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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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Endogenous androgens modulate angiogenic/progenitor cell mobilization in response to ischemia. (A and B) Ulex+/AcLDL+ cells in the bone marrow (A) and spleen (B). Cells were seeded at a density of 5 × 106 cells/mm2 and after 4 d were assessed for the ability to ingest 4 µg/ml AcLDL (Invitrogen) and to bind 10 µg/ml FITC–UEA-1 (Sigma-Aldrich) via fluorescence microscopy. (C and D) SCA-1+/CXCR4+ cells in the bone marrow (C) and spleen (D). Hematopoietic progenitor cells were assessed using methylcellulose assays. (E and F) Granulocyte/macrophage CFUs in the bone marrow (E) and spleen (F). (G and H) Erythroid CFUs in the bone marrow (G) and spleen (H; n = 6/group; combined data from three independent experiments are shown). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using ANOVA. All data are expressed as means ± SEM.
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fig6: Endogenous androgens modulate angiogenic/progenitor cell mobilization in response to ischemia. (A and B) Ulex+/AcLDL+ cells in the bone marrow (A) and spleen (B). Cells were seeded at a density of 5 × 106 cells/mm2 and after 4 d were assessed for the ability to ingest 4 µg/ml AcLDL (Invitrogen) and to bind 10 µg/ml FITC–UEA-1 (Sigma-Aldrich) via fluorescence microscopy. (C and D) SCA-1+/CXCR4+ cells in the bone marrow (C) and spleen (D). Hematopoietic progenitor cells were assessed using methylcellulose assays. (E and F) Granulocyte/macrophage CFUs in the bone marrow (E) and spleen (F). (G and H) Erythroid CFUs in the bone marrow (G) and spleen (H; n = 6/group; combined data from three independent experiments are shown). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using ANOVA. All data are expressed as means ± SEM.

Mentions: It is now clear that the mobilization of various angiogenic/progenitor cells from the bone marrow to sites of ischemia is critical for neovascularization (Sieveking and Ng, 2009). Because the spleen serves as a reservoir for peripheral stem/progenitor cells (Heeschen et al., 2003), to further elucidate the mechanisms of androgen-induced angiogenesis in hindlimb ischemia, the early mobilization of angiogenic/progenitor cells was assessed in both the bone marrow and the spleen. Mononuclear cells (MNCs) staining positive for ulex lectin and uptake of Dil-acetylated low density lipoprotein (AcLDL) represent a heterogeneous population of cells with angiogenic activity (Asahara et al., 1997). Although the precise role of these cells in angiogenesis has been debated, these cells have been reported to augment angiogenesis in a paracrine fashion through the secretion of various growth factors (Heil et al., 2004; Sieveking et al., 2008). In females, estrogen has also been demonstrated to mobilize bone marrow–derived angiogenic/progenitor cells that participate in cardiovascular regeneration (Iwakura et al., 2006). Similarly, the findings of this study show that androgens modulate the mobilization of angiogenic cells. Orchidectomy markedly decreased the numbers of ulex+/AcLDL+ cells in the bone marrow (22.8 ± 1 vs. 13.4 ± 0.9 cells/200× field for control vs. orchidectomized mice, respectively; P < 0.001 using ANOVA; Fig. 6 A) and the spleen (20.6 ± 0.8 vs. 15.2 ± 0.7 cells/200× field for control vs. orchidectomized mice, respectively; P < 0.001 using ANOVA; Fig. 6 B). DHT treatment rescued castration-mediated decline in ulex+/AcLDL+ angiogenic cells in both the spleen (17.8 ± 0.9 cells/200× field; P > 0.05 using ANOVA) and the bone marrow (31.7 ± 5 cells/200× field; P < 0.001 using ANOVA). SDF-1α is a key chemokine in the migration and homing of bone marrow–derived progenitor cells to sites of ischemia, and is itself a ligand for the receptor CXCR4 (Jin et al., 2006); therefore, we also assessed the levels of CXCR4+/Sca-1+ progenitor cells after induction of hindlimb ischemia. Consistent with increased expression of SDF-1α, orchidectomized mice receiving DHT had significantly elevated numbers of CXCR4+/Sca-1+ cells present in the bone marrow at day 3 after ischemia (0.8 ± 0.2% vs. 0.4 ± 0.07% of MNCs for orchidectomized + DHT vs. controls, respectively; P < 0.05 using ANOVA; Fig. 6 C). Trends similar to those seen for ulex+/AcLDL+ cells were observed in the spleen for CXCR4+/Sca-1+ cells (i.e., decrease with castration and increase with DHT treatment), but these were not statistically significant (Fig. 6 D). The mobilization of cells from the bone marrow to the periphery is a highly coordinated, time-dependent event. Therefore, because sampling took place at day 3, it is possible that the significantly elevated numbers of Sca-1+/CXCR4+ cells in the bone marrow would not correspond to higher levels in the periphery until a later time point. Overall, our findings suggest that androgens play a role in the proliferation and mobilization of circulating angiogenic cell populations in the context of ischemia.


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)

Endogenous androgens modulate angiogenic/progenitor cell mobilization in response to ischemia. (A and B) Ulex+/AcLDL+ cells in the bone marrow (A) and spleen (B). Cells were seeded at a density of 5 × 106 cells/mm2 and after 4 d were assessed for the ability to ingest 4 µg/ml AcLDL (Invitrogen) and to bind 10 µg/ml FITC–UEA-1 (Sigma-Aldrich) via fluorescence microscopy. (C and D) SCA-1+/CXCR4+ cells in the bone marrow (C) and spleen (D). Hematopoietic progenitor cells were assessed using methylcellulose assays. (E and F) Granulocyte/macrophage CFUs in the bone marrow (E) and spleen (F). (G and H) Erythroid CFUs in the bone marrow (G) and spleen (H; n = 6/group; combined data from three independent experiments are shown). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using ANOVA. All data are expressed as means ± SEM.
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Related In: Results  -  Collection

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fig6: Endogenous androgens modulate angiogenic/progenitor cell mobilization in response to ischemia. (A and B) Ulex+/AcLDL+ cells in the bone marrow (A) and spleen (B). Cells were seeded at a density of 5 × 106 cells/mm2 and after 4 d were assessed for the ability to ingest 4 µg/ml AcLDL (Invitrogen) and to bind 10 µg/ml FITC–UEA-1 (Sigma-Aldrich) via fluorescence microscopy. (C and D) SCA-1+/CXCR4+ cells in the bone marrow (C) and spleen (D). Hematopoietic progenitor cells were assessed using methylcellulose assays. (E and F) Granulocyte/macrophage CFUs in the bone marrow (E) and spleen (F). (G and H) Erythroid CFUs in the bone marrow (G) and spleen (H; n = 6/group; combined data from three independent experiments are shown). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using ANOVA. All data are expressed as means ± SEM.
Mentions: It is now clear that the mobilization of various angiogenic/progenitor cells from the bone marrow to sites of ischemia is critical for neovascularization (Sieveking and Ng, 2009). Because the spleen serves as a reservoir for peripheral stem/progenitor cells (Heeschen et al., 2003), to further elucidate the mechanisms of androgen-induced angiogenesis in hindlimb ischemia, the early mobilization of angiogenic/progenitor cells was assessed in both the bone marrow and the spleen. Mononuclear cells (MNCs) staining positive for ulex lectin and uptake of Dil-acetylated low density lipoprotein (AcLDL) represent a heterogeneous population of cells with angiogenic activity (Asahara et al., 1997). Although the precise role of these cells in angiogenesis has been debated, these cells have been reported to augment angiogenesis in a paracrine fashion through the secretion of various growth factors (Heil et al., 2004; Sieveking et al., 2008). In females, estrogen has also been demonstrated to mobilize bone marrow–derived angiogenic/progenitor cells that participate in cardiovascular regeneration (Iwakura et al., 2006). Similarly, the findings of this study show that androgens modulate the mobilization of angiogenic cells. Orchidectomy markedly decreased the numbers of ulex+/AcLDL+ cells in the bone marrow (22.8 ± 1 vs. 13.4 ± 0.9 cells/200× field for control vs. orchidectomized mice, respectively; P < 0.001 using ANOVA; Fig. 6 A) and the spleen (20.6 ± 0.8 vs. 15.2 ± 0.7 cells/200× field for control vs. orchidectomized mice, respectively; P < 0.001 using ANOVA; Fig. 6 B). DHT treatment rescued castration-mediated decline in ulex+/AcLDL+ angiogenic cells in both the spleen (17.8 ± 0.9 cells/200× field; P > 0.05 using ANOVA) and the bone marrow (31.7 ± 5 cells/200× field; P < 0.001 using ANOVA). SDF-1α is a key chemokine in the migration and homing of bone marrow–derived progenitor cells to sites of ischemia, and is itself a ligand for the receptor CXCR4 (Jin et al., 2006); therefore, we also assessed the levels of CXCR4+/Sca-1+ progenitor cells after induction of hindlimb ischemia. Consistent with increased expression of SDF-1α, orchidectomized mice receiving DHT had significantly elevated numbers of CXCR4+/Sca-1+ cells present in the bone marrow at day 3 after ischemia (0.8 ± 0.2% vs. 0.4 ± 0.07% of MNCs for orchidectomized + DHT vs. controls, respectively; P < 0.05 using ANOVA; Fig. 6 C). Trends similar to those seen for ulex+/AcLDL+ cells were observed in the spleen for CXCR4+/Sca-1+ cells (i.e., decrease with castration and increase with DHT treatment), but these were not statistically significant (Fig. 6 D). The mobilization of cells from the bone marrow to the periphery is a highly coordinated, time-dependent event. Therefore, because sampling took place at day 3, it is possible that the significantly elevated numbers of Sca-1+/CXCR4+ cells in the bone marrow would not correspond to higher levels in the periphery until a later time point. Overall, our findings suggest that androgens play a role in the proliferation and mobilization of circulating angiogenic cell populations in the context of ischemia.

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