eight different E2F genes (E2F1–E2F8). The individual E2Fs can act in direct opposition to one another to promote either cell proliferation or cell cycle exit and terminal differentiation [7]. Moreover, Zhan et al. [9] have reported that E2F transcription factor 5 (E2F5) modulates the expressions of genes involved in cell cycle progression through directly binding to genes promoters. At present, E2F5 is demonstrated to facilitate the development of multiple human malignant tumors [10-12]. Interestingly, Xu et al. [13] have reported that miR 132 could inhibit high glucose induced vascular smooth muscle cells (VSMCs) proliferation and migration via targeting E2F5. However, little is known about the function E2F5 plays in regulating VSMCs phenotype switching in DAS.

In the present study, our findings demonstrated that the downregulation of E2F5 could repress VSMCs phenotype switching and ultimately inhibit the progression of DAS. The findings may provide the basis for the development of E2F5 based therapeutic approaches for DAS.

METHODS

Animals and treatment

Apoprotein E (ApoE)-knockout mice (aged 6 to 8 weeks) on a C57BL/6J background were purchased from Gempharmatech (Nanjing, China). ApoE-knockout mice were maintained under the 12-hour dark-light cycle with unrestricted access to standard rodent chow and water. Mice were systematically divided into control, DAS, DAS+ adeno-associated virus carrying negative control small hairpin RNA (AAV-shNC), and DAS+AAV-shE2F5 groups. The healthy C57BL/6J mice were used as a control group. ApoE-knockout mice received an intraperitoneal injection of streptozotocin (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) for 5 consecutive days, and fasting blood glucose level was monitored. The model was established when the blood glucose was ≥11.1 mmol/L. Then, the mice were fed the high-fat diet (HFD; 0.15% cholesterol and 21% fat) for 12 weeks. At the 4th week after HFD, the mice in HFD+AAV-shNC and HFD+AAV-shE2F5 groups were injected with AAV-shNC or AAV-shE2F5 (100 μL, 1.0×10¹³ viral genomes/mL) in phosphate-buffered saline (PBS) via the tail vein. The mice in control and HFD groups were injected with the same volume of PBS. After the mice sacrificed, blood and aorta tissues were collected. The Institutional Review Board of The First Affiliated Hospital of Shandong First Medical University approved the research (2020-(S089)). The animal experimental was performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Detection of serum biochemical index

Serum was prepared and subjected to analysis of levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) using the corresponding assess kits.

Histological section

The aortic tissue was immediately collected and preserved utilizing 4% paraformaldehyde following the mice were sacrificed. Then, the aortic tissues were embedded in paraffin and cut into 5-μm thick sections. Sections were dyed with hematoxylin for 10 minutes, counterstained using eosin for 2 minutes, dehydrated, soaked with xylene and sealed with resin. At last, images were captured using a light microscope. For observing lipid accumulation, aorta was preserved utilizing 4% paraformaldehyde, and then 8-μm thick sections were made. Subsequently, the sections were dyed using Oil red O working solution (5 mg/mL Oil red O stock solution: ddH₂O=3:2), and then stained using haematoxylin. In the end, a light microscope was employed for visualizing the sections.

Western blot analysis

The lysates from the cells and aortic tissues were extracted using radioimmunoprecipitation assay (RIPA) buffer (R0010, Solarbio, Beijing, China). The sample containing 40 μg protein was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% mini-gels. After gel electrophoresis, the separated proteins were transferred to polyvinylidene fluoride membranes (Beyotime, Shanghai, China), which were then blocked in 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) for half an hour. Afterwards, the blots were incubated for 24 hours at 4°C with the respective primary antibodies, including anti-E2F5 (BA0813-2, BOSTER, Wuhan, China), anti-α-smooth muscle actin (anti-α-SMA) (BM3902, BOSTER), anti-SM22 (A03962-2, BOSTER), anti-osteopontin (anti-OPN) (ab218237, Abcam, Cambridge, UK), anti-ClyclinE (SAB4503516, Sigma-Aldrich), anti-cyclin-dependent kinase 2 (anti-CDK2) (BM3926, BOSTER), wnt1 (ab15251, Abcam), β-catenin (ab224803, Abcam), and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (BM3874, BOSTER). After secondary antibody labeled with horseradish peroxidase incubates for 60 minutes, the blots were developed with the ECL Western blotting substrate (Solarbio).

Quantitative reverse transcription polymerase chain reaction

TRIzol reagent (Invitrogen, Waltham, MA, USA) was employed for extracting total RNA. The HiScript IV RT SuperMix for quantitative polymerase chain reaction (qPCR) (+gDNA wiper) (Vazyme, Nanjing, China) was used for cDNA synthesis. Taq Pro Universal SYBR qPCR Master Mix (Vazyme) was employed for quantitative reverse transcription polymerase chain reaction (qRT-PCR). GAPDH were used as the control. E2F5 forward 5´-TGGGCTTGCTTACCACCAAA-3´ and reverse 5´-GGTATCTGCAGCCGCTTTGA-3´; GAPDH forward 5´-AGGTCGGTGTGAACGGATTT-3´ and reverse 5´-TTCCCATTCTCGGCCTTGAC-3´.

VSMCs culture and treating

Primary cultured VSMCs were obtained from the C57BL/6 mice thoracic aorta by enzymatic dispersion according to the modified method of Zhang et al. [14]. VSMCs were grown in Dulbecco’s Modified Eagle Medium supplemental with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution, and maintained in 5% CO₂ at 37˚C within a humidified atmosphere. The si-E2F5 and negative control siRNA (si-NC) which were supplied by Ribobio (Guangzhou, China), and CyclinE overexpression plasmids pcDNA-CyclinE (CyclinE-OE) which was purchased from FulenGen (Guangzhou, China) were transfected to VSMCs for 2 days employing Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Before further operation, VSMCs were cultured in serum-free medium for 24 hours. After that, VSMCs were stimulated with fresh medium containing 30 mM glucose (high glucose [HG]) for 24 hours, and then treated with oxidized low-density lipoprotein (ox-LDL; 100 μg/mL) for 24 hours. Meanwhile, VSMCs were treated with BML-284 (a Wnt pathway activator, Med-ChemExpress, Monmouth Junction, NJ, USA) for 48 hours.

Cell counting kit-8 assay

VSMCs were cultured in a 96-well culture plate for 48 hours. Afterwards, 10 μL of cell counting kit-8 (CCK-8) solution (A311-01, Vazyme, Wuhan, China) was added into each well, followed by incubation for 2 hours. In the end, the absorbance was read at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Flow cytometry analysis

VSMCs at the logarithm growth period were harvested via centrifugation and fixed in ice-cold 70% ethanol overnight. After that, RNase A was added to VSMCs and incubated for half an hour at room temperature, followed by addition of propidium iodide (Beyotime, Shanghai, China) in the dark for 15 minutes. Finally, DNA content was analyzed using flow cytometry on the FACScan flow cytometry system (Becton Dickinson, Franklin Lakes, NJ, USA).

5-ethynyl-2´-deoxyuridine assay

VSMCs at a density of 5×10⁴ were seeded into 24-well plates and incubated for 48 hours. Afterwards, VSMCs were treated with 10 μM 5-ethynyl-2’-deoxyuridine (EdU) for additional 2 hours at room temperature. Following fixed in 4% paraformaldehyde, VSMCs were dyed using 4´,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). At last, EdU-positive-stained VSMCs were counted under an Eclipse Ni-U fluorescence microscope (NIKON, Tokyo, Japan).

Wound healing assay

VSMCs were cultured in 6-well plates and a 200-μL sterile plastic tip was utilized for scratching wound when cell confluence reached 90%. Following washed three times, VSMCs were cultured in serum-free medium for 24 hours. Finally, we collected images and analyzed the scratched area in each group under a light microscope.

Transwell assay

VSMCs were plated in the upper transwell chamber (BD Biosciences, San Jose, CA, USA). A 200 μL of serum-free medium was filled into the upper cavity of the transfer well. The bottom chamber filled 600 μL of medium with 10% FBS. After incubation for 2 days, the migrated VSMCs were fixed utilizing methanol and then dyed utilizing crystal violet (Solarbio). Lastly, the cells were observed by using a light microscope.

Statistical analysis

We used SPSS software 22.0 (IBM Co., Armonk, NY, USA) to analyze the experimental data. The results are presented as mean±standard deviations. Differences between groups were compared by Student’s t-test or analysis of variance. P<0.05 was considered statistically significant.

RESULTS

Downregulation of E2F5 alleviated diabetes-related atherosclerosis in mice

Firstly, the expression of E2F5 was evaluated in DAS mice. As showed in Fig. 1A and B, E2F5 expression was significantly elevated in DAS group when compared with control group. H&E staining manifested that the atherosclerotic injury was notably enhanced in DAS group mice relative to control group (Fig. 1C). The downregulation of E2F5 mitigated the atherosclerotic injury in mice (Fig. 1C). The Oil red O staining indicated the significant increase in the plaque of aorta in DAS group mice relative to the control group mice (Fig. 1D). When compared with DAS+AAV-shNC group, mice in DAS+AAV-shE2F5 group exhibited a significant decrease in plaque area in aorta (Fig. 1D). Blood samples from HFD-fed diabetic ApoE^−/− mice manifested an increase in TC, TG, and LDL-C level and a decrease in HDL-C level (Fig. 1E). Moreover, the downregulation of E2F5 significantly reversed the above abnormal levels of TC, TG, HDL-C, and LDL-C (Fig. 1E). Collectively, these data proved that the downregulation of E2F5 could alleviate diabetes-related atherosclerosis in mice.

Downregulation of E2F5 inhibited the phenotypic transformation of VSMCs in DAS mice

The results of Western blot showed that the decreased contractile markers level of α-SMA and SM22 and increased synthetic markers level of OPN were observed in aortic tissues of DAS mice (Fig. 2A). When compared with DAS+AAV-shNC group, the expression of α-SMA and SM22 was markedly elevated in DAS+AAV-shE2F5 group, but OPN expression was remarkedly reduced (Fig. 2A). Furthermore, Western blot analysis demonstrated the upregulated expressions of CyclinE and CDK2 in aortic tissues of DAS mice were significantly attenuated after injecting AAV-shE2F5 (Fig. 2B). These experimental results suggested that the downregulation of E2F5 could inhibit the phenotypic transformation of VSMCs in DAS mice.

The knockdown of E2F5 inhibited the phenotypic transformation of VSMCs in vitro

As Fig. 3A manifested, E2F5 was highly expressed after the induction of HG and ox-LDL. On the contrary, E2F5 silencing notably reversed the upregulated E2F5 expression (Fig. 3A). The results of CCK-8 and EdU assays manifested that the treatment of HG and ox-LDL significantly promoted VSMCs proliferation (Fig. 3B and C). In comparison with HG+ox-LDL+si-NC group, VSMCs proliferative capacity in HG+ox-LDL+si-E2F5 group was notably reduced (Fig. 3B and C). Additionally, the increased S-phase cell arrest of VSMCs induced by HG and ox-LDL significantly reversed after the treatment of si-E2F5 (Fig. 3D). Subsequently, we explored the function of E2F5 on cell cycle-related proteins (CyclinE and CDK2) and discovered that the increased CyclinE and CDK2 expressions induced by HG and ox-LDL were neutralized after the treatment of si-E2F5 (Fig. 3E). During atherosclerosis, the contractile phenotype of VSMCs, characterized by poor synthesis and rare proliferation, is transformed into a synthetic phenotype characterized by excessive proliferation and migration [15,16]. Hence, we subsequently explored E2F5 roles on the migration capacity of VSMCs. Both the wound healing and transwell experiments data demonstrated the enhanced migration ability of VSMCs induced by HG and ox-LDL was dramatically reversed by the knockdown of E2F5 (Fig. 4A and B). Furthermore, the level of α-SMA and SM22 was reduced, but OPN level was elevated after the treatment of HG and ox-LDL (Fig. 4C). In comparison with HG+ox-LDL+si-NC group, α-SMA and SM22 expression was markedly elevated in HG+ox-LDL+si-E2F5 group, but OPN expression was remarkedly reduced (Fig. 4C). Together, these results indicated that the knockdown of E2F5 could inhibit the phenotypic transformation of VSMCs in vitro.

CyclinE overexpression reversed the inhibitory effect of E2F5 silencing on phenotypic transformation of VSMCs

As Fig. 5A showed, the expression of CyclinE and CDK2 was remarkedly higher in HG+ox-LDL+si-E2F5+CyclinE-OE group than that in HG+ox-LDL+si-E2F5 group. When compared with HG+ox-LDL+si-E2F5 group, the proliferation, S-phase cell numbers, as well as the migration of VSMCs were significantly elevated in HG+ox-LDL+si-E2F5+CyclinE-OE group (Fig. 5B-D). Moreover, the expression of α-SMA and SM22 in HG+ox-LDL+si-E2F5+CyclinE-OE group was markedly reduced compared with HG+ox-LDL+si-E2F5 group, while the expression of OPN was notably elevated (Fig. 5E). These above observations indicated that CyclinE overexpression could reverse the inhibitory effect of E2F5 silencing on phenotypic transformation of VSMCs.

E2F5 knockdown repressed VSMCs phenotype switching through inactivating Wnt/β-catenin pathway

To elucidate the mechanism by which E2F5 affects the phenotypic transformation of VSMCs, we searched for signaling pathways that E2F5 can affect in the previous studies. Malgundkar et al. [17] shows that E2F5 promotes the malignancy of ovarian cancer via the regulation Wnt pathway. Given that Wnt/β-catenin plays important role in the phenotypic transformation of VSMCs [18], we speculate that E2F5 may affect the phenotypic transformation of VSMCs by influencing the Wnt/β-catenin pathway. As shown in Fig. 6A, E2F5 silencing significantly reversed the increased wnt1 and β-catenin expressions caused by HG and ox-LDL. Moreover, the expressions of wnt1 and β-catenin were notably upregulated in HG+ox-LDL+si-E2F5+BML-284 group compared with HG+ox-LDL+si-E2F5 group (Fig. 6A). These data revealed that E2F5 silencing might inhibit the activation of Wnt/β-catenin pathway. To verify this, BML-284 was applied to perform the rescue experiments. When compared with HG+ox-LDL+si-E2F5 group, the proliferation, S-phase cell numbers, as well as the migration of VSMCs were significantly elevated in HG+ox-LDL+si-E2F5+BML-284 group (Fig. 6B-D). Furthermore, the levels of α-SMA and SM22 in HG+ox-LDL+si-E2F5+BML-284 group were notably decreased compared with HG+ox-LDL+si-E2F5 group, while the level of OPN was significantly increased (Fig. 6E). These above observations revealed that the downregulation of E2F5 could repress VSMCs phenotype switching through inactivating Wnt/β-catenin pathway.

DISCUSSION

Atherosclerosis is the main cause of death in the world and is driven by a variety of risk factors, including diabetes, which leads to an increased atherosclerotic burden [5]. The incidence of atherosclerosis in diabetes people has been reported to be several times higher than that of non-diabetes people, with the onset time being early, the process being fast and the death rate being high [19]. However, the molecular mechanisms for DAS progression has not been fully elucidated. This study is the first to report the effect of E2F5 in promoting VSMCs phenotype switching, which could ultimately accelerate the progression of DAS. These data indicated that E2F5 could exert some proatherosclerotic effects in the context of diabetes.

VSMCs are a major component of the arterial media and are essential for the good function of the vascular system. Recently, lineage-tracing and single cell genomics have proved that VSMCs are a major cellular component of atherosclerotic plaques [20-22]. VSMCs have obvious phenotypic plasticity in blood vessels, transitioning between contractile/differentiated and synthetic/proliferative phenotypes under the stimulus of physiological and pathological conditions, including atherosclerosis [23]. Contractile VSMCs are characterized by poor synthesis and rare proliferation, while synthetic VSMCs possess higher proliferation and migration capacities [24]. In addition, VSMCs switching from the contractile phenotype to the synthetic phenotype was often related to the upregulated levels of synthetic markers (OPN), and the downregulated levels of contractile markers (α-SMA and SM22) [25]. Hence, suppressing VSMCs phenotype switching could be a potential strategy for managing DAS.

In recent years, the high VSMCs proliferation and VSMCs phenotype switch in DAS were been observed [26,27]. E2F5 participates the modulation of multiple cellular processes, such as cell proliferation, apoptosis, differentiation, and DNA damage [28]. E2F5 exerts a cancer-promoting role in various human malignant tumors through increasing cancers cell proliferation and migration [10-12]. It is worth that miR 132 could inhibit high glucose induced VSMCs proliferation and migration via targeting E2F5 [13]. Therefore, we explored whether E2F5 might accelerate DAS development through regulating VSMCs phenotype switching. In vivo, E2F5 was highly expressed in DAS, and the downregulation of E2F5 could alleviate the symptoms of DAS in mice. We found that the knockdown of E2F5 also inhibited VSMCs proliferation and migration. Furthermore, E2F5 knockdown was been verified to notably elevate α-SMA and SM22 levels, and reduced OPN expression in both animal and cell models, further suggesting that E2F5 promoted DAS progression through accelerating the phenotypic transformation of VSMCs. E2F5 regulates the transition from the G1 to S phase by directly regulating CyclinE-CDK2 [29]. In current research, we proved that E2F5 silencing could reverse the increased S-phase cell arrest of VSMCs and the increased CyclinE and CDK2 expressions induced by HG and ox-LDL. In addition, we also confirmed that CyclinE overexpression could reverse the inhibitory effect of E2F5 silencing on phenotypic transformation of VSMCs.

Moreover, we found that knockdown E2F5 could decrease TC, TG, and LDL-C level and increase HDL-C level in the DAS model. These results may be due to the involvement of E2F5 in the regulation of lipid metabolism. Urs et al. [30] discover that compared with preadipocytes, several genes involved in lipid metabolism are overexpressed in adipocytes, including fatty acid binding protein, adipose differentiation-related protein, adipose most abundant transcript 1 and E2F5 transcriptional factor. This finding reveals that E2F5 is involved in the transformation of preadipocytes to adipocytes. In addition, E2F5 as a transcriptional factor may affect lipid metabolism through regulating the expression of key enzymes involved in lipid metabolism. Through the prediction of website animalTFDB, it was found that E2F5 could bind the promoter of acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1) which participate in the synthesis of fatty acids. These predicted results will be further validated in future experiments.

Dysregulation of Wnt/β-catenin pathway has been involved in the pathogenesis of numerous cardiovascular diseases, including atherosclerosis [31]. The proliferative effects of Wnt/β-catenin pathway in VSMCs have been comprehensively reported by Lyon et al. [32]. A study by Xue et al. [33] has demonstrated that cell migration-inducing hyaluronan binding protein (CEMIP) can regulate the proliferation and migration of VSMCs in atherosclerosis by activating the Wnt/β-catenin pathway. Moreover, Rong et al. [18] have confirmed that solute carrier family 6 member 6 (SLC6A6) overexpression could suppress neointimal formation by inhibiting VSMC proliferation and migration via Wnt/β-catenin signaling and maintaining the VSMC contractile phenotype. Interestingly, E2F5 has been proved to activate the Wnt/β-catenin signaling [17]. Therefore, we next explored whether E2F5 might promote phenotypic transformation of VSMCs by activating the Wnt/β-catenin pathway, which was demonstrated in our study.

Collectively, the results of our research suggested that E2F5 downregulation could repress VSMCs phenotype switching through inactivating Wnt/β-catenin pathway, and ultimately inhibit the progression of DAS. Our findings suggest that targeting E2F5 in DAS may be a viable option.

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: M.D., Q.X.

Acquisition, analysis, or interpretation of data: M.D., J.W., Q.X.

Drafting the work or revising: M.D., J.W., L.S., G.Y.

Final approval of the manuscript: all authors.

FUNDING

This work was supported by funding from the Natural Science Foundation of Shandong Province (ZR2020QH022) and the National Natural Science Foundation Cultivation Fund of Shandong Provincial Qianfoshan Hospital (QYPY2020NSFC0801).

ACKNOWLEDGMENTS

None

Fig. 1.

The downregulation of E2F transcription factor 5 (E2F5) alleviated diabetes-related atherosclerosis in mice. The expression of E2F5 in diabetic atherosclerosis (DAS) mice was evaluated utilizing Western blot (A) and quantitative reverse transcription polymerase chain reaction (B). (C) Representative picture of hematoxylin and eosin (HE) staining (×50). (D) Representative picture of Oil red O staining (×50). (E) The serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were assessed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; AAV, adeno-associated virus; shNC, negative control small hairpin RNA. ^aP<0.05, ^bP<0.05.

Fig. 2.

The downregulation of E2F transcription factor 5 (E2F5) inhibited the phenotypic transformation of vascular smooth muscle cells in diabetic atherosclerosis (DAS) mice. (A) Western blot for detecting α-smooth muscle actin (α-SMA), SM22, and osteopontin (OPN) expression in aortic tissues in different groups. (B) Western blot for detecting CyclinE and cyclin-dependent kinase 2 (CDK2) expression. AAV, adeno-associated virus; shNC, negative control small hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^aP<0.05, ^bP<0.05.

Fig. 3.

The downregulation of E2F transcription factor 5 (E2F5) suppressed vascular smooth muscle cells (VSMCs) proliferation and cell cycle. (A) Western blot for detecting E2F5 expression in VSMCs in different groups. (B) Cell counting kit-8 (CCK-8) and (C) 5-ethynyl-2´-deoxyuridine (EdU) assays were employed for assessing the proliferative ability of VSMCs (×200). (D) Flow cytometry was employed for testing VSMCs cell cycle. (E) Western blot for detecting CyclinE and cyclin-dependent kinase 2 (CDK2) expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HG, high glucose; ox-LDL, oxidized low-density lipoprotein; si-NC, negative control siRNA; DAPI, 4´,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.05.

Fig. 4.

The downregulation of E2F transcription factor 5 (E2F5) suppressed vascular smooth muscle cells (VSMCs) migration. (A) Wound healing (×40) and (B) transwell assays were utilized for evaluating the migrative ability of VSMCs in different groups (×100). (C) Western blot for detecting α-smooth muscle actin (α-SMA), SM22, and osteopontin (OPN) expression in VSMCs in different groups. HG, high glucose; ox-LDL, oxidized low-density lipoprotein; si-NC, negative control siRNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^aP<0.05, ^bP<0.05.

Fig. 5.

CyclinE overexpression reversed the inhibitory effect of E2F transcription factor 5 (E2F5) silencing on phenotypic transformation of vascular smooth muscle cells (VSMCs). (A) Western blot for detecting CyclinE and cyclin-dependent kinase 2 (CDK2) expression in VSMCs in different groups. (B) 5-ethynyl-2´-deoxyuridine (EdU) assay was employed for assessing the proliferative ability of VSMCs (×200). (C) Flow cytometry was employed for testing VSMCs cell cycle. (D) Transwell assay was utilized for evaluating the migrative ability of VSMCs (×100). (E) Western blot for detecting α-smooth muscle actin (α-SMA), SM22, and osteopontin (OPN) expression in VSMCs. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HG, high glucose; ox-LDL, oxidized low-density lipoprotein; si-NC, negative control siRNA; OE, overexpression; DAPI, 4´,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.05.

Fig. 6.

E2F transcription factor 5 (E2F5) knockdown repressed vascular smooth muscle cells (VSMCs) phenotype switching through inactivating Wnt/β-catenin pathway. (A) Western blot for detecting wnt1 and β-catenin expression in VSMCs in different groups. (B) 5-ethynyl-2´-deoxyuridine (EdU) assay was employed for assessing the proliferative ability of VSMCs (×200). (C) Flow cytometry was employed for testing VSMCs cell cycle. (D) Transwell assay was utilized for evaluating the migrative ability of VSMCs (×100). (E) Western blot for detecting α-smooth muscle actin (α-SMA), SM22, and osteopontin (OPN) expression in VSMCs. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HG, high glucose; ox-LDL, oxidized low-density lipoprotein; si-NC, negative control siRNA; BML-284, a Wnt pathway activator; DAPI, 4´,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.05, ^cP<0.05.

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Di M, Wang J, Sun L, Yang G, Xu Q. E2F5 Accelerates Vascular Smooth Muscle Cells Phenotype Switching in Diabetic Atherosclerosis through Activating Wnt/β-Catenin Pathway. Diabetes Metab J. 2025 Sep 1. doi: 10.4093/dmj.2024.0588. Epub ahead of print.

Received: Sep 25, 2024; Accepted: Apr 21, 2025

DOI: https://doi.org/10.4093/dmj.2024.0588.

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