Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

Abstract

Introduction: The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic.

Aim: To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells.

Methods: Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10-15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H₂O₂) was measured using the Amplex Red assay and intracellular H₂O₂ using a fluorescent sensor. In addition, ROS superoxide (O₂•-) was measured using a O₂•- chemiluminescence enhancer. Both H₂O₂ and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO^-) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [¹⁴C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C₁₆-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells' dysfunction.

Main outcome measures: Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway.

Results: Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O₂•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis.

Strengths and limitations: This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O₂•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED.

Conclusions: This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED. Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721-1733.

Keywords: Endothelial Damage; Erectile Dysfunction; Oxidative Stress; Radiation; Reactive Oxygen Species.