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A sex-specific role for androgens in angiogenesis.

Sieveking DP, Lim P, Chow RW, Dunn LL, Bao S, McGrath KC, Heather AK, Handelsman DJ, Celermajer DS, Ng MK - J. Exp. Med. (2010)

Bottom Line: Androgen receptor (AR) antagonism or gene knockdown abrogated these effects in male ECs.In vivo, castration dramatically reduced neovascularization of Matrigel plugs.Androgen treatment fully reversed this effect in male mice but had no effect in female mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Heart Research Institute, Sydney 2042, Australia. sievekingd@hri.org.au

ABSTRACT
Mounting evidence suggests that in men, serum levels of testosterone are negatively correlated to cardiovascular and all-cause mortality. We studied the role of androgens in angiogenesis, a process critical in cardiovascular repair/regeneration, in males and females. Androgen exposure augmented key angiogenic events in vitro. Strikingly, this occurred in male but not female endothelial cells (ECs). Androgen receptor (AR) antagonism or gene knockdown abrogated these effects in male ECs. Overexpression of AR in female ECs conferred androgen sensitivity with respect to angiogenesis. In vivo, castration dramatically reduced neovascularization of Matrigel plugs. Androgen treatment fully reversed this effect in male mice but had no effect in female mice. Furthermore, orchidectomy impaired blood-flow recovery from hindlimb ischemia, a finding rescued by androgen treatment. Our findings suggest that endogenous androgens modulate angiogenesis in a sex-dependent manner, with implications for the role of androgen replacement in men.

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DHT augments key angiogenic events in vitro in male ECs via the AR. (A and B) Boyden chamber (cells were stained with Ulex lectin [UEA-1] and DAPI) and (C) scratch wounding assays of migration by male ECs pretreated with DHT or vehicle (0.1% EtOH) for 24 h ± HF, assessed after 6 and 24 h, respectively. Bar, 50 µm. (D and E) Vascular network formation by male ECs exposed to DHT or vehicle ± HF for 72 h. Tubule area was quantified using image analysis software (ImageJ; available at http://rsbweb.nih.gov/ij/). Bar, 100 µm. (F) Proliferation of male ECs exposed to DHT or vehicle ± HF for 24 h, assessed by direct cell counting (n = 4 independent experiments for A–F, respectively). (G and H) Matrigel assays of male ECs transfected with siRNA targeted to the AR (G) or pretreated with the ER blocker ICI182780 and treated with DHT (H; n = 3 independent experiments). **, P < 0.01; and ***, P < 0.001 versus control using ANOVA. All data are expressed as means ± SEM. For each independent experiment, cells from a different donor were used.
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fig1: DHT augments key angiogenic events in vitro in male ECs via the AR. (A and B) Boyden chamber (cells were stained with Ulex lectin [UEA-1] and DAPI) and (C) scratch wounding assays of migration by male ECs pretreated with DHT or vehicle (0.1% EtOH) for 24 h ± HF, assessed after 6 and 24 h, respectively. Bar, 50 µm. (D and E) Vascular network formation by male ECs exposed to DHT or vehicle ± HF for 72 h. Tubule area was quantified using image analysis software (ImageJ; available at http://rsbweb.nih.gov/ij/). Bar, 100 µm. (F) Proliferation of male ECs exposed to DHT or vehicle ± HF for 24 h, assessed by direct cell counting (n = 4 independent experiments for A–F, respectively). (G and H) Matrigel assays of male ECs transfected with siRNA targeted to the AR (G) or pretreated with the ER blocker ICI182780 and treated with DHT (H; n = 3 independent experiments). **, P < 0.01; and ***, P < 0.001 versus control using ANOVA. All data are expressed as means ± SEM. For each independent experiment, cells from a different donor were used.

Mentions: Male ECs treated with DHT demonstrated a dose-dependent increase in key angiogenic processes in vitro (Fig. 1). EC migration was assessed by two different methods. Cells were pretreated with varying doses of DHT (0, 4, 40, and 400 nM) and subsequently assessed using a Boyden chamber assay or a scratch wounding assay. Using the Boyden chamber assay, 24 h of DHT administration induced dose-dependent male EC migration (220 ± 22, 262 ± 38, and 336 ± 39% vs. 100% control value for DHT at 4, 40, and 400 nM, respectively; P < 0.001 using analysis of variance [ANOVA]; Fig. 1 B). Similar results were obtained using a scratch wounding assay. Migration by male ECs was augmented in a dose-dependent fashion (138 ± 7, 280 ± 23, and 306 ± 32%; P < 0.01 using ANOVA; Fig. 1 C). Interestingly, in both assays, addition of the AR antagonist hydroxyflutamide (HF) abrogated DHT-mediated EC migration (96 ± 6 and 94 ± 6% vs. 100% control value for 40 and 400 nM DHT + HF, respectively; P > 0.05; Fig. 1, B and C). DHT effects on tubulogenesis were assessed using both Matrigel and co-culture methods (Sieveking et al., 2008). Chronic exposure to DHT (72 h) induced a dose-dependent increase in male EC tubulogenesis (Fig. 1, D and E), as did 24 h of exposure (Fig. S1). Similarly, DHT augmented male EC proliferation in a dose-dependent fashion after 48 h (Fig. 1 F). Again, addition of HF abolished all androgen-mediated effects on these key angiogenic events, consistent with an AR-dependent mechanism (Fig. 1, B, C, E, and F).


A sex-specific role for androgens in angiogenesis.

Sieveking DP, Lim P, Chow RW, Dunn LL, Bao S, McGrath KC, Heather AK, Handelsman DJ, Celermajer DS, Ng MK - J. Exp. Med. (2010)

DHT augments key angiogenic events in vitro in male ECs via the AR. (A and B) Boyden chamber (cells were stained with Ulex lectin [UEA-1] and DAPI) and (C) scratch wounding assays of migration by male ECs pretreated with DHT or vehicle (0.1% EtOH) for 24 h ± HF, assessed after 6 and 24 h, respectively. Bar, 50 µm. (D and E) Vascular network formation by male ECs exposed to DHT or vehicle ± HF for 72 h. Tubule area was quantified using image analysis software (ImageJ; available at http://rsbweb.nih.gov/ij/). Bar, 100 µm. (F) Proliferation of male ECs exposed to DHT or vehicle ± HF for 24 h, assessed by direct cell counting (n = 4 independent experiments for A–F, respectively). (G and H) Matrigel assays of male ECs transfected with siRNA targeted to the AR (G) or pretreated with the ER blocker ICI182780 and treated with DHT (H; n = 3 independent experiments). **, P < 0.01; and ***, P < 0.001 versus control using ANOVA. All data are expressed as means ± SEM. For each independent experiment, cells from a different donor were used.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822613&req=5

fig1: DHT augments key angiogenic events in vitro in male ECs via the AR. (A and B) Boyden chamber (cells were stained with Ulex lectin [UEA-1] and DAPI) and (C) scratch wounding assays of migration by male ECs pretreated with DHT or vehicle (0.1% EtOH) for 24 h ± HF, assessed after 6 and 24 h, respectively. Bar, 50 µm. (D and E) Vascular network formation by male ECs exposed to DHT or vehicle ± HF for 72 h. Tubule area was quantified using image analysis software (ImageJ; available at http://rsbweb.nih.gov/ij/). Bar, 100 µm. (F) Proliferation of male ECs exposed to DHT or vehicle ± HF for 24 h, assessed by direct cell counting (n = 4 independent experiments for A–F, respectively). (G and H) Matrigel assays of male ECs transfected with siRNA targeted to the AR (G) or pretreated with the ER blocker ICI182780 and treated with DHT (H; n = 3 independent experiments). **, P < 0.01; and ***, P < 0.001 versus control using ANOVA. All data are expressed as means ± SEM. For each independent experiment, cells from a different donor were used.
Mentions: Male ECs treated with DHT demonstrated a dose-dependent increase in key angiogenic processes in vitro (Fig. 1). EC migration was assessed by two different methods. Cells were pretreated with varying doses of DHT (0, 4, 40, and 400 nM) and subsequently assessed using a Boyden chamber assay or a scratch wounding assay. Using the Boyden chamber assay, 24 h of DHT administration induced dose-dependent male EC migration (220 ± 22, 262 ± 38, and 336 ± 39% vs. 100% control value for DHT at 4, 40, and 400 nM, respectively; P < 0.001 using analysis of variance [ANOVA]; Fig. 1 B). Similar results were obtained using a scratch wounding assay. Migration by male ECs was augmented in a dose-dependent fashion (138 ± 7, 280 ± 23, and 306 ± 32%; P < 0.01 using ANOVA; Fig. 1 C). Interestingly, in both assays, addition of the AR antagonist hydroxyflutamide (HF) abrogated DHT-mediated EC migration (96 ± 6 and 94 ± 6% vs. 100% control value for 40 and 400 nM DHT + HF, respectively; P > 0.05; Fig. 1, B and C). DHT effects on tubulogenesis were assessed using both Matrigel and co-culture methods (Sieveking et al., 2008). Chronic exposure to DHT (72 h) induced a dose-dependent increase in male EC tubulogenesis (Fig. 1, D and E), as did 24 h of exposure (Fig. S1). Similarly, DHT augmented male EC proliferation in a dose-dependent fashion after 48 h (Fig. 1 F). Again, addition of HF abolished all androgen-mediated effects on these key angiogenic events, consistent with an AR-dependent mechanism (Fig. 1, B, C, E, and F).

Bottom Line: Androgen receptor (AR) antagonism or gene knockdown abrogated these effects in male ECs.In vivo, castration dramatically reduced neovascularization of Matrigel plugs.Androgen treatment fully reversed this effect in male mice but had no effect in female mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Heart Research Institute, Sydney 2042, Australia. sievekingd@hri.org.au

ABSTRACT
Mounting evidence suggests that in men, serum levels of testosterone are negatively correlated to cardiovascular and all-cause mortality. We studied the role of androgens in angiogenesis, a process critical in cardiovascular repair/regeneration, in males and females. Androgen exposure augmented key angiogenic events in vitro. Strikingly, this occurred in male but not female endothelial cells (ECs). Androgen receptor (AR) antagonism or gene knockdown abrogated these effects in male ECs. Overexpression of AR in female ECs conferred androgen sensitivity with respect to angiogenesis. In vivo, castration dramatically reduced neovascularization of Matrigel plugs. Androgen treatment fully reversed this effect in male mice but had no effect in female mice. Furthermore, orchidectomy impaired blood-flow recovery from hindlimb ischemia, a finding rescued by androgen treatment. Our findings suggest that endogenous androgens modulate angiogenesis in a sex-dependent manner, with implications for the role of androgen replacement in men.

Show MeSH
Related in: MedlinePlus