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

Abstract

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.

Keywords: carotid stenosis; endothelium; mitochondria; prevention; radiation therapy; statin.

Figure 1

Pravastatin and atorvastatin preserve endothelial function in vivo following head-and-neck IR. (A–C) Effects of statins on endothelium-dependent relaxation of the carotid artery in response to acetylcholine (ACh). C57BL/6J mice were treated with (A) vehicle, (B) pravastatin (Prava), or (C) atorvastatin (Ator) after head-and-neck irradiation (12Gy) or sham treatment, and relaxation was tested at 24 and 240 hr after irradiation. (D–F) Effects of statins on endothelium-independent relaxation of the carotid artery in response to sodium nitroprusside (SNP), in mice treated as in A, B, and C, respectively. (G–I) Effects of statins on endothelium-dependent relaxation of mesenteric resistance arteries (MRAs), in mice treated as in A, B, and C, respectively. n=5 mice per group. p values were determined using repeated measures 2-way ANOVA followed by Tukey's post-Hoc test.

Figure 2

When administered before IR in vitro, pravastatin and atorvastatin prevent IR-induced ROS production and loss of NO production. All panels compare HCAECs subjected to irradiation (4 Gy). (A,B) Change in cellular ROS levels, as determined by CM-H₂DCFDA fluorescence at (A) 24 and (B) 240 h after IR, in cells treated with vehicle, pravastatin (Prava), or atorvastatin (Ator) starting at 18 h before irradiation (4 Gy). HCAECs treated with TEMPO or H₂O₂ served as negative and positive controls, respectively. (C–F) NO production in cells pretreated with pravastatin (C,D) or atorvastatin (E,F) and in response to PDGF (20 ng/ml), as determined by DAF2-DA fluorescence. NO levels are normalized to baseline (i.e., levels before PDGF addition) and plotted as fold-change relative to untreated cells. HCAECs treated with L-NNA and SNP served as controls. Images were taken at (C,E) 24 and (D,F) 240 h after irradiation. n = 4 independent experiments. p values by Kruskal-Wallis test.

Figure 3

Pravastatin protects against IR-induced mitochondrial damage or hyperpolarization in vitro. All panels compare cells subjected to irradiation (4 Gy) after pretreatment with pravastatin (Prava, 10 mM) starting at 18 hr before irradiation. (A,B) Representative images and signal integrated density of mitoSOX fluorescence normalized to mitoTracker fluorescence in HCAECs at (A) 24 and (B) 240 hr after IR. (C,D) mtDNA lesions in DNA extracted from HUVECs at (C) 24 hr and (D) 240 hr after IR. (E,F) Representative images and integrated density of mitochondrial membrane potential in HCAECs, as determined by TMRM fluorescence, at (E) 24 hr and (F) 240 hr after irradiation. Analysis per cell, n = 4 independent experiments. p values were determined by Kruskal-Wallis test.

Figure 4

Atorvastatin does not protect against IR-induced mitochondrial damage or hyperpolarization in vitro. All panels compare HCAECs subjected to irradiation (4 Gy) after pretreatment with atorvastatin (Ator, 5 mM) or vehicle. Parameters assessed are: (A,B) Representative images and signal integrated density of MitoSOX fluorescence normalized to MitoTracker fluorescence at (A) 24 and (B) 240 hr after irradiation, in cells treated with atorvastatin (5 mM) or vehicle starting 18 hr before irradiation. (C,D) Damage to mtDNA as assessed by PCR assay. mtDNA lesions at (C) 24 and (D) 240 hr after irradiation, in HUVECs treated with atorvastatin or vehicle starting 18 hr before IR. (E,F) Representative images and integrated density of mitochondrial membrane potential, as determined by TMRM fluorescence, at (E) 24 and (F) 240 hr after irradiation. Analysis per cell, n = 4 independent experiments, p values by Kruskal-Wallis test.

Figure 5

Pravastatin prevents IR-induced inflammatory signaling. All panels compare HCAECs subjected to irradiation (4 Gy) after pretreatment with pravastatin (Prava, 10 μM, overnight) or vehicle. (A,B) Quantitative (q)RT-PCR for NFκB-p50 at (A) 24 and (B) 240 h after irradiation. (C,D) qRT-PCR for NFκB-p65 at (C) 24 and (D) 240 h after irradiation. (E,F) qRT-PCR for TNFα at (E) 24 and (F) 240 h after irradiation. p values by Kruskal-Wallis test.

Figure 6

Atorvastatin does not prevent IR-induced inflammatory signaling. All panels compare HCAECs subjected to irradiation (4 Gy) after pretreatment with atorvastatin (Ator, 5 μM, overnight) or vehicle. (A,B) Quantitative (q)RT-PCR for NFκB-p50 at (A) 24 and (B) 240 h after irradiation. (C,D) qRT-PCR for NFκB-p65 at (C) 24 and (D) 240 h after irradiation. (E-F) qRT-PCR for TNFα at (E) 24 and (F) 240 h after irradiation. p values by Kruskal-Wallis test.

Figure 7

Pravastatin, but not atorvastatin, prevents irradiation-induced reduction of mtDNA transcription and ETC activity. All panels compare HCAECs subjected to irradiation (4 Gy) after pretreatment with atorvastatin (Ator, 5mM, overnight), pravastatin (Prava, 10mM, overnight) or vehicle. (A–D) Effects of pretreatment with pravastatin (Prava, 10mM, overnight) on transcriptional activity. (A,B) Quantitative (q)RT-PCR for cytochrome c oxidase I (MT-COI) at (A) 24 and (B) 240 hr after irradiation. (C–D) qRT-PCR for NADH-ubiquinone oxidoreductase chain 1 (MT-ND1) at (C) 24 and (D) 240 hr after irradiation. (E–H) Effects of pretreatment with atorvastatin (Ator, 5 mM, overnight) on transcriptional activity. (E,F) qRT-PCR for MT-COI at (E) 24 and (F) 240 hr after irradiation. (G,H) qRT-PCR for MT-ND1 at (G) 24 and (H) 240 hr after irradiation. (I,J) Activity of ETC complex 1, as assessed by fluorometric assay at (I) 24 and (J) 240 hr after irradiation. p values by Kruskal-Wallis test.