T/G HA-VSMCs were obtained from American Type Culture Collection (Rockefeller, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C with 5% CO₂. The cells were transfected with control siRNA (siC), Rab5a siRNA (siR; a pool of four siRNAs; Dharmacon Research, Lafayette, CO, USA), siC combined with PDGF (siC + P; 20 ng/ml; R&D Biosystems, Minneapolis, MN, USA) and siR combined with PDGF (siR + P; 20 ng/ml) prior to experiments. Transfection was performed using DharmaFECT transfection reagent in serum-free medium (GE Healthcare Life Sciences, Chalfont, UK) following manufacturer's protocol.

Following treatment with siRNA and/or PDGF for 24 h, the total RNA from cells was obtained using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The RNA (25 nM) was subsequently reverse transcribed using the RevertAid First-Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). PCR amplification was performed with the SYBR Green Premix Ex Taq kit (Takara Bio., Inc., Dalian, China). Primer sequences are listed in Table I. The PCR program included denaturation at 95°C for 10 sec, amplification with 40 cycles at 95°C for 5 sec and 60°C for 31 sec, and a final 2 min extension at 72°C. Finally, the 2^−ΔΔCq method (15) was used to calculate the expression levels of genes, normalized against the levels of GAPDH.

Following treatment for 48 h with siRNA and/or PDGF, the cells were lysed in radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Inc., Jiangsu, China) containing 1 mM phenylmethanesulfonyl fluoride. The supernatants were acquired by centrifugation at 13,440 × g for 15 min at 4°C. The proteins (30 µg/lane) were separated and subsequently transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 2 h. Following blocking, the membranes were incubated with specific primary antibodies overnight at 4°C and then incubated with horserdish peroxidase-conjugated secondary antibodies (cat. nos. A0181; A0216; A0208; 1:1,000; Beyotime Institute of Biotechnology, Inc.) at room temperature for 2 h. The antibodies used were as follows: anti-Rab5a (cat. no. 2143; 1:1,000), anti-survivin (cat. no. 2803), anti-caspase-3 (cat. no. 9662; 1:1,000), anti-Beclin1 (cat. no. 3738; 1:1,000), anti-Light chain (LC)3-I/II (cat. no. 12741; 1:1,000), anti-phosphorylated (p-) (cat. no. 4060; 1:1,000) and total (t-)protein kinase B (Akt) (cat. no. 4691; 1:1,000), anti-p-mammalian target of rapamycin (mTOR; cat. no 5536; 1:1,000) and anti-t-mTOR (cat. no. 2972; 1:1,000), and p-extracellular signal-regulated kinase (ERK cat. no. 4370; 1:1,000) and t-ERK (cat. no. 4695; 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA. Anti-α-smooth muscle actin (SMA; cat. no. SAB2500963; 0.5 µg/ml) and anti-calponin (cat. no. C2687; 1:10,000) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-osteopontin (cat. no. sc-10591; 1:200) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and anti-GAPDH (cat. no. AF0006; 1:1,000) was purchased from Beyotime Institute of Biotechnology, Inc. Following antibody incubations, an enhanced chemiluminescence assay (PerkinElmer, Waltham, MA, USA) was used to detect the proteins. GAPDH was used as a loading control.

The proliferation of VSMCs was detected using a sulforhodamine B (SRB) assay. A total of 3.0×10³ T/G HA-VSMCs were seeded into 96-well plates. The next day, the cells were assigned into the following four groups: i) siC group, cells treated with control siRNA; ii) siC + P group, cells treated with control siRNA and PDGF; iii) siR group, cell treated with Rab5a siRNA; and iv) siR+P group, cells treated with Rab5a siRNA and PDGF. Following treatment for 24, 48 and 72 h, the cells were fixed in 30% (w/v) trichloroacetic acid and were subsequently stained with 0.4% SRB (Sigma-Aldrich) diluted with 1% acetic acid for 30 min at 25°C. The excess SRB was rinsed off using 1% (v/v) acetic acid and the proteins were dissolved in 10 mM Tris solution. Finally, a microplate reader (iMark; Bio-Rad, Hercules, CA, USA) was used to measure the value of SRB at a wavelength of 570 nm.

The migration of T/G HA-VSMCs was detected using a Transwell system with 24-well inserts and 8 µm pores (Corning Costar, Lowell, MA, USA). Matrigel (30 µl; BD Biosciences, San Jose, CA, USA) was used to coat the inserts and 600 µl complete medium was added into the lower chamber. Following treatment for 48 h, trypsin was used to detach the cells and the cells were resuspended in fresh medium (0.2 ml) without FBS at a concentration of 1×10⁴ cells. These cell suspensions were seeded into the upper chamber. Following incubation for 24 h, the cells in the upper chamber were removed with cotton swabs, while non-migrating cells were gently removed and rinsed out with phosphate-buffered saline (PBS) three times. Cells in the lower chamber were fixed with methanol at room temperature for 30 min and were subsequently stained using hematoxylin at room temperature for 5 min. Migrating cells were counted in six randomly selected representative fields of each insert by light microscopy (BX51; Olympus, Tokyo, Japan).

Flow cytometry was performed to detect the cell cycle and apoptosis in each group. Following treatment for 48 h, the cells were trypsinized, collected and fixed with pre-cooled 70% ethanol overnight at 4°C. The cells were subsequently centrifuged at 1,120 × g for 10 min at 4°C and the ethanol was washed off with PBS. The cells were trypsinized for 30 min at 37°C. Propidium iodide (PI; 50 µg/ml; Sigma-Aldrich) was used to stain the cells for 30 min at 25°C for cell cycle analysis.

Cell apoptosis analysis was performed using a Vybrant^® Apoptosis assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). Following treatment for 48 h, the cells were co-stained with annexin V and PI, according to the manufacturer's protocol. The results were detected using flow cytometry (FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA). The data regarding cell cycle and apoptosis were acquired using CellQuest 7.0 software (Beckman Coulter, Brea, CA, USA) and subsequently analyzed using ModFit3.0 software (Verity Software House, Maine, ME, USA).

To observe the characteristics of autophagic cell death, acidic vesicle organelles (AVOs) were stained with acridine orange (Sigma-Aldrich). Following treatment for 48 h, the cells in different groups were stained with acridine orange (1 µg/ml) for 15 min at 37°C in the dark. The cells were subsequently rinsed with PBS, collected with trypsin and then resuspended in PBS, supplemented with 1% FBS. Finally, the fluorescence emission was detected using CellQuest 7.0 software.

To further reveal the autophagy of cells in each group, the cells were co-transfected with pEGFP-LC3 (National Institute for Basic Biology, Okazaki, Japan) using Lipofectamine2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h, the cells were fixed in 95% ethanol in the dark and fluorescence was detected by fluorescent microscopy (BX51; Olympus).

TEM was performed to observe the formation of autophagosomes. Following treatment for 48 h, the cells in each group were fixed in 3% glutaraldehyde at 25°C for 2 h. The cells were subsequently fixed in 1% osmium tetroxide, and were sectioned and embedded in LX112 plastic. Finally, uranyl acetate combined with lead citrate was used to stain the ultrathin sections, and electron micrographs were obtained by TEM (Philips Medical Systems, BG Eindhoven, The Netherlands).

Statistical analysis was performed using SPSS version 15.0 software (SPSS Inc., Chicago, IL, USA). All data were expressed as the mean ± standard deviation. Comparisons among different groups were analyzed by one-way analysis of variance, while comparisons between two groups were analyzed by Student's t-test. P≤0.05 was considered to indicate a statistically significantly difference.

The knockdown efficiency of Rab5a siRNA transfection and the effects of PDGF on T/G HA-VSMCs were determined using RT-qPCR and western blotting. As a result, transfection with siRNA against Rab5a led to a significant decrease in the mRNA and protein expression levels of Rab5a (P<0.01); however, the expression of Rab5a in the siR + P group was relatively close to the siC group (Fig. 1). These data revealed that Rab5a was effectively downregulated by Rab5a siRNA transfection and the reduced expression trend of Rab5a can be rescued by the intervention of PDGF in T/G HA-VSMCs.

The expression of molecular markers of the T/G HA-VSMC phenotype, including α-SMA, calponin, osteopontin and vimentin, were detected to evaluate the phenotypic transition of T/G HA-VSMCs in different groups. As a result, the mRNA expression levels of α-SMA and calponin were significantly increased, while osteopontin and vimentin were significantly reduced in the siR group compared with the siC group (P<0.01). However, the expression of these proteins was restored to normal levels by the intervention of PDGF, which was relatively close to siC (Fig. 2A). Similarly, the changes in the protein expression levels of α-SMA, calponin and osteopontin were consistent with that of the mRNA level (Fig. 2B). These results indicated that Rab5a may promote the phenotypic transition of T/G HA-VSMCs.

In addition to the phenotypic transition of T/G HA-VSMCs, the proliferation, cell cycle and migration of T/G HA-VSMCs were investigated in the different groups. Compared with the siC group, the growth of the cells was significantly reduced in the siR group at 24 (P<0.05), 48 (P<0.01) and 72 h (P<0.01). This growth trend was rescued by the intervention of PDGF in the siR + P group (Fig. 3A). According to the cell cycle analysis, the percentage of cells in the siR group was significantly decreased in the G0/G1 phase and increased at the G2/M and S phases; however, the percentage of cells at the G0/G1, G2/M and S phases were not significantly changed in the siR + P group compared with the siC group (Fig. 3B). These data indicated that Rab5a modulated T/G HA-VSMC proliferation at different phases of the cell cycle. In addition, compared with the siC group, the average number of migrated cells in the siR group was revealed to be significantly decreased (P<0.01). However, the percentage of migrated cells was not markedly changed in the siR + P group. Additionally, more migrated cells were observed in the siC + P compared with the siC group (P<0.05; Fig. 3C). These results indicated that the knockdown of Rab5a can inhibit T/G HA-VSMCs migration and that PDGF treatment can reverse the decreased migration level in Rab5a siRNA transfected cells.

The early apoptotic rate was quantitatively analyzed by flow cytometry and the results demonstrated that cells transfected with siRNA against Rab5a exhibit a significantly higher apoptotic rate compared with the siC group (P<0.01), while the percentage of apoptotic cells was significantly reduced by the intervention of PDGF (Fig. 4A). In addition, the expression of the apoptotic proteins survivin and caspase-3 was detected by western blotting. These results revealed reduced expression of survivin and increased expression of cleaved caspase-3 in the siR group compared with the siC group, while a combination treatment with PDGF and siRNA (siCon or siRab) transfection did not alter the expression levels of survivin and caspase-3 (Fig. 4B).

The autophagy in T/G HA-VSMCs from each group was first detected by flow cytometry. As a result, the percentage of cells with AVOs were significantly decreased (P<0.01), while autophagy returned to a normal degree following treatment with PDGF (Fig. 5A). In addition, the autophagy in these cells was also analyzed by fluorescence localization of LC3. As a result, the percentage of cells with punctate fluorescence was significantly reduced in the siR group (P<0.05), while no difference was observed in the other three groups (Fig. 5B). Additionally, VSMCs were visualized by TEM to examine the formation of autophagosomes. It was revealed that Rab5a siRNA transfection caused a significantly reduced number of autophagosomes with typical scattered double-membrane vacuolar structures (P<0.05), while the number of phagosomes in cells treated with PDGF remained unchanged compared with the siC group (Fig. 5C). Furthermore, the expression levels of the autophagic proteins Beclin1, LC3-I and LC3-II were also analyzed by western blotting. As an autophagy-related protein, Beclin1 is required for the initiation of autophagy. Additionally, LC3-II formation is also a confirmed marker of autophagy. As the formation of LC3-II is transient and the protein can be rapidly degraded in the lysosome, a conversion of LC3-I to LC3-II may also indicate autophagy in cells. Compared with the siC group, the protein expression of Beclin1 and the conversion of LC3-I to LC3-II was significantly reduced in the siR group, whereas it was elevated to a normal level following treatment with PDGF (Fig. 5D).

To assess the mechanism of Rab5a siRNA transfection-related autophagy, the PI3K/AKT/mTOR and ERK1/2 signaling pathways in T/G HA-VSMCs were assessed by western blotting. The result revealed that compared with the siC group, the expression levels of p-AKT, p-mTOR and p-ERK were markedly reduced in the siR group, while PDGF recovered the normal expression of these proteins. In addition, no differences were observed in the expression levels of t-AKT, t-mTOR and t-ERK among these four groups (Fig. 6). The results suggested that the MAPK/ERK pathway may be involved in Rab5a-related autophagy, which did not occur via the inhibition of the classical PI3K/AKT/mTOR pathway.