Mechanisms by which statins protect endothelial cells from radiation-induced injury in the carotid artery

Background The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. However, the mechanisms by which statins protect the vasculature from irradiation injury remain poorly understood. Objectives Identify the mechanisms by which the hydrophilic and lipophilic statins pravastatin and atorvastatin preserve endothelial function after irradiation. Methods Cultured human coronary and umbilical vein endothelial cells irradiated with 4 Gy and mice subjected to 12 Gy head-and-neck irradiation were pretreated with statins and tested for endothelial dysfunction, nitric oxide production, oxidative stress, and various mitochondrial phenotypes at 24 and 240 h after irradiation. Results Both pravastatin (hydrophilic) and atorvastatin (lipophilic) were sufficient to prevent the loss of endothelium-dependent relaxation of arteries after head-and-neck irradiation, preserve the production of nitric oxide by endothelial cells, and suppress the cytosolic reactive oxidative stress associated with irradiation. However, only pravastatin inhibited irradiation-induced production of mitochondrial superoxide; damage to the mitochondrial DNA; loss of electron transport chain activity; and expression of inflammatory markers. Conclusions Our findings reveal some mechanistic underpinnings of the vasoprotective effects of statins after irradiation. Whereas both pravastatin and atorvastatin can shield from endothelial dysfunction after irradiation, pravastatin additionally suppresses mitochondrial injury and inflammatory responses involving mitochondria. Clinical follow-up studies will be necessary to determine whether hydrophilic statins are more effective than their lipophilic counterparts in reducing the risk of cardiovascular disease in patients undergoing radiation therapy.

Antibodies for NFκB-p65 and for GAPDH were purchased from Cell Signaling (CST 8284S and CST 5174S).

Cell counts
Wells of 6-well plates were seeded with 30,000 HUVECs and the cultures were grown to 80%-90% confluency. Then, atorvastatin or pravastatin was added at a concentration of 5 M or 10 M. After incubation overnight (approximately 12 hours), the HUVECs were rinsed with ECM, trypsinized and counted using an automated cell counter (Beckman Coulter).

MTT assay
HUVECs were seated in 96-well plate at a density of 1,000 cells per well and cultured for 48 hr before performing MTT toxicity assay. Cells were incubated with statins dissolved in DMSO or DMSO overnight.
The next morning, the MTT stock solution was prepared at a concentration of 5mg/ml in PBS. 10 μl of MTT stock solution with 90 μl of media was added into each well for 2 hr. Then, media were aspirated, cells rinsed with PBS and intracellular MTT formazan crystals dissolved in 50 μl DMSO.
Absorbance was measured in a microtiter plate reader at a wavelength of 570 nm.

Immunoblot
Western blot analysis for NFκB-p65 was performed in cell lysates of HUVECs cells subjected to irradiation (4Gy) in the presence or absence of Pravastatin (10μM) or Atorvastatin (5μM). Briefly, the cells were harvested, lysed in RIPA buffer supplemented with proteinase and phosphatase inhibitors, and then sonicated using a sonicator. After centrifugation for 10 min at 10,000 rpm, the total protein was quantified using the BCA assay and 20 μg of protein per sample were loaded into SDS-PAGE gels. The proteins were then transferred to PVDF membranes, incubated in 5% milk and then, with primary antibodies for NFκB-p65 (1:1000) and GAPDH (1:5000) as a loading control. Blots were washed 3 times for 10 min with 0.05% Tween-20 in TBS, incubated for 1 hr at room temperature with the respective secondary antibodies, and then washed again. The blots were then developed using ECL chemiluminescent substrate according to the manufacturer's instructions.