Fig 1.
Autophagy and mitophagy were increased in PA-treated cells.
A. Primary human aortic endothelial cells (HAECs) were treated 0 or 0.3mM palmitic acid (PA) for 24 hours. Treated cells were stained with mitochondria marker Mito, autophagy marker LC3, and lysosome marker Lamp1. The numbers of mitochondria co-localized with LC3 or with Lamp1 per cell were quantified. Representative immunostaining images and quantification of 3 independent experiments demonstrated increased co-localization of mitochondria with LC3 and Lamp1 in PA treated cells. At least 25–30 cells per experiment were analyzed. B. HAECs were treated with 0 or 0.3mM PA for 24h and 5nM bafilomycin A1 during the last 6h prior to fixation. Representative electron microscopy photomicrographs showed the formation of autophagosomes containing heterogeneous cytoplasmic materials (black arrows) and mitophagosomes containing mitochondria fragments (white arrows). Asterisk indicated normal mitochondria. At least 10–15 cells per condition were imaged. Quantification showed the number of autophagosome and mitophagosome was increased by PA treatment. C. HAECs were treated with 0 to 0.5mM PA for 24h. Representative images of western blot and quantification from 3 independent experiments showed dual effects of PA on PINK1 and Parkin expression. */#P< 0.001 vs. PA (0mM). D. HAECs were treated with 0 or 0.3mM PA for 24h. Representative immunostaining images of 3 independent experiments demonstrated co-localization of PINK1 and Parkin with mitochondria in perinuclear area in PA treated cells.
Fig 2.
PINK1 and Parkin were involved in mitophagy in PA-treated cells.
A. HAECs were transfected with PINK1 or Parkin siRNA followed by treatment with PA for 24 hours. Representative western blots and quantitative analysis of 3 independent experiments showed successful silencing of PINK1 and Parkin. B. Representative immunostaining images and quantitative analysis of 3 independent experiments showed that PINK1 or Parkin siRNA reduced the number of mitochondria co-localized with LC3 per cell. At least 25–30 cells per condition were analyzed. *P< 0.001 vs. PA (0mM). C. HAECs were transfected with PINK1 or Parkin or control plasmid followed by treatment with PA for 48 hours. Representative western blot of 3 independent experiments showed successful overexpression of PINK1 and Parkin. D. Representative immunostaining and quantitative analysis of 3 independent experiments showed PA induced LC3 expression and the number of mitochondria co-localized with LC3 per cell which was markedly increased by overexpression of PINK1 or Parkin. At least 25–30 cells per experiment were analyzed. *P< 0.001 vs. PA (0mM). E. Representitive immunostaining and quantitative analysis of 3 independent experiments show that silencing PINK1 or Parkin partially prevented PA induced mitochondria co-localized with Lamp1. At least 25–30 cells per experiment were analyzed. F. Representative electron microscopy photomicrographs show decreased mitophagy with increased mitochondria fragments in PINK1 or Parkin deficient cells. Black arrows indicated autophagosomes, white arrows indicated mitophagosomes and arrowheads indicated mitochondria fragments.
Fig 3.
PINK1-Parkin protected mitochondrial integrity.
HEACs were treated with PA (A) for 24 hours or CCCP (positive control) (B) for 6 hours. Active and total mitochondria were stained with TMRM and MitoTracker, respectively. Fluorescent intensity from 20 randomly selected 20 x microscopic fields per group was captured and analyzed. TMRM and Mito ratio was compared. Representative images and quantitative analysis of 3 independent experiments showed that CCCP and PA reduced mitochondrial membrane potential. C. HAECs were transfected with PINK1 or Parkin siRNA followed by treatment with PA (0 and 0.3mM) for 24 hours. Fluorescent intensity from 20 randomly selected 20 x microscopic fields per group was captured and analyzed. TMRM and MitoTracker ratio was compared. Representative images and quantitative analysis of 3 independent experiments showed that PINK1 or Parkin siRNA amplified PA-induced reduction in mitochondrial membrane potential. *P< 0.001 vs. PA (0mM). D. HAECs were transfected with PINK1 or Parkin or control plasmid followed by treatment with PA (0, 0.3 and 0.5mM) for 48 hours. Fluorescent intensity from 20 randomly selected 20 x microscopic fields per group was captured and analyzed. TMRM and Mito ratio was compared. Representative images and quantitative analysis of 3 independent experiments showed that PINK1 or Parkin overexpression protected PA-induced reduction in mitochondrial membrane potential.
Fig 4.
PINK1-Parkin prevented PA-induced ROS accumulation and ATP reduction.
HAECs were transfected with PINK1 or Parkin siRNA followed by treatment with PA for 24 hours. A. The treated cells were incubated with 50 nM MitoTracker (red) and 10 μM DCFH-DA (green) diluted in serum free culture medium at 37°C for 30 min in the darkness. Intracellular ROS and mitoROS accumulation was compared between the groups. Representative images and quantitative analysis of 3 independent experiments indicated that PA induced intracellular ROS and mitoROS accumulation was increased by PINK1 or Parkin siRNA. B HAECs were transfected with PINK1 or Parkin or control plasmid followed by treatment with PA for 48 hours. The treated cells were incubated with 10 μM DCFH-DA (green) diluted in serum free culture medium at 37°C for 30 min in the darkness. Intracellular ROS accumulation was compared between the groups. Representative images and quantitative analysis of 3 independent experiments indicated that PA induced intracellular ROS accumulation was decreased by PINK1 or Parkin overexpression. C. ATP levels were measured. Quantitative analysis of 3 independent experiments indicated PINK1 or Parkin siRNA aggravated CCCP-induced ATP reduction.
Fig 5.
PINK1-Parkin prevented PA-induced endothelial death.
A. HAECs were transfected with PINK1 or Parkin siRNA followed by treatment with PA for 24 hours. Representative images and quantification of 3 independent TUNEL analysis showed that PINK1 or Parkin siRNA increased PA-induced apoptotic cell death. B. Representative electron microscopy photomicrographs of PA-treated cells show that PINK1 or Parkin siRNA increased apoptotic or necrotic bodies.
Fig 6.
PINK1 and Parkin were up-regulated in the vascular wall of the obese and diabetic mice.
(A). Four week old wild-type male WT mice were either fed with chow diet (CD, n = 6) or high fat diet (HFD, n = 12) for 12 weeks. Body weight levels and free fatty acid (FFA) were compared between the groups. (B). Representative immunostaining showed increased PINK1 and Parkin in aortic wall and in endothelial cells (enlarged in the square box) in HFD fed mice. L indicated lumen. Red dot line indicated internal elastic plate of aorta. Negative controls for the PINK1 and Parkin immunostaining were shown on the right. (C). Eight week old wild-type male mice received daily IP injection of 50 mg/kg body weight streptozotocin (STZ, n = 8) or vehicle (n = 5) for five consecutive days. Aortas were collected 4 weeks after the STZ injection. Blood glucose level was compared between the groups. (D) Representative immunostaining showed increased PINK1 and Parkin in the aortic wall and in endothelial cells (enlarged in the square box) in STZ treated mice. L indicated lumen. Red dot line indicated internal elastic plate of aorta. Negative controls for the PINK1 and Parkin immunostaining were the same of (B).