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Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin.

Ekstrand M, Gustafsson Trajkovska M, Perman-Sundelin J, Fogelstrand P, Adiels M, Johansson M, Mattsson-Hultén L, Borén J, Levin M - PLoS ONE (2015)

Bottom Line: Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS.Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Clinical Medicine/Wallenberg Laboratory, University of Gothenburg and Sahlgrenska University Hospital, SE-413 45, Gothenburg, Sweden.

ABSTRACT

Objective: The first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin.

Approach and results: To address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).

Conclusions: Aortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

No MeSH data available.


Related in: MedlinePlus

Real-time imaging of intracellular and extracellular ROS ex vivo.(A) Fresh aortas in organ bath were depicted through microscope optics connected to a photon counting camera. (B). Photon counting image of two aortas after addition of extracellular ROS probe isoluminol. The left aorta had extensive atherosclerosis and right aorta limited atherosclerosis. (C) Luminescence intensity (y-axis) over time in atherosclerotic aortic arch of the left aorta (A—red line) and background (B—blue line). Time points for addition of isoluminol, SOD and catalase, and luminol is indicated at the y-axis. (D) Extracellular ROS levels (left grey area) were quantified by subtracting the SOD/catalase signal from the isoluminol signal. Intracellular ROS (right grey area) were quantified by subtracting the SOD/catalase blocked signal from the luminol signal.
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pone.0130898.g001: Real-time imaging of intracellular and extracellular ROS ex vivo.(A) Fresh aortas in organ bath were depicted through microscope optics connected to a photon counting camera. (B). Photon counting image of two aortas after addition of extracellular ROS probe isoluminol. The left aorta had extensive atherosclerosis and right aorta limited atherosclerosis. (C) Luminescence intensity (y-axis) over time in atherosclerotic aortic arch of the left aorta (A—red line) and background (B—blue line). Time points for addition of isoluminol, SOD and catalase, and luminol is indicated at the y-axis. (D) Extracellular ROS levels (left grey area) were quantified by subtracting the SOD/catalase signal from the isoluminol signal. Intracellular ROS (right grey area) were quantified by subtracting the SOD/catalase blocked signal from the luminol signal.

Mentions: Two freshly dissected aortas were put in organ bath with oxygenated (21% oxygen 5% CO2 in nitrogen) Krebs Ringer solution (Sigma-Aldrich, MO, USA) at 37°C. The experiment set up and a typical registration is shown in Fig 1.


Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin.

Ekstrand M, Gustafsson Trajkovska M, Perman-Sundelin J, Fogelstrand P, Adiels M, Johansson M, Mattsson-Hultén L, Borén J, Levin M - PLoS ONE (2015)

Real-time imaging of intracellular and extracellular ROS ex vivo.(A) Fresh aortas in organ bath were depicted through microscope optics connected to a photon counting camera. (B). Photon counting image of two aortas after addition of extracellular ROS probe isoluminol. The left aorta had extensive atherosclerosis and right aorta limited atherosclerosis. (C) Luminescence intensity (y-axis) over time in atherosclerotic aortic arch of the left aorta (A—red line) and background (B—blue line). Time points for addition of isoluminol, SOD and catalase, and luminol is indicated at the y-axis. (D) Extracellular ROS levels (left grey area) were quantified by subtracting the SOD/catalase signal from the isoluminol signal. Intracellular ROS (right grey area) were quantified by subtracting the SOD/catalase blocked signal from the luminol signal.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476734&req=5

pone.0130898.g001: Real-time imaging of intracellular and extracellular ROS ex vivo.(A) Fresh aortas in organ bath were depicted through microscope optics connected to a photon counting camera. (B). Photon counting image of two aortas after addition of extracellular ROS probe isoluminol. The left aorta had extensive atherosclerosis and right aorta limited atherosclerosis. (C) Luminescence intensity (y-axis) over time in atherosclerotic aortic arch of the left aorta (A—red line) and background (B—blue line). Time points for addition of isoluminol, SOD and catalase, and luminol is indicated at the y-axis. (D) Extracellular ROS levels (left grey area) were quantified by subtracting the SOD/catalase signal from the isoluminol signal. Intracellular ROS (right grey area) were quantified by subtracting the SOD/catalase blocked signal from the luminol signal.
Mentions: Two freshly dissected aortas were put in organ bath with oxygenated (21% oxygen 5% CO2 in nitrogen) Krebs Ringer solution (Sigma-Aldrich, MO, USA) at 37°C. The experiment set up and a typical registration is shown in Fig 1.

Bottom Line: Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS.Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Clinical Medicine/Wallenberg Laboratory, University of Gothenburg and Sahlgrenska University Hospital, SE-413 45, Gothenburg, Sweden.

ABSTRACT

Objective: The first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin.

Approach and results: To address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).

Conclusions: Aortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

No MeSH data available.


Related in: MedlinePlus