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+
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; ) 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; ). DHT treatment rescued castration-mediated decline in ulex+
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+
progenitor cells after induction of hindlimb ischemia. Consistent with increased expression of SDF-1α, orchidectomized mice receiving DHT had significantly elevated numbers of CXCR4+
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; ). Trends similar to those seen for ulex+
cells were observed in the spleen for CXCR4+
cells (i.e., decrease with castration and increase with DHT treatment), but these were not statistically significant (). 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+
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.
Finally, in light of the well-established effects of androgens on erythropoiesis (Kennedy and Gilbertsen, 1957
), we assessed erythroid progenitor cell levels, as these cells have been demonstrated to augment angiogenesis (Sasaki et al., 2007
). Gonadal status had no effect on the numbers of granulocyte/macrophage progenitors present in the bone marrow or the spleen (). However, consistent with the enhanced recovery from ischemia observed in orchidectomized mice treated with DHT, erythroid progenitors were significantly elevated in these mice in both the bone marrow (29.5 ± 2 vs. 20.5 ± 1 erythroid CFUs/105
MNCs for orchidectomized + DHT vs. control, respectively; P < 0.01 using ANOVA; ) and the spleen (10.6 ± 1 vs. 5.5 ± 1 erythroid CFUs/105
MNCs for orchidectomized + DHT vs. control, respectively; P < 0.01 using ANOVA; ). These data suggest that the enhanced recovery from hindlimb ischemia seen in orchidectomized males receiving DHT may be mediated in part by increased mobilization of erythroid progenitors. In toto, the data presented in this report suggest that endogenous androgens are necessary in the coordination of neovascularization in response to critical ischemia and that consequent proangiogenic effects of DHT in this context are mediated in part through increased angiogenic/progenitor cell mobilization.
In summary, we report a sex-specific role for androgens in angiogenesis. Androgens stimulate key angiogenic events in male but not female cells in vitro, and these sex-specific proangiogenic effects are mediated via the AR. In addition, we report that endogenous androgens modulate angiogenesis in males but not females in vivo. Moreover, in males, endogenous androgens are involved in the coordination and enhancement of neovascularization in the context of ischemic injury. Our findings suggest that androgens regulate vascular regeneration in a sex-dependent manner. Given the age-related decline of androgens, our findings have implications for the role of androgen replacement in men. Additionally, these data may explain in part some of the observed sex differences in the outcomes of CVD.