Aging impairs VEGF-mediated, androgen-dependent regulation of angiogenesis

Abstract

There is a progressive impairment of vascular repair mechanisms with advancing age concomitant with a steady decline in circulating androgen levels in men. Emerging evidence indicates androgens regulate angiogenesis; however, little research has focused on the impact of age upon androgen-mediated regulation of angiogenic mechanisms. Human dermal fibroblasts from young (<30 years) and older (>65 years) men were incubated with DHT, with or without androgen receptor antagonist hydroxyflutamide, or phosphoinositide 3-kinase inhibitor. Fibroblast-conditioned medium was used to stimulate angiogenic functions in human umbilical vein endothelial cells. Nuclear fractionation and fluorescence microscopy were used to study androgen receptor (AR) distribution. Conditioned medium from fibroblasts of young men, but not old men, treated with DHT produced a 3-fold increase in human umbilical vein endothelial cell tubulogenesis and 2-fold increase in migration via increased vascular endothelial growth factor (VEGF) expression and secretion, predominantly of VEGF145. DHT-induced VEGF secretion from fibroblasts of young men was AR-dependent and increased AKT phosphorylation, which was abrogated by phosphoinositide 3-kinase inhibition. By contrast, fibroblasts from older men were unresponsive to DHT and lacked androgen-mediated enhancement in VEGF production. These findings were associated with reduced AR nuclear translocation in old fibroblasts. The failure of DHT-induced paracrine stimulation of angiogenesis in fibroblasts from older men is likely due to defective nuclear translocation of AR. This first demonstration of androgen resistance (or insensitivity) acquired by human fibroblasts with aging suggests that pharmacological testosterone therapy for old men may be less effective in enhancing angiogenesis and facilitating tissue regeneration mechanisms reliant on paracrine release of VEGF.

Figure 1.

DHT mediates paracrine regulation of EC function. A, Relative number of migrated HUVECs per Transwell, toward conditioned media (CM) from untreated control vs 48 hours DHT (40nM) treatment of fibroblasts from young and old men. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control; n = 3 donors for young and old performed in duplicate over 3 individual experiments with data presented as means ± SEM. B, Relative number of tubule branch points per 4000 HUVECs treated for 24 hours with CM from control and 40nM DHT-treated young and old male fibroblasts. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control; n = 3 donors for young and old performed in quadruplicate over 3 individual experiments with data presented as means ± SEM. Representative micrographs of HUVEC tubule formation after 4 hours on Matrigel after 24 hours pretreatment with CM from control and 40nM DHT-treated fibroblasts from young and old men. Bar, 50 μm. C, Relative absorbance of HUVECs incubated in young and old DHT-treated fibroblast CM after 24 hours; n = 3 donors for young and old performed in quadruplicate over 3 individual experiments with data presented as means ± SEM.

Figure 2.

The effect of DHT treatment on fibroblast VEGF production. A, Quantitative RT-PCR on VEGF mRNA levels in fibroblasts from young and old men after 48 hours of 40nM DHT treatment. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control. B, The level of VEGF protein in CM from fibroblasts after 48 hours of DHT treatment using ELISA. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control. C, VEGF145 isoform mRNA levels in fibroblasts from young men after 48 hours of 40nM DHT treatment analyzed by quantitative RT-PCR. Statistical analysis was performed using Student's t test. *, P < .05 vs young control. D, The levels of VEGF145 protein in CM from young fibroblasts after 48 hours DHT treatment using ELISA. Statistical analysis was performed using Student's t test. ***, P < .001 vs young control. E, Relative number of tubule branch points per 4000 HUVECs treated for 24 hours with rhVEGF145 at indicated concentrations. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. ###, P < .001 vs untreated control; ***, P < .001; 73.7 pg/mL vs 52.9 pg/mL rhVEGF145-treated HUVECs. n = 3 donors of HUVECs performed in quadruplicate. Data presented as means ± SEM. F, Relative fold increase of migrated HUVECs per transwell, toward rhVEGF145 after 24 hours treatment. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. ###, P < .001, 73.7 pg/mL rhVEGF145-treated HUVECs vs untreated; #, P < .05, 500 pg/mL rhVEGF145-treated HUVECs vs untreated; *** P < .001, 73.7 pg/mL vs 52.9 pg/mL rhVEGF145-treated HUVECs; n = 3 donors of HUVECs performed in duplicate with data presented as means ± SEM.

Figure 3.

The effect of DHT treatment on cytokine production. A–F, The level of epidermal growth factor (EGF), basic FGF (FGFb), PDGF, nerve growth factor (NGF), TNF-α, and TGFβ, respectively in CM of fibroblasts from young and old men after 48 hours DHT treatment; n = 3 donors for young and old performed in triplicate over 3 individual experiments (means ± SEM).

Figure 4.

DHT-mediated increase in paracrine regulation of EC function by fibroblasts from young men is via an AR-dependent increase in VEGF. A, Level of VEGF in fibroblast CM measured by ELISA after 48 hours treatment with 40nM DHT in the presence or absence of 4μM HF. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. *, P < .05 vs control. B, Level of VEGF protein in fibroblasts after 24 hours transfection with 250 pmol of either control or AR siRNA, and after another 48 hours, cells were treated for 48 hours with 40nM DHT. Western blot demonstrating the level of AR knockdown 72 hours after transfection. *, P < .05 DHT vs control scrambled siRNA transfected cells. C, Relative level of HUVEC migration toward fibroblast CM after 48 hours DHT (40nM) treatment in the presence or absence of 4μM HF or 1 μg/mL VEGF-neutralizing antibody. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. *, P < .05 vs control. D, Tubule branch points per 4000 HUVECs after 24 hours HUVEC pretreatment with fibroblast CM after fibroblasts were treated for 48 hours with 40nM DHT in the presence or absence of 4μM HF or 1 μg/mL VEGF-neutralizing antibody. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. ***, P < .001 vs control; n = 4 donors performed in triplicate over 3 individual experiments (means ± SEM).

Figure 5.

PI3-kinase pathway activation in DHT-mediated VEGF production. A, Level of VEGF in fibroblast CM measured by ELISA after 48 hours treatment with 40nM DHT in the presence or absence of 10μM PI3-kinase inhibitor (LY294002). Statistical analysis was performed using one-way ANOVA with Bonferroni correction. *, P < .01 vs untreated control; n = 3 donors performed in triplicate over 3 individual experiments (means ± SEM). B, The level of pAKT(Ser473)/AKT expressed in fibroblasts from young men after 48 hours treatment with 40nM DHT in the presence or absence of 4μM HF or 10μM PI3-kinase inhibitor (LY294002). Data are normalized to vehicle control; n = 3. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. *, P < .05, DHT vs untreated control; #, P < .01, DHT plus HF vs untreated control plus HF. C, The level pAKT(Tyr308)/AKT expressed in fibroblasts from young men treated for 48 hours with DHT with or without the addition of LY294002. D, The level of pAKT(Ser473)/AKT expressed in young fibroblasts transfected with scrambled siRNA or AR siRNA after 48 hours treatment with 40nM DHT; n = 3. Data are normalized to untreated cells transfected with scrambled siRNA or AR siRNA. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. *, P < .05, DHT vs control scrambled siRNA transfected cells. E, The level of pAKT(Ser473)/AKT expressed in fibroblasts from young and old men after 48 hours treatment with 40nM DHT. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control; n = 3 donors performed in duplicate over 3 individual experiments (means ± SEM).

Figure 6.

AR expression in fibroblasts from young and old men. A, AR mRNA expression in fibroblasts from young and old men. Statistical analysis was performed using Student's t test. *, P < .05 vs young. B, Protein expression of AR in fibroblasts from young and old men; n = 3 donors performed in triplicate over 3 individual experiments (means ± SEM). C, Relative AR transcriptional activities of young and old fibroblasts after overnight treatment with 40nM DHT. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control; n = 3 donors performed in triplicate over 3 individual experiments (means ± SEM).

Figure 7.

The effect of DHT treatment on AR localization in fibroblasts from young and old men. Panel A, Cytoplasmic (C) and nuclear (N) expression of AR after subcellular fractionation of fibroblasts from young men subjected to 6 hours treatment with 40nM DHT. Statistical analysis was performed using Student's t test. *, P < .01 vs N control; n = 4 donors performed in quadruplicate over 3 individual experiments (means ± SEM). LSD1 and tubulin were used to demonstrate equal separation of nuclear and cytoplasmic fractions, respectively. Panel B, Cytoplasmic (C) and nuclear (N) expression of AR after subcellular fractionation of fibroblasts from old men subjected to 6 hours treatment with 40nM DHT. Statistical analysis was performed using Student's t test; n = 4 donors performed in quadruplicate over 3 individual experiments (means ± SEM). Panel C, Immunofluorescence localization of AR labeled with FITC (green) in untreated and 6 hours DHT-treated (40nM) fibroblasts from young men. Nuclei are stained blue with DAPI. Panel D, Immunofluorescence localization of AR labeled with FITC (green) in untreated and 6 hours DHT-treated (40nM) fibroblasts from old men. Nuclei are stained blue with DAPI. Bar, 30 μm. Panel E, Pixel fluorescence intensity of nuclear AR in young and old fibroblasts after 6 hours treatment with 40nM DHT. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. *, P < .05 vs young control.