BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

Abstract

Atherosclerosis is a chronic systemic inflammatory disease that causes severe cardiovascular events. B cell lymphoma 2-associated athanogene (BAG3) was proven to participate in the regulation of tumor angiogenesis, neurodegenerative diseases, and cardiac diseases, but its role in atherosclerosis remains unclear. Here, we aim to investigate the role of BAG3 in atherosclerosis and elucidate the potential molecular mechanism. In this study, ApoE^-/- mice were given a tail-vein injection of BAG3-overexpressing lentivirus and fed a 12-week high-fat diet (HFD) to investigate the role of BAG3 in atherosclerosis. The overexpression of BAG3 reduced plaque areas and improved atherosclerosis in ApoE^-/- mice. Our research proves that BAG3 promotes autophagy in vitro, contributing to the suppression of EndMT in human umbilical vein endothelial cells (HUVECs). Mechanically, autophagy activation is mediated by BAG3 via the interaction between BAG3 and its chaperones HSP70 and HSPB8. In conclusion, BAG3 facilitates autophagy activation via the formation of the chaperone-assisted selective autophagy (CASA) complex interacting with HSP70 and HSPB8, leading to the inhibition of EndMT during the progression of atherosclerosis and indicating that BAG3 is a potential therapeutic target for atherosclerosis.

Keywords: BAG3; CASA complex; EndMT; atherosclerosis; autophagy.

Figure 1

Overexpression of BAG3 reduces atherosclerotic lesions in ApoE^−/− mice. (A–D) The levels of serum TG, TC, HDL-C and LDL-C in mice (n = 6). (E) Hematoxylin–eosin (H&E) and Oil Red O staining of aortic root sections showing the atherosclerotic lesions and lipid deposition (scale bar indicates 200 μm) (n = 3). (F,G) Representative en face images of Oil Red O staining of aortas; the lesion area of the whole aorta was quantified (n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Figure 2

BAG3 prevents EndMT in vivo and vitro. (A) Morphological changes post the indicated treatments were observed under the microscope (scale bar indicates 100 µm) (n = 3). (B–E) CD31, BAG3 and α-SMA protein expressions were detected using Western blotting. Fold changes are shown (n = 5). (F,G) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm) (n = 3). (H) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red) in the endothelium. The nuclei were stained blue with DAPI. (scale bar indicates 25 μm) (n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Figure 3

BAG3 positively regulates autophagy in HUVECs. (A–C) The LC3-II/LC3-I ratio and p62 protein expressions were detected using Western blotting. Fold changes are shown (n = 5). (D) Electron micrographs of HUVECs submitted to the indicated treatments, including autophagosomes and autolysosomes (red arrow) (scale bar indicates 2 µm and 500 nm) (n = 3). (E) Representative images and quantification of GFP-mRFP-LC3II puncta in HUVECs. (scale bar indicates 25 µm) (n = 5). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Figure 4

Inhibition of autophagy induces EndMT in HUVECs. (A) Morphological changes post the indicated treatments were observed under the microscope (scale bar indicates 100 µm) (n = 3). (B–F) ATG5, CD31, and α-SMA protein expressions and the LC3-II/LC3-I ratio were detected using Western blotting. Fold changes are shown (n = 5). (G,H) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm) (n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Figure 5

BAG3 suppresses EndMT by inducing autophagy in HUVECs. (A–D) CD31 and α-SMA protein expressions and the LC3-II/LC3-I ratio were detected using Western blotting. Fold changes are shown (n = 5). (E,F) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm) (n = 3). (G) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red). The nuclei were stained blue with DAPI. (scale bar indicates 50 µm) (n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Figure 6

BAG3 blocks ox-LDL-induced EndMT through formation of CASA complex with HSP70 and HSPB8 in HUVECs. (A) Co-IP analysis for the interaction of BAG3 with HSP70 or HSPB8. HUVEC extracts were immunoprecipitated with anti-BAG3 antibody and probed with anti-HSP70 or anti-HSPB8 antibody. (n = 3). (B–H) BAG3, HSP70, HSPB8, CD31, and α-SMA protein expressions and the LC3-II/LC3-I ratio were analyzed using Western blotting. Fold changes are shown (n = 5). (I) Morphological changes post the indicated treatments were observed under a microscope (scale bar indicates 100 µm). (J,K) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm). (n = 3). (L) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red). The nuclei were stained blue with DAPI (scale bar indicates 50 µm). The data are presented as the mean ± S.E.M. ^ns p > 0.05, * p < 0.05, ** p < 0.01.