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Protective Effects of Diet and Sex on Cell Death and Intracellular Calcium in Resistance Arteries during Oxidative Stress

Reactive oxygen species are implicated in vascular dysfunction and the development of cardiovascular disease. However, chronic exposure to oxidative stress during advanced age protects endothelial cells (ECs) of the superior epigastric artery (SEA) in male mice from hydrogen peroxide (H2O2)‐induced Ca2+ entry and cell death (PMC4422569), and we have found similar protection in SEA smooth muscle cells (SMCs). We hypothesized that a Western style diet (WD) high in fat and fructose, by promoting chronic oxidative stress, would also improve vascular cell survival during exposure to H2O2. Because females appear more resilient to oxidative stress compared to males, we also investigated the effect of sex on cell death and intracellular calcium ([Ca2+]i) with WD. Male and female C57BL/6 mice (age, 4 weeks; n=4–5/group) were fed WD (TestDiet 58Y1; calories: 46% fat, 17.5% sucrose, 17.5% high fructose corn syrup; 19% protein), or Standard diet (Formulab Diet 5008; calories: 17% Fat, 56% carbohydrate, 27% protein) for 20 weeks. A mouse was then anesthetized (isoflurane) and the SEA (inner diameter, 100–150 μm) was isolated, pressurized to 100 cm H2O in physiological salt solution (pH 7.4, 37°C), and exposed to H2O2 (200 μM) for 50 minutes. Hoechst 33342 (1 μM) stained nuclei of all cells while propidium iodide (2 μM) stained nuclei from dead cells; SMC nuclei (n=121±3 per SEA) were oriented perpendicular to the vessel axis and EC nuclei (n=125±2 per SEA) were oriented parallel to the vessel axis. With Standard diet, SEAs from males had greater (P<0.05) cell death (SMCs=21±1%, ECs=6±1%) versus SEAs from females (SMCs=9±2%, ECs=2%). Consuming WD attenuated SMC death in SEAs from both males (to 11±6%) and females (to 3±1%) (P<0.05 for each) as well as in ECs from males (to 2%) with no further effect in ECs from females (2%). Because an aberrant rise in [Ca2+]i can initiate cell death, [Ca2+]i responses to H2O2 were evaluated using Fura‐2 fluorescence (F340/F380 ratios). With no effect of diet or sex on [Ca2+]i at rest, H2O2 increased [Ca2+]i in SEAs from males fed Standard diet (ΔF340/F380=0.57±0.04) by an 8‐fold greater (P<0.05) extent than in SEAs from females (ΔF340/F380=0.07±0.05). SEAs of males fed a WD exhibited a reduced [Ca2+]i response to H2O2 (ΔF340/F380=0.45±0.08) (P<0.05), however this effect of diet was not observed in SEAs of females (ΔF340/F380=0.19±0.06). We conclude that ECs and SMCs in resistance arteries of females are more resilient to acute H2O2 exposure compared to males and that WD promotes resilience to oxidative stress most effectively in males.