Plin5 inhibits proliferation and migration of vascular smooth muscle cell through interacting with PGC-1α following vascular injury

Abstract

Abnormal proliferation and migration of vascular smooth muscle cell (VSMC) is a hallmark of vascular neointima hyperplasia. Perilipin 5 (Plin5), a regulator of lipid metabolism, is also confirmed to be involved in vascular disorders, such as microvascular endothelial dysfunction and atherosclerosis. To investigate the regulation and function of plin5 in the phenotypic alteration of VSMC, -an animal model of vascular intima hyperplasia was established in C57BL/6 J and Plin5 knockdown (Plin5^±) mice by wire injure. Immunohistochemical staining was used to analyze neointima hyperplasia in artery. Ki-67, dihydroethidium immunofluorescence staining and wound healing assay were used to measure proliferation, reactive oxygen species (ROS) generation and migration of VSMC, respectively. Plin5 was downregulated in artery subjected to vascular injury and in VSMC subjected to platelet-derived growth factor (PDGF)-BB. Plin5 knockdown led to accelerated neointima hyperplasia, excessive proliferation and migration of VSMC after injury. In vitro, we observed increased ROS content in VSMC isolated from Plin5^± mice. Antioxidative N-acetylcysteine (NAC) inhibited VSMC proliferation and migration induced by PDGF-BB or plin5 knockdown. More importantly, plin5-peroxlsome proliferator-activated receptor-γ coactivator (PGC)-1α interaction was also attenuated in VSMC after knockdown of plin5. Overexpression of PGC-1α suppressed PDGF-BB-induced ROS generation, proliferation, and migration in VSMC isolated from Plin5^± mice. These data suggest that plin5 serves as a potent regulator of VSMC proliferation, migration, and neointima hyperplasia by interacting with PGC-1α and affecting ROS generation.

Keywords: PGC-1α; Plin5; ROS; VSMC; neointima hyperplasia.

Graphical abstract

Figure 1.

Plin5 is down-regulated in proliferating VSMC and injured artery. The relative mRNA (a) and protein (b) levels of plin5 in common carotid arteries from C57BL/6 J mice at day 7, 14 and 28 after injury were analyzed by qRT-PCR and immunoblotting (n = 5). The relative mRNA (c) and protein (d) levels of plin5 were determined by qRT-PCR and immunoblotting in VSMC after 0, 12, 24 and 48 h of DMSO or PDGF-BB (30 ng/mL) treatment (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.

Figure 2.

Knockdown of plin5 promotes neointima hyperplasia after vascular injury. The relative mRNA (a) (n = 4) and protein (b) (n = 4) levels of plin5 in common carotid arteries from WT or Plin5^± mice. (c) Representative H&E staining of carotid arteries from WT or Plin5^± mice at day 28 after sham operation or wire injury (left) and corresponding quantification for ratio of intima/media (right) were shown (n = 5). Magnification 200 × . (d) Immunohistochemistry staining of Ki-67 (brown) in sections of carotid arteries from WT or Plin5^± mice at day 28 after sham operation or wire injury (left) and corresponding quantification for Ki-67 positive cells within neointima (right) were shown (n = 4). Magnification 200 × . *P < 0.05, **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.

Figure 3.

VSMC proliferation and migration in vitro is elevated after plin5 deletion. (a) After DMSO or PDGF-BB (30 ng/mL) treatment for 48 h, VSMC isolated from WT or Plin5^± mice was stained with Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67 positive VSMC (right) were shown (n = 5). Magnification 400 × . (b) VSMC isolated from WT or Plin5^± mice was incubated with DMSO or PDGF-BB (30 ng/mL) for 48 h. Then, the absorbance at 450 nm was obtained (n = 4). (c) After 24 h of DMSO or PDGF-BB (30 ng/mL) treatment, migration of VSMC isolated from WT and Plin5^± mice was measured via wound healing assay. Representative images (left panel) and corresponding quantification of healing rates (right panel) were shown (n = 5). Magnification 100 × . (d) After 8 h of DMSO or PDGF-BB (30 ng/mL) treatment, migration of VSMC isolated from WT and Plin5^± mice was measured via transwell assay. Representative images (left panel) and corresponding quantification of migration cells (right panel) were shown (n = 4). Magnification 100 × . **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.

Figure 4.

NAC reverses elevated proliferation and migration of VSMC induced by plin5 knockdown. (a) After DMSO or PDGF-BB (30 ng/mL) treatment for 48 h, VSMC isolated from WT or Plin5^± mice was stained with DHE (red). Representative images (left) and corresponding quantification of DHE fluorescence (right) were shown (n = 5). Magnification 400 × . (b) VSMC isolated from WT or Plin5^± mice was incubated with DMSO or NAC (10 nmol/L; 8 h) and received 48 h of PDGF-BB (30 ng/mL) treatment. Then, VSMC was stained with Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67 positive VSMC (right) were shown (n = 5). Magnification 400 × . (c) Migration of VSMC was measured via wound healing assay. Representative images (left) and corresponding quantification of healing rates (right) were shown (n = 5). Magnification 100 × . *P < 0.05, **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.

Figure 5.

Declined plin5-PGC-1α interaction causes increased ROS level in VSMC. VSMC isolated from WT or Plin5^± mice after 48 h of DMSO or PDGF-BB (30 ng/mL) treatment was subjected to immunoprecipitation (IP) using anti-PGC-1α antibody or control IgG. (a) Inputs and immunocomplexes were analyzed by immunoblotting. (b) VSMC isolated from WT or Plin5^± mice was transfected with Ad-Con or Ad-Pgc1α. VSMC was next incubated with DMSO or NAC (10 nmol/L) for 8 h and PDGF-BB (30 ng/mL) for 48 h. Then, VSMC was stained with DHE (red). Representative images (left) and corresponding quantification of DHE fluorescence (right) were shown (n = 5). Magnification 400 × . **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.

Figure 6.

Plin5 knockdown causes increased VSMC proliferation and migration induced by PDGF-BB in a Pgc1α-dependent manner. VSMC isolated from WT or Plin5^± mice was transfected with Ad-Con or Ad-Pgc1α. VSMC was next incubated with PDGF-BB (30 ng/mL) for 48 h. (a) VSMC was stained with Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67 positive VSMC (right) were shown (n = 5). Magnification 400 × . (b) Migration of VSMC was measured via wound healing assay. Representative images (left) and corresponding quantification of healing rates (right) were shown (n = 5). Magnification 100 × . **P < 0.01 and ***P < 0.001 denote statistical comparison between the two marked groups, respectively. Data are shown as mean ± S.D.