Galectin-3 induces vascular smooth muscle cells calcification via AMPK/TXNIP pathway

Reagents

The Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Carlsbad, CA, USA). Galectin-3 and compound C (AMPK inhibitor) were purchased from Abcam (Cambridge, UK). 3-MA was obtained from Sigma Aldrich (St. Louis, MO, USA). Verapamil was purchased from Hefeng Pharmaceutical (Shanghai, China). The primary antibody against phospho-AMPK, AMPK, phospho-mTOR, mTOR and GAPDH were acquired from the Cell Signaling Technology Inc (Danvers, MA, USA). The primary antibodies against TXNIP, LC3B and Runx2 purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-galectin-3, anti-P62, anti-NLRP3, anti-caspase-1, anti-IL-1β, anti-OPN and anti-BMP2 were obtained from Abcam. All other chemicals were from commercial sources.

Cell culture

A primary culture of mouse SMCs was established by explant outgrowth of a segment of human umbilical cord retrieved at the time of caesarean section [20]. Endothelial cells were removed by scraping the luminal surface of the vessel with a cotton swab, and the adventitia was mechanically stripped away. To induce calcification, VSMCs were maintained in DMEM with L-glutamine, sodium pyruvate, 10 mmol/L β-glycerophosphate, 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO₂ incubator. We performed our further experimentations by using the cells at a density of 10⁵/well in six-well plates.

SiRNA interference

siRNA method was performed to inhibit TXNIP, AMPKα and Atg 5 expression. Briefly, VSMCs were transfected with TXNIP, AMPKα or Atg 5 siRNA with Lipofectamine^® 2000 according to the manufacturer’s instructions. The process of transfection was performed in the absence of antibiotics. Following 24h incubation, the cells were used for other experiments. siRNA was synthesized by Biotend (Shanghai, China).

Alizarin Red staining

The arterial samples were fixed with 4% formaldehyde and embedded in paraffin. Then, the paraffin-embedded tissues were cut into 4 μm-thick sections for subsequent analysis. After deparaffinization and rinsing in ethanol, sections were rinsed in distilled water for 5 minutes. The slices were dyed with 1% Alizarin Red S solution for 30 min and then photographed. VSMCs were fixed with 4% paraformaldehyde for 5 min at room temperature, washed three times with Ca²⁺-free PBS, and then stained with 1% Alizarin red solution (Beyotime, Shanghai, China) for 30 min at 37°C to visualize the matrix calcium deposition. The excess Alizarin red S reagent were washed out with Ca²⁺-free PBS and the samples were photographed.

ROS determination

Intracellular ROS level was determined with the dihydroethidium (DHE) reagent (Sigma-Aldrich, USA). The treated cells were washed with PBS solution and incubated with 10 μM DHE for 30 min at 37°C in darkness. Then, VSMCs were washed twice using PBS and examined by fluorescence microscopy.

Animals and in vivo experiments

The animal experiments were conducted on healthy male C57BL/6 mice weighing 20–25 g at 6 weeks of age, the mice were kept in a 12h dark/light cycle room with constant temperature (22–25°C). All experiments on mice were approved by the committee of Ruijing Hospital Academy of Medical Sciences. All the mice were randomly divided into four groups (n = 6 for each group): (I) control group; (II) galectin-3 treatment group (GAL); (III) galectin-3 and verapamil cotreatment group. Galectin-3 (20 μg) was intraperitoneally injected or the same volume of vehicle (saline) was conducted on every 3 days for a total of 4 weeks. Verapamil (100 mg/kg) was intraperitoneally injected every day. All mice were euthanized 4 weeks after the initial injection of galectin-3.

Western blot

Cells were lysed with 100 mM phenylmethanesulfonyl fluoride. Protein concentrations were measured with the BCA Protein Assay. The lysates (20 μg) were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Danvers, MA, USA). The membrane was blocked with 5% nonfat dry milk in TBST buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 0.1% Tween-20) for 1h at room temperature. The membrane was then incubated with diluted primary antibodies at 4°C overnight, and then washed twice with TBST buffer and incubated for 1 h with secondary antibody at room temperature. Image J was used to quantity the protein by assessing band intensity.

Statistical analysis

Data were presented as the mean ± SEM and analyzed by one-way ANOVA followed by the Student-Newman–Keuls post hoc analyses when appropriate. Non-parametric ANOVA (Kruskal-Wallis test) was used, when the data were not passed the normality test. P < 0.05 was considered statistically significant. All experiments were performed at least three times.

Data availability

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

Galectin-3 induced aorta and VSMCs calcification

In order to investigate the effects of galectin-3 on VSMCs calcification, VSMCs were cultured in DMEM without serum for 12 h, after that, the serum-starved VSMCs were treated with different concentration of galectin-3 for 24 h, we observed the expression of osteogenic markers, including BMP2, Runx2 and OPN. As shown in Figure 1A and 1B, the expression of BMP2, Runx2 and OPN was upregulated after galectin-3 treatment. As TXNIP, an important protein of inflammation, may also participate in vascular calcification [7], we observed TXNIP expression after the treatment of different concentration of galectin-3 (0, 1.25, 2.5, 5, 10 and 25 μg/ml), 10 μg/ml galectin-3 most effectively increased TXNIP expression. We then used 10 μg/ml galectin-3 to deal with VSMCs for different times, TXNIP was obviously upregulated by galectin-3 treatment for 3 h, and was increased significantly until 24 h galectin-3 treatment (Figure 1C–1F). Since TXNIP was also increased after galectin-3 treatment, we next determined the effect of TXNIP in galectin-3-induced aorta and VSMCs calcification. In vivo, we injected verapamil and (or) galectin-3 into mice and observed calcium deposition in aortic tissues via Alizarin Red Staining. There was no calcified deposition in aortas of control group, galectin-3 significantly increased positive staining among elastic fibers of aortal media. Verapamil, a TXNIP inhibitor, effectively decreased galectin-3-induced artery calcification (Figure 1G).

Figure 1. Galectin-3 induced aorta and VSMCs calcification. Cells were treated with galectin-3 over a range of concentrations (0, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml) for 24 h, VSMCs osteogenic differentiation proteins (BMP2, Runx2 and OPN) were measured by Western blot, the quantification result is shown in the right panel (A and B). VSMCs were treated with galectin-3 over a range of concentrations (0, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml) for 24 h, TXNIP expression was measured by Western blot (C), the quantification result is shown in the right panel (D). 10 μg/ml galectin-3 was used to deal with VSMCs for different times (0, 3 h, 6 h, 12 h, 24 h), TXNIP expression was measured by Western blot (E), the quantification result is shown in the right panel (F). Band density of native VSMCs was defined as a control and considered to 1. Representative images of Alizarin Red staining of aorta (G). All experiments were performed at least three times. ^*P < 0.05 compared with control.

TXNIP mediated galectin-3-induced VSMCs autophagy and osteogenic differentiation

In atherosclerotic plaque, there is an increase expression of autophagy related proteins [21]. In order to explore the possible mechanisms of how TXNIP mediated galectin-3-induced VSMCs osteogenic differentiation, we focus on the autophagy process. We found that after galectin-3 treatment for 24 h, there was a time-dependent increase in LC3-II formation and decrease in p62 expression. A significant increase in LC3-II was observed 6 h after galectin-3 treatment, in contrast, a selective substrate of the autophagy degrading pathway p62 was decreased after galectin-3 treatment (Figure 2A and 2B). We furtherly verified TXNIP effect on autophagy, TXNIP siRNA decreased galectin-3-induced upregulation of LC3-II/LC3-I. Our results suggest that TXNIP mediates galectin-3-induced autophagy in VSMCs.

Figure 2. Galectin-3 induced VSMCs calcification and autophagy via TXNIP. VSMCs were treated with 10 μg/ml galectin-3 for different times (0, 6 h, 12 h, 24 h). The protein expression level of LC3-II/LC3-I and P62 were measured by western blot, the results quantifications were shown in the right panel (A and B). After transfection with either control or TXNIP siRNA for 24 h, VSMCs were incubated in the absence or presence of 10 μg/ml galectin-3 for 24 h, the expression of LC3-II/LC3-I and VSMCs osteogenic differentiation proteins (BMP2, Runx2 and OPN) was measured by western blot, the results quantifications were shown in the right panel (C and D). Band density of native VSMCs was defined as a control and considered to 1. After transfection with either control or TXNIP siRNA for 24 h, VSMCs were incubated in the absence or presence of 10μg/ml galectin-3 for 7 d, Alizarin red staining was used to observe the calcium deposition (E). Data were obtained from three independent experiments. ^*P < 0.05 vs. control; ^#P < 0.05 vs. galectin-3.

We then verified the role of TXNIP in galectin-3-induced VSMCs calcification. We examined BMP2, Runx2 and OPN expression in VSMCs after TXNIP siRNA treatment. As shown in Figure 2C and 2D, TXNIP knockdown has little effect on BMP2, Runx2 and OPN expression in basal condition, but it significantly attenuated galectin-3-induced upregulation of these proteins. These results suggested that TXNIP mediated galectin-3-induced expression of VSMCs osteogenic differentiation. Alizarin red staining was used to observe the calcium deposition. Alizarin red staining showed that galectin-3 significantly increased calcium content in VSMCs and that TXNIP siRNA blocked calcium deposition in cells (Figure 2E).

TXNIP knockdown decreases galectin-3-induced ROS and inflammation factors expression in VSMCs

TXNIP binding to NLRP3 inflammasome is involved in regulating inflammation [14], besides, inflammation plays a key role in vascular calcification. In order to verify TXNIP effect on the expression of NLRP3 inflammasome (NLRP3 and caspase-1) and IL-1β in galectin-3 treatment VSMCs, the protein and mRNA level of NLRP3, caspase-1, and IL-1β were observed by using Western blot and qRT-PCR respectively. As shown in Figure 3A and 3B, galectin-3 significantly increased NLRP3, caspase-1, and IL-1β expression. The VSMCs with verapamil and galectin-3 treatment showed less expression of NLRP3, caspase-1, and IL-1β compared with galectin-3 treated VSMCs. The result was similar in Figure 3C, which showed increased ROS production after galectin-3 treatment was reversed by TXNIP siRNA treatment in VSMCs.

Figure 3. TXNIP mediated galectin-3-induced ROS and inflammation factors expression in VSMCs. VSMCs were treated with 10 μg/ml galectin-3 and (or) TXNIP siRNA for 24 h, The protein expression level of NLRP3, caspase-1, and IL-1β were measured by western blot, the results quantifications were shown in the right panel (A and B). Band density of native VSMCs was defined as a control and considered to 1. DHE staining was used to observe the ROS production (C). Data were obtained from three independent experiments. ^*P < 0.05, vs. the control; ^#P < 0.05 vs. galectin-3.

Autophagy mediated galectin-3-induced VSMCs osteogenic differentiation

Until now, the role of autophagy in VSMCs osteogenic differentiation is still unclear, we would like to explore the relationship between autophagy and VSMCs osteogenic differentiation. We hypothesized that autophagy might mediate galectin-3-induced VSMCs differentiation. To test this hypothesis, we used autophagy inhibitors, 3-MA (100 mmol/L) or Atg5 siRNA, to treat VSMCs before galectin-3 stimulation. 3-MA or Atg5 siRNA not only effectively decreased the LC3-II protein expression (Figure 4A and 4B) but also inhibited galectin-3-induced upregulation of BMP2, Runx2 and OPN expression (Figure 4C and 4D).

Figure 4. Autophagy mediated galectin-3-induced VSMCs calcification. After pre-treatment with 100 mmol/L 3-MA for 1 h, the VSMCs were then treated with 10 μg/ml galectin-3 for 24 h, BMP2, Runx2 and OPN expression were measured by Western blot, the results quantifications were shown in the right panel (A and B). After treatment with Atg5 siRNA for 24 h, the VSMCs were then treated with 10 μg/ml galectin-3 for 24 h, BMP2, Runx2 and OPN expression were measured by Western blot, the results quantifications were shown in the right panel (C and D). Band density of native VSMCs was chosen as a reference and set to 1. Data were obtained from three independent experiments. ^*P < 0.05, vs. the control.

AMPK signaling pathway mediated galectin-3-induced TXNIP expression and VSMCs osteogenic differentiation

AMPK has also been regarded as a mTOR-independent manner to induce the up-regulation of autophagic activity, besides, phosphorylation of AMPK has also been shown to mediate VSMCs differentiation [22]. Here, we examined mTOR and AMPK signaling pathways after galectin-3 treatment. We used 10 μg/ml galectin-3 to deal with VSMCs for 60 min, p-mTOR was effectively suppressed after galectin-3 treatment. These results furtherly confirmed that galectin-3 activated autophagy process in VSMCs (Figure 5A and 5B). We also observed AMPK signaling pathway after galectin-3 treatment, galectin-3 significantly increased p-AMPK expression, and there was no difference of total AMPK level after galectin-3 treatment (Figure 5A and 5B).

Figure 5. AMPK signaling pathway mediated galectin-3-induced TXNIP and VSMCs osteogenic differentiation. Cells were treated with 10 μg/ml galectin-3 over a range of times (0–60 min), and the expression of AMPK, p-AMPK, mTOR, and p-mTOR were measured by Western blot. Western blot results are shown, quantification of the results is given in the right panel (A and B). Band density of native VSMCs was defined as a control and considered to 1. Data were obtained from three independent experiments. ^*P < 0.05 compared with control. After pre-treatment with 1 μmol/L compound C for 1 h, VSMCs were then treated with 10 μg/ml galectin-3 for 24 h, the expression of TXNIP and VSMCs osteogenic differentiation proteins was measured by western blot (C). Quantification of the results were shown in the right panel (D). After pre-treatment with AMPK siRNA for 24 h, VSMCs were then treated with 10 μg/ml galectin-3 for 24 h, the expression of TXNIP and VSMCs osteogenic differentiation proteins was measured by western blot (E). Quantification of the results were shown in the right panel (F). Band density of native VSMCs was defined as a control and considered to 1. DHE staining was used to observe the ROS production (G). Data were obtained from three independent experiments. ^*P < 0.05 vs. control.

We then verified the role of AMPK signaling pathway in galectin-3-induced TXNIP expression and VSMCs osteogenic differentiation. we pretreated VSMCs with a specific AMPK inhibitor compound C (1 μmol/L) or AMPKα specific siRNA, and then examined TXNIP expression. Either compound C or AMPKα specific siRNA significantly enhanced galectin-3-induced upregulation of TXNIP (Figure 5C–5F). To furtherly explore whether galectin-3 induced VSMCs osteogenic differentiation via AMPK, we also pretreated VSMCs with compound C or AMPKα siRNA before galectin-3 treatment. As shown in Figure 5C and 5E, AMPK signal blockade furtherly increased the expression of OPN, Runx2 and BMP2 in galectin-3-treated VSMCs, besides, we also observed ROS production via DHE staining, AMPK inhibition can also promote galectin-3-induced ROS expression (Figure 5G).