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Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells.

Tian L, Chen K, Cao J, Han Z, Gao L, Wang Y, Fan Y, Wang C - Mol Med Rep (2015)

Bottom Line: The oxLDL‑induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α‑actin, smooth muscle contractile proteins.The oxLDL‑induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis.Concordant with these results, oxLDL‑treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β‑catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200011, P.R. China.

ABSTRACT
Oxidized low-density lipoprotein (oxLDL) is involved in the pathological phenotypic transformation of vascular smooth muscle cells in atherosclerosis. Galectin‑3 also has an important role in atherosclerosis. However, little is currently known regarding the effects of galectin‑3 on the oxLDL‑induced phenotypic transformation of vascular smooth muscle cells. In the present study, primary culture human umbilical vascular smooth muscle cells were treated with various oxLDL concentrations (0‑50 µg/ml) for 72 h, and phenotypic changes were subsequently recorded. The results of the present study suggested that oxLDL increases the expression levels of galectin‑3, and induces the phenotypic transformation of vascular smooth muscle cells. The oxLDL‑induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α‑actin, smooth muscle contractile proteins. The oxLDL‑induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis. Concordant with these results, oxLDL‑treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β‑catenin. Silencing of galectin‑3 by small interfering RNA reversed the phenotypic transformation and functional changes observed in the oxLDL‑treated cells, suggesting these changes were dependent on the activation of galectin‑3. In addition, galectin‑3 knockdown decreased the protein expression levels of β‑catenin in both the cytoplasm and nucleus; however, the mRNA expression levels of β‑catenin remained unchanged. These results suggest that galectin‑3 is responsible for the phenotypic transformation of human umbilical vascular smooth muscle cells, and the canonical Wnt/β-catenin signaling pathway may be involved in this process.

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Galectin-3 (gal-3) silencing inhibits the expression of β-catenin. Following transfection with either control or gal-3-specific small interfering (si)RNA for 24 h, human umbilical smooth muscle cells (HUSMCs) were incubated for 48 h in the presence or absence of 50 μg/ml oxidized low-density lipoprotein (oxLDL). β-catenin expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. β-catenin representative (A and B) western blotting and (C) RT-qPCR results are shown. GAPDH expression was used to normalize protein expression levels. (B) The respective densitometric measurement results are given. The band density of HUSMCs transfected with either scramble siRNA was defined as the control and set to 1. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. the control. #P<0.05, vs. oxLDL. (D) The confocal images of HUSMC transfected with either control or gal-3-specific siRNA using anti-β-catenin (red) antibodies, followed by DAPI nuclear counter staining (blue) are shown. The merged images containing all markers are also shown (magnification, ×630).
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f4-mmr-12-04-4995: Galectin-3 (gal-3) silencing inhibits the expression of β-catenin. Following transfection with either control or gal-3-specific small interfering (si)RNA for 24 h, human umbilical smooth muscle cells (HUSMCs) were incubated for 48 h in the presence or absence of 50 μg/ml oxidized low-density lipoprotein (oxLDL). β-catenin expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. β-catenin representative (A and B) western blotting and (C) RT-qPCR results are shown. GAPDH expression was used to normalize protein expression levels. (B) The respective densitometric measurement results are given. The band density of HUSMCs transfected with either scramble siRNA was defined as the control and set to 1. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. the control. #P<0.05, vs. oxLDL. (D) The confocal images of HUSMC transfected with either control or gal-3-specific siRNA using anti-β-catenin (red) antibodies, followed by DAPI nuclear counter staining (blue) are shown. The merged images containing all markers are also shown (magnification, ×630).

Mentions: To further elucidate the molecular mechanism underlying the gal-3-mediated oxLDL-induced activation and transformation of HUSMCs, the present study investigated whether Wnt/β-catenin signaling was involved in the process. The basis for these experiments was provided by the well-documented role of gal-3 in Wnt/β-catenin signaling (22), as well as the association between Wnt/β-catenin signaling and phenotypic changes in murine VSMCs (23). Following treatment with oxLDL, the protein expression levels of β-catenin increased; however, the mRNA expression levels of β-catenin remained unchanged. The present study subsequently investigated the expression levels of β-catenin using RT-qPCR and western blotting, following gal-3 knockdown. Silencing of gal-3 significantly reduced the oxLDL-induced protein expression levels of β-catenin (Fig. 4A and B). However, no changes were observed in the mRNA expression levels of β-catenin following treatment with gal-3 siRNA (Fig. 4C). In the gal3 siRNA-transfected and control cells, the subcellular distribution of β-catenin was examined by immunofluorescence staining and confocal microscopy. Reduced expression levels of β-catenin were observed not only in the cytoplasm but also in the nucleus (Fig. 4D). These data suggest that gal-3 has an important role in oxLDL-induced increases in β-catenin expression.


Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells.

Tian L, Chen K, Cao J, Han Z, Gao L, Wang Y, Fan Y, Wang C - Mol Med Rep (2015)

Galectin-3 (gal-3) silencing inhibits the expression of β-catenin. Following transfection with either control or gal-3-specific small interfering (si)RNA for 24 h, human umbilical smooth muscle cells (HUSMCs) were incubated for 48 h in the presence or absence of 50 μg/ml oxidized low-density lipoprotein (oxLDL). β-catenin expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. β-catenin representative (A and B) western blotting and (C) RT-qPCR results are shown. GAPDH expression was used to normalize protein expression levels. (B) The respective densitometric measurement results are given. The band density of HUSMCs transfected with either scramble siRNA was defined as the control and set to 1. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. the control. #P<0.05, vs. oxLDL. (D) The confocal images of HUSMC transfected with either control or gal-3-specific siRNA using anti-β-catenin (red) antibodies, followed by DAPI nuclear counter staining (blue) are shown. The merged images containing all markers are also shown (magnification, ×630).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581830&req=5

f4-mmr-12-04-4995: Galectin-3 (gal-3) silencing inhibits the expression of β-catenin. Following transfection with either control or gal-3-specific small interfering (si)RNA for 24 h, human umbilical smooth muscle cells (HUSMCs) were incubated for 48 h in the presence or absence of 50 μg/ml oxidized low-density lipoprotein (oxLDL). β-catenin expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. β-catenin representative (A and B) western blotting and (C) RT-qPCR results are shown. GAPDH expression was used to normalize protein expression levels. (B) The respective densitometric measurement results are given. The band density of HUSMCs transfected with either scramble siRNA was defined as the control and set to 1. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. the control. #P<0.05, vs. oxLDL. (D) The confocal images of HUSMC transfected with either control or gal-3-specific siRNA using anti-β-catenin (red) antibodies, followed by DAPI nuclear counter staining (blue) are shown. The merged images containing all markers are also shown (magnification, ×630).
Mentions: To further elucidate the molecular mechanism underlying the gal-3-mediated oxLDL-induced activation and transformation of HUSMCs, the present study investigated whether Wnt/β-catenin signaling was involved in the process. The basis for these experiments was provided by the well-documented role of gal-3 in Wnt/β-catenin signaling (22), as well as the association between Wnt/β-catenin signaling and phenotypic changes in murine VSMCs (23). Following treatment with oxLDL, the protein expression levels of β-catenin increased; however, the mRNA expression levels of β-catenin remained unchanged. The present study subsequently investigated the expression levels of β-catenin using RT-qPCR and western blotting, following gal-3 knockdown. Silencing of gal-3 significantly reduced the oxLDL-induced protein expression levels of β-catenin (Fig. 4A and B). However, no changes were observed in the mRNA expression levels of β-catenin following treatment with gal-3 siRNA (Fig. 4C). In the gal3 siRNA-transfected and control cells, the subcellular distribution of β-catenin was examined by immunofluorescence staining and confocal microscopy. Reduced expression levels of β-catenin were observed not only in the cytoplasm but also in the nucleus (Fig. 4D). These data suggest that gal-3 has an important role in oxLDL-induced increases in β-catenin expression.

Bottom Line: The oxLDL‑induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α‑actin, smooth muscle contractile proteins.The oxLDL‑induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis.Concordant with these results, oxLDL‑treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β‑catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200011, P.R. China.

ABSTRACT
Oxidized low-density lipoprotein (oxLDL) is involved in the pathological phenotypic transformation of vascular smooth muscle cells in atherosclerosis. Galectin‑3 also has an important role in atherosclerosis. However, little is currently known regarding the effects of galectin‑3 on the oxLDL‑induced phenotypic transformation of vascular smooth muscle cells. In the present study, primary culture human umbilical vascular smooth muscle cells were treated with various oxLDL concentrations (0‑50 µg/ml) for 72 h, and phenotypic changes were subsequently recorded. The results of the present study suggested that oxLDL increases the expression levels of galectin‑3, and induces the phenotypic transformation of vascular smooth muscle cells. The oxLDL‑induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α‑actin, smooth muscle contractile proteins. The oxLDL‑induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis. Concordant with these results, oxLDL‑treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β‑catenin. Silencing of galectin‑3 by small interfering RNA reversed the phenotypic transformation and functional changes observed in the oxLDL‑treated cells, suggesting these changes were dependent on the activation of galectin‑3. In addition, galectin‑3 knockdown decreased the protein expression levels of β‑catenin in both the cytoplasm and nucleus; however, the mRNA expression levels of β‑catenin remained unchanged. These results suggest that galectin‑3 is responsible for the phenotypic transformation of human umbilical vascular smooth muscle cells, and the canonical Wnt/β-catenin signaling pathway may be involved in this process.

Show MeSH
Related in: MedlinePlus