Diverging antioxidative responses to IGF-1 in cultured human skin fibroblasts versus vascular endothelial cells

Abstract

Insulin-like growth factor 1 (IGF-1) stimulates cell proliferation and is crucial for maintenance of somatic tissues. However, this effect is associated with the inhibition of FOXO transcription factors and downregulation of antioxidative enzymes. In this study, we compared the responses of primary dermal fibroblasts and human umbilical vein endothelial cells with IGF-1 treatment. We found that IGF-1 primarily downregulated enzymatic antioxidants in skin fibroblasts. However, human umbilical vein endothelial cells were protected from an IGF-1-mediated decrease in antioxidative capacity. Moreover, IGF-1 also activated endothelial nitric oxide synthase in human umbilical vein endothelial cells. These observations suggest a dichotomous role for IGF-1, which provides for growth and repair needs of the soma, while attenuating the effect of oxidative stress on the vasculature by activating endothelial nitric oxide synthase. This increases the production of nitric oxide, an antiproliferative and, under certain circumstances, an antioxidant agent. Findings could help clarify the role of IGF-1 in aging and longevity of lower organisms, short-lived mammals, and humans.

Figure 1.

Insulin-like growth factor 1 (IGF-1) effect on transcription of antioxidant enzymes. Catalase (A), glutathione peroxidase (B), and superoxide dismutase (C –D) transcripts were measured by real-time quantitative PCR. Mean ± SEM of the change (Δ) in relative transcript expression upon IGF-1 treatment is plotted; experiments were performed in duplicate in five cell strains of serum-starved fibroblasts and human umbilical vein endothelial cells between passages 4–9. Effects indicate slopes obtained from linear models of log₁₀ IGF-1 dose versus change in relative transcript expression; *p < .05, **p < .01 for trend (exact p values delineated in text).

Figure 2.

Antioxidant enzyme levels and activities in response to insulin-like growth factor 1 (IGF-1) treatment. A –F, Protein levels were determined by Western immunoblot analysis. Integrated optical density (IOD) of each enzyme band was normalized to β-actin and α-tubulin loading controls, and the change (Δ) in relative IOD upon IGF-1 treatment is plotted. Shown are catalase (A), glutathione peroxidase (B), and superoxide dismutase (SOD; C –D); mean ± SEM of responses from five strains of each cell type after serum starvation are plotted. Effects indicate slopes obtained from linear models of log₁₀ IGF-1 dose versus change in relative IOD; *p < .05, **p < .01 for trend (exact p values delineated in text). E –F, Western blots of antioxidative enzymes and loading controls from a representative fibroblast strain (GM05399, panel E) and human umbilical vein endothelial cell (HUVEC) strain (newborn donor #2, panel F). G, Total SOD activity (units per mL) in supernatants from five strains of fibroblasts and HUVECs after overnight serum starvation was normalized to total protein levels in milligrams; mean ± SEM of change (Δ) in activity upon 24 hour IGF-1 treatment (75 ng/mL) is plotted. Experiments were performed in duplicate in each cell strain, and mean changes were tested for significance (**p = .001).

Figure 3.

Insulin-like growth factor 1 (IGF-1) effect on antioxidative capacity (AOC) in response to oxidative challenge. AOC was measured by a flow cytometric method to detect changes in the ratio of reactive oxygen species indicator (rhodamine) fluorescence at baseline versus upon oxidative challenge with H₂O₂ at each IGF-1 dose. Mean ± SEM of the change (Δ) in AOC ratio in five serum-starved fibroblast and human umbilical vein endothelial cell (HUVEC) strains upon treatment with increasing doses of IGF-1 is plotted. Effects indicate slopes obtained from linear models of log₁₀ IGF-1 dose versus AOC ratio change (*p = .031); responses were statistically significantly different by a test of interaction.

Figure 4.

Insulin-like growth factor 1 (IGF-1) effect on endothelial nitric oxide synthase (eNOS) transcripts and active eNOS levels in fibroblasts and human umbilical vein endothelial cells (HUVECs). A, Relative eNOS expression in five strains of fibroblasts and HUVECs as determined by real-time quantitative PCR. B –C, Levels of phosphorylated eNOS (Ser1177) in serum-starved HUVECs in response to IGF-1 treatment. Phospho-eNOS levels in five HUVEC strains were quantified by densitometry and normalized to tubulin expression. Treatment response differed significantly by repeated measures ANOVA; 100 ng/mL IGF-1 treatment was different by Bonferroni post-test (p < .001). A representative Western blot of phospho-eNOS and α-tubulin loading control from one HUVEC strain after IGF-1 treatment is shown in C.