Animals used in this study were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The procedures were in accordance with the Ethical Standards of the Committee on Animal Experimentation of China Medical University (project identification code, SCXK-2013-0001).

The Real-Time polymerase chain reaction (qPCR) system was purchased from Applied Biosystems (Foster City, CA, USA). Click-iT Nascent RNA Capture kit was purchased from Invitrogen (Carlsbad, CA, USA). Western blot analysis-related equipment was purchased from Invitrogen. EdU Alexa Fluor 555 Imaging kit was purchased from Invitrogen. Tetramethylrhodamine methyl ester (TMRE) was purchased from Molecular Probes (Eugene, OR, USA). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), cycloheximide (CHX), and actinomycin D were purchased from Sigma (St. Louis, MO, USA). BSA and AGEs-BSA were obtained from Merck-Millipore (Darmstadt, Germany). A fluorescence microscope (CKX41-F32FL) was purchased from Olympus (Tokyo, Japan). Microchemi 4.2 was purchased from DNR Bio-Imaging Systems, Ltd. (Jerusalem, Israel). Transwell-related equipment (8-µm pore) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Microplate reader was purchased from Bio-Rad (Hercules, CA, USA). Antibodies for BAG3 (sc-292154) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-47724) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lentiviral vectors were purchased from GeneChem (Shanghai, China).

Neonatal rats (1–2 days old) were sacrificed by cervical dislocation, disinfected with 75% alcohol, and then moved to a clean bench. The thoracic aorta was excised and the inner/outer layers of blood vessels were removed. Primary neonatal rat VSMCs were then isolated as described in our previous study (14). VSMCs were cultured in complete medium including 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/ml-100 µg/ml) at 37°C, in 5% CO₂ and a humidified atmosphere as previously described (14,27). Media were changed every other day. After primary cells achieved 80–90% confluence, 0.25% trypsin was added into the culture plate for digestion. Thirty seconds later, serum-containing medium was used to terminate the digestion. Subsequently, a part of the cells was moved into a new culture dish. After attachment to the wall, the cells were incubated with complete medium, which was passage 2 of VSMCs. Using the methods, cells between passages 2 to 8 were obtained and applied for the next experiments.

The construction of the BAG3 plasmids was carried out by GeneChem. The cells were transfected with Lipofectamine 2000 reagent (Invitrogen) as previously described (28). The lentiviral plasmids which contained short hairpin RNA (shRNA) against rat BAG3 or control shRNA were labelled with green fluorescent protein (GFP) and used in the knockdown experiments. There were five shRNA oligo-nucleotides specific for rat BAG3, i.e. shBAG3#1, #2, #3, #4 and #5. The titers of control shRNA and shBAG3#1, #2, #3, #4 and #5 were 8×10⁸, 4×10⁸, 4×10⁸, 3×10⁸, 3×10⁸ and 4×10⁸ TU/ml, respectively. Cells were seeded into 6-well plate and incubated with vector supernatants at a multiplicity of infection (MOI) of 100 for 12 h. Then the old culture medium was removed and replaced with DMEM with 10% FBS. After being cultured for 2 days, the cells were observed under fluorescence microscopy and transduction efficiency was calculated using the following formula: Transduction efficiency = GFP⁺ cells/total cells. After digestion, transduced cells were cultured for another 2 days. To confirm that the transduction was successful, the mRNA and protein expression levels of BAG3 were analyzed by quantitative PCR (qPCR) and western blot analysis, respectively.

Western blot analysis was performed as described in our previous studies (14,29). Briefly, the cells were solubilized in a radio-immunoprecipitation assay (RIPA) lysis buffer for 30 min, and then total protein concentrations were measured by a BCA protein assay kit (Beyotime, Shanghai, China). After heat denaturation, the samples were analyzed on a 12 or 14% Tris-glycine gradient gel, and then transferred to PVDF membranes and blocked with 5% nonfat milk in Tris-buffered solution (TBS) for 1.5 h at room temperature. The membranes were incubated with primary antibody overnight at 4°C. After washing three times with TBS, the membranes were incubated with secondary antibodies for 1.5 h at room temperature. After the washing steps, immunoreactive binding was detected with enhanced chemiluminescence (ECL) (Amersham Biosciences, Piscataway, NJ, USA). The band intensity was quantified using ImageJ 1.47 software and GAPDH served as a control.

MTT assay was used to determined cell viability. VSMCs were seeded in a 96-well plate at a density of 4×10³ cells/well. After being cultured for 48 h, the cells were incubated with MTT solution (final concentration, 5 mg/ml) for 4 h at 37°C. Then the culture media containing MTT were removed and replaced with 100 µl DMSO. Then the plate was gently rotated on a linear and orbital shaker for 5 min to completely dissolve the precipite. The absorbance was measured with a microplate reader at a wavelenght of 570 nm. The percentage of cell viability was calculated according to the following formula: Cell viability (%) = optical density (OD) of the treatment group/OD of the control group ×100%.

As described in our previous studies (14,30) and according to the manufacturer's instructions, the DNA synthesis rate in VSMCs was determined by EdU incorporation analysis using Click-iT™ EdU Alexa Fluor 555 Imaging kit. Briefly, the cells were incubated with EdU-labeling solution for 8 h at 37°C, and then fixed with 4% cold formaldehyde for 30 min at room temperature. After permeabilization with 1% Triton X-100, the cells were reacted with Click-iT reaction cocktails (Invitrogen) for 30 min. Subsequently, the DNA contents of the cells were stained with Hoechst 33258 for 30 min. Finally, EdU-labeled cells were counted using ImageJ 1.47 software and normalized to the total number of Hoechst-stained cells. At least 500 cells in each experiment were counted, and EdU-positive cells are expressed as a percentage of the total cells.

Cells at 80–90% confluence were wounded with a 200-µl pipette tip and incubated with BSA or AGEs for 24 h. Multiple views of the leading edge of the scratch were photographed under a microscope at 0 and 24 h. The experiments were performed three times independently.

For the Transwell migration assay, cells were seeded at a density of 2×10⁶ cells in the upper chamber. The lower chamber was filled with BSA or AGEs. After being cultured for 24 h, the cells on the upper chamber were removed by gentle abrasion with a cotton swab, and the cells on the lower chamber were fixed and stained with Hoechst 33258. Three experiments were performed independently. The cells that had passed through the filter were photographed under a fluorescence microscope with ultraviolet light. Hoechst-labeled cells in five representative microscopic fields were counted using ImageJ 1.47 software.

The total RNA was extracted using the Qiagen RNeasy Mini kit. After the determination of concentration, the synthesis of cDNA was performed. With a primer design software we synthesized the sense and antisense primers of each fragment: BAG3 sense, 5′-CATCCAGGAGTGCTGAAAGTG-3′ and antisense primer, 5′-TCTGAACCTTCCTGACACCG-3′; GAPDH sense, 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense primer, 5′-TGGTGAAGACGCCAGTGGA-3′. qPCR was run and analyzed with the 7500 Real-Time-PCR system. Results were normalized against those of GAPDH and are presented as arbitrary unit.

Click-iT Nascent RNA capture kit was used to detect newly synthesized RNA according to the manufacturer's instructions. 5-Ethymyl uridine (EU) is an alkyne-modified uridine analog and it is efficiently and naturally incorporated into nascent RNA. Cells were incubated in 0.2 mM of EU for 4 h and total RNA labeled with EU was isolated using Trizol reagent (Invitrogen). Then EU-labeled RNA was biotinylated in a Click-iT reaction buffer with 0.5 mM of biotin azide and subsequently captured on streptavidin magnetic beads.

We used TMRE (Ex/Em, 549/573 nm) to detect changes in mitochondrial membrane potential, as described in our previous study (31). Briefly, unfixed live cells were incubated with 100 nM TMRE in the dark for 30 min at 37°C in 5% CO₂. After being washed, the cells were analyzed under a fluorescence microscope. The fluorescence intensity of TMRE staining was quantified using ImageJ.

The formation of intracellular ROS was measured with the DCFH-DA (Ex/Em, 485/530 nm) method, as described in our previous study (31). DCFH-DA transforms into the fluorescent compound dichlorofluorescein (DCF) upon oxidation by ROS. Briefly, the cells were incubated with DCFH-DA at a final concentration of 5 mM at 37°C in 5% CO₂ in darkness for 40 min. After being washed, the cells were analyzed under a fluorescence microscope. The fluorescence intensity of DCFH-DA staining was quantified using ImageJ.

Data were obtained from at least three individual experiments. Continuous variables were expressed as the mean ± SD and tested by one-way ANOVA or Student's t-test. All the statistical analyses were performed using SPSS statistics for Windows (version 17.0; SPSS, Chicago, IL, USA), and p-values <0.05 were considered statistically significant.

The cells were treated with different concentrations of AGEs (25, 50, 100 and 200 µg/ml) and BSA (2.5, 5, 10 and 20 µg/ml) for 24 h, respectively. We found that AGEs increased the BAG3 mRNA (Fig. 1A) and protein (Fig. 1B) expression in the VSMCs in a dose-dependent manner using qPCR and western blot analysis, respectively. Next, we addressed whether transcriptional and translational inhibitors (actinomycin D and CHX, respectively) could modulate the effect of AGEs on the expression of BAG3 in VSMCs. The results of qPCR showed that actinomycin D significantly reduced the expression of BAG3 mRNA induced by AGEs while CHX had no effects (Fig. 1C), which indicated that AGEs mainly increased the expression of BAG3 mRNA by increasing the RNA synthesis rather than inhibiting the RNA translation. Next, to further support these observations, we evaluated the effect of AGEs on BAG3 mRNA expression using Click-iT Nascent RNA capture kit to isolate newly synthesized RNA. The results of qPCR indicated that AGEs significantly increased BAG3 mRNA synthesis at 24 h with the maximal stimulation effect at a concentration of 100 µg/ml (Fig. 1D). Then, the cells were incubated with acti-nomycin D and 100 µg/ml AGEs or 10 µg/ml BSA for different times (0, 1, 2, 4, 8 and 24 h). We found that AGEs increased the expression of BAG3 nascent mRNA in a time-dependent manner (Fig. 1E). Therefore, in the next experiment, we used the dose (100 µg/ml AGEs/10 µg/ml BSA) and time (24 h) to determine the molecular mechanism of AGE-induced proliferation and migration of VSMCs.

To further investigate the involvement of BAG3 in VSMC proliferation, we generated lentiviral vectors containing shRNAs against BAG3 (shBAG3) to knock down BAG3 expression in the VSMCs. Measurement of the GFP⁺ cells under fluorescence microscopy demonstrated that the transduction efficiency by lentiviral vectors at 100 MOI was 80–90% (Fig. 2A). The results of qPCR (Fig. 2B) and western blot analysis (Fig. 2C) demonstrated that two shRNAs (shBAG3#3 and shBAG3#5) significantly decreased the mRNA and protein expression of BAG3 in VSMCs, respectively. Importantly, results from MTT assay (Fig. 2D) and EdU staining (Fig. 2E and F) consistently demonstrated that forced knockout of BAG3 significantly reduced the cell viability and proliferation of VSMCs.

To investigate the potential involvement of BAG3 in the proliferation of VSMCs induced by AGEs, VSMCs containing shRNA against BAG3 were treated with 100 µg/ml AGEs or 10 µg/ml BSA for 24 h. The results of the MTT assay (Fig. 3A) and EdU staining (Fig. 3B) demonstrated that BAG3 knockout could significantly decrease the proliferation of VSMCs induced by AGEs.

Cells were treated with 100 µg/ml AGEs or 10 µg/ml BSA for 24 h. The wound-healing assay showed that AGEs significantly increased the migration of VSMCs compared with that noted in control group (Fig. 4A). The Transwell assay demonstrated that the number of invasive cells in the AGE-treated group was significantly higher than the number in the control group (Fig. 4B and C). We then continued to investigate the potential involvement of BAG3 in the migration of VSMCs. The results of the wound-healing assay (Fig. 4D) and Transwell assay (Fig. 4E) demonstrated that BAG3 knockout significantly decreased the migration of the VSMCs.

The results of DCHF-DA assay indicated that BAG3 knockout obviously reduced the ROS generation in the VSMCs (Fig. 5A and B). ROS mainly result from mitochondrial respiratory chain complexes in mitochondria (32). We then investigated the potential effect of BAG3 on the mitochondria. The results of TMRE showed that BAG3 knockout reversed the decrease in mitochondrial membrane potential induced by AGEs (Fig. 5C and D).

ROS, such as superoxide anions and hydrogen peroxide, play a crucial role in regulating the proliferation and migration of VSMCs. We tested the hypothesis that the stimulative effect of AGEs on the proliferation and migration of VSMCs involved ROS production. We next used NAC, a potent antioxidant, to investigate the potential effect of oxidative stress on the VSMCs. Cells were incubated with 100 µg/ml NAC and 100 µg/ml AGEs or 10 µg/ml BSA for 24 h. The results of the DCFH-DA assay indicated that NAC could completely prevented the generation of the intracellular ROS level after AGE stimulation (Fig. 6A and B). Furthermore, the results from the EdU staining (Fig. 6C and D) and MTT assay (Fig. 6E) consistently demonstrated that NAC significantly reduced the proliferation of VSMCs induced by AGEs. Results from the wound healing (Fig. 6F) and Transwell assays (Fig. 6G and H) consistently demonstrated that NAC significantly reduced the migration of VSMCs induced by AGEs. Overall, our results indicated that intracellular ROS generation has an essential role in AGE-induced proliferation and migration of VSMCs.