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.

Figure 1.

NOX activation in response to radiotherapy in BAECs. Using confocal microscopy and staining endothelial cells with anticeramide antibody (red) and anti-p47phox (green), we showed radiation-induced co-localization of ceramide with p47phox as early as 1 minute postradiation, with a decrease after 5 minutes. The images were taken by microscope at 400× magnification. BAECs = bovine aortic endothelial cells; NOX = NADPH oxidase.

Figure 2.

Sildenafil and NOX-inhibitor DPI (10 μM) inhibit radiation-induced apoptosis in BAECs. Endothelial apoptosis was scored 8 hours after irradiation using the bis-benzimide trihydrochloride staining. Panel A shows sildenafil doses between 1 and 10 μM added 5 minutes before irradiation to 10 Gy. Panel B shows sildenafil 5 μM added between 5 and 60 minutes before irradiation to 10 Gy. Panel C shows sildenafil 5 μM added 5 minutes before irradiation to 10–20 Gy. Panel D shows DPI (10 μM) added 30 minutes before irradiation to 10–15 Gy. Data are expressed as mean ± standard error from three independent experiments. *P < .05 of sildenafil-treated BAECs compared with untreated BAECs. BAECs = bovine aortic endothelial cells; DPI = diphenyleneiodonium; NOX = NADPH oxidase.

Figure 3.

Sildenafil inhibits general ROS production in BAECs irradiated to 10 Gy. Sildenafil (5 μM) was added 5 minutes before exposure to 10 Gy. Cellular ROS production assessed using the DCF-DA after irradiation with 10 Gy and 30 minutes incubation subsequently. Data are expressed as mean ± standard error from 3 independent experiments. *P < .05 of sildenafil-treated BAECs compared with untreated BAECs. BAECs = bovine aortic endothelial cells; DCF-DA = 2’,7 ‘dichlorofluorescein diacetate assay; ROS = reactive oxygen species.

Figure 4.

Sildenafil and NOX-inhibitor DPI (10 mM) inhibit O2•− production in BAECs after exposure to 10 Gy. Sildenafil and DPI (10 μM) were added 5 and 30 minutes before exposure to 10 Gy, respectively. Data are expressed as mean ± standard error from 3 independent experiments. **P < .01 of sildenafil-treated and DPI-treated BAECs compared with untreated BAECs. *P < .05 of DPI-treated BAECs compared with untreated BAECs. BAECs = bovine aortic endothelial cells; DPI = diphenyleneiodonium; NOX = NADPH oxidase.

Figure 5.

Sildenafil at 5 μM has no effect on extracellular or on intracellular H₂O₂ in BAECs after exposure to 10 Gy. Sildenafil and DPI (10 μM), were added 5 and 30 minutes before exposure to 10 Gy, respectively. DPI (10 μM) inhibits intracellular H₂O₂ fluorescence intensity at 2 minutes after exposure to 10 Gy. Panel A shows extracellular H₂O₂ was measured using the Amplex Red assay (Molecular Probes) after 10 Gy irradiation and 30 minutes of incubation. Panel B shows intracellular H₂O₂ was measured using HyPer transfection (Evrogen). Periodic images of transfected cells were taken at baseline and after 10 Gy irradiation. Data are expressed as mean ± standard error from 3 independent experiments. *P < 0.05 DPI-treated BAECs compared with untreated BAECs. Panel C shows that sildenafil attenuates the production of intracellular peroxynitrite in BAECs after exposure to 10 Gy. Peroxynitrite levels were quantified by the chemiluminescent reporter PNCL, 10 minutes after 10 Gy of irradiation. Data are expressed as mean ± standard error from 3 independent experiments. *P < .05. BAECs = bovine aortic endothelial cells; DPI = diphenyleneiodonium; H₂O₂ = hydrogen peroxide.

Figure 6.

Sildenafil inhibits ASMase activity and ceramide generation in BAECs after exposure to 10 Gy. Panel A shows ASMase activity quantified by radioemzymatic assay using [14C-methylcholine] sphingomyelin. Panel B shows ceramide was measured using the diacylglycerol kinase assay after 10 Gy of irradiation. Data are expressed as mean ± standard error from 3 independent experiments. *P < .05 sildenafil-treated BAECs compared with untreated BAECs. ASMase = acid sphingomyelinase; BAECs = bovine aortic endothelial cells.

Figure 7.

Proposed sildenafil interaction within radiation-induced endothelial cell dysfunction via ASMaseeceramideeNOX axis. This schematic model was adopted from work published by our collaborators (and with their and the journal permission) in response to cytokine activation in BAECs. In response to radiation, ASMase containing lysosomes are driven to fuse with the cell membrane through soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins. ASMase is released and upon encountering its substrate, sphingomyelin, hydrolyses it to produce ceramide, within the outer leaflet of the plasma membrane. Ceramide, having fusigenic properties initiates the generation of CRMs. Subsequently, NOX subunits, such as gp91phox and p47phox, are aggregated, resulting in activated NOX via this process, and producing O₂•−. O₂•− may activate ASMase in a feed-forward mechanism, positive enhancing CRMs clustering and forming amplifications of this process. O₂•− coupling with NO generates another ROS, peroxynitrite (OONO-) but, most importantly depletes NO levels. Altogether, these processes constitute a redox signaling network or signalosome, resulting in endothelial dysfunction and impairment of endothelium-dependent vasodilation in coronary arteries. Sildenafil significantly inhibited ROS production in general: the immediate O₂•− production and the subsequent OONO-generation, in endothelial cells, by stopping the feed-forward activation of ASMase, ceramide generation, and NOX activation. ASMase =acid sphingomyelinase; BAECs = bovine aortic endothelial cells; CRMs = ceramide-rich microdomains; NOX = NADPH oxidase.