Our findings reveal that exposure of neonatal female mice to a single dose of testosterone is sufficient to increase the adult susceptibility to AngII-induced AAAs. Notably, exposure of neonatal females to testosterone promoted a regional increase in abdominal aortic AT1aR mRNA abundance in adult females, a mechanism that was suggested to contribute to sexual dimorphism of AngII-induced AAAs between adult male and female mice.24, 25
To gain insights into the cell targets of testosterone during development to increase aortic AT1aR mRNA abundance and enhance AngII-induced AAAs, we performed studies in female mice lacking AT1aR in SMCs. SMC-specific deficiency of AT1aR reduced neonatal effects of testosterone to promote AngII-induced AAAs. Notably, neonatal exposures of female mice to testosterone also increased AngII-induced atherosclerosis and ascending aortic aneurysms, even though these vascular pathologies do not exhibit sexual dimorphism between adult males and females. However, SMC deficiency of AT1aR had no effect on augmented atherosclerosis or ascending aortic aneurysms in adult females exposed to testosterone as neonates. Mechanisms for site-specific effects of testosterone to regulate AT1aR mRNA expression and AngII-induced reactive oxygen species production include differential expression of androgen receptors in SMCs from abdominal compared to thoracic aortas. Moreover, heritable effects of testosterone to promote AT1aR expression in abdominal aortic SMCs from female, but not male mice demonstrate sexual dimorphism of testosterone regulation of AT1aR expression. These results demonstrate that distinct mechanisms mediate effects of neonatal testosterone exposures to promote 3 different AngII-induced vascular pathologies in adult females. Moreover, these studies are the first to demonstrate that transient exposure of neonatal females to testosterone abrogates the sexual dimorphism phenotype of AAAs.
Exposure of neonatal females to testosterone, designed to mimic a surge of testosterone in males shortly after birth,28, 29
has been used extensively by many investigators to study sexual dimorphism of brain structure and function. Permanent changes in brain neurons of females androgenized during development are considered to be “organizational”, allowing for male-like behaviors that persist to adulthood.27
In contrast to studies focused on brain, little is known about developmental effects of testosterone on other traits. We focused on effects of testosterone during neonatal development on regional variations in AT1aR abundance in aortas since previous studies demonstrated a critical requirement of AT1aR for AngII-induced AAAs, atherosclerosis and ascending aortic aneurysms in male mice.18, 22
Moreover, our previous results demonstrated higher expression of AT1aR in abdominal compared to thoracic aortas of male, but not female mice.26
In this study we demonstrated that administration of testosterone to neonatal female mice initiated greater abdominal aortic AT1aR mRNA abundance and markedly enhanced AAA formation in adult females. Interestingly, unlike males,25, 26
exposure of neonatal females to testosterone resulted in long-lasting effects that persisted into adulthood and did not require the continued presence of high concentrations of testosterone in serum. In SMCs cultured from abdominal aortas of female, but not male mice, testosterone promotion of AT1aR expression was heritable, supporting epigenetic mechanisms contribute to sexual dimorphism of testosterone’s effects. Androgen receptors, which exhibited a region-specific increase in abdominal compared to thoracic aortic SMCs in the present study, regulate transcription of target genes by accessing DNA in complexes with co-activators and co-repressors, many of which have enzymatic activities for histone modification.40, 41
Previous studies demonstrated that administration of a histone deacetylase (HDAC) inhibitor to neonatal male mice blocked masculinization of the adult male brain.42
Moreover, administration of an HDAC inhibitor to neonatal female mice that were androgenized by testosterone blocked masculinization of the adult brain.42
These findings suggest that epigenetic effects of androgen receptor stimulation during neonatal development may have contributed to long-lasting increases in vascular disease susceptibility of adult females. Moreover, regional differences in androgen receptor expression in aortic SMCs most likely contributed to site-specific increases in AT1aR expression in abdominal aortas of adult females administered testosterone as neonates.
Our findings demonstrate that male mice experiencing normal surges of testosterone during the neonatal period administered the single dose of testosterone did not respond the same as females exhibiting striking increases in AngII-induced vascular diseases. Thus, it is unlikely that the dose of testosterone administered in these studies was supra-physiologic. Rather, these results suggest that neonatal male and female mice respond differently to testosterone. This hypothesis is supported by augmented AngII-induced atherosclerosis and ascending aortic aneurysms in adult females administered testosterone as neonates, even though adult male and female mice do not display sex differences in these AngII-initiated vascular diseases. Moreover, even though male mice exhibited no response to the higher dose of testosterone during neonatal development, a 4-fold lower dose of testosterone administered to neonatal females promoted their adult susceptibility to AngII-induced vascular diseases. Finally, abdominal aortic SMCs from female, but not male mice exhibited heritability of testosterone regulation of AT1aR. Taken together, these results suggest that responses to testosterone differed between male and female mice. Recent studies support sex chromosomes influence sexual dimorphism of AngII-induced hypertension.43
Similarly, sexual dimorphism of AngII-bradycardiac baroreflex responses were ascribed to differences in sex chromosomes in a 4 core genotype model allowing for dissection of effects of gonadal sex from sex chromosome complement.44
Differences in the complement of genes in XX versus XY cells, or X-linked gene differences may have contributed to differential responses to testosterone between male and female mice.
Our previous results demonstrated medial elastin degradation was an early event in AAA formation in adult male ApoE-/- mice.36
Results from this study demonstrate that testosterone increases AT1aR mRNA expression in primary cultured mouse abdominal aortic SMCs. Moreover, this is the first demonstration that deficiency of AT1aR in SMCs decreased neonatal effects of testosterone to promote AngII-induced AAAs, but had no effect on 2 other vascular pathologies induced by infusion of AngII. Interestingly, in this study, AAAs that did form in female mice with smooth muscle cell-specific AT1aR deficiency had similar size and pathologic characteristics to those of wild type controls. Thus, as opposed to downstream signaling pathways, testosterone may influence initiating events in the formation of AngII-induced AAAs by promoting AT1aR expression in pivotal cell types, including vascular SMCs. Apoptosis of SMCs contribute to AngII-induced AAAs, since administration of a caspase inhibitor reduced medial apoptosis and significantly decreased AAA formation.45
Moreover, genetic deficiency of cyclophilin A, a chaperone protein abundantly expressed in SMCs, abolished AngII-induced AAAs.46
Since effects of testosterone to increase AT1aR mRNA abundance were heritable in SMCs specifically from abdominal aortas of female mice, these results suggest that testosterone exerts epigenetic effects to regulate AT1aR expression in a site-specific and sexually dimorphic manner. Mechanisms for site-specific effects of testosterone to increase abdominal aortic AT1aR expression include greater androgen receptor expression in abdominal aortic SMCs. These results suggest that promotion of AT1aR expression in abdominal aortic SMCs by testosterone influenced the initiating event in AngII-induced AAAs. Of note, increased AT1aR mRNA abundance by testosterone exposures in SMCs isolated from abdominal aortas resulted in enhanced functional responses to AngII.
The diversity of SMC embryonic origins has been suggested to contribute to region-specific aortic pathologies, including those induced by infusion of AngII.47, 48
For example, thoracic and abdominal aortic SMCs respond distinctively to transforming growth factor-β (TGF-β).30
Inhibition of TGF-β has a beneficial effect in a mouse model of ascending aortic aneurysms while inhibition of TGF-β promotes aortic dissection of AngII-induced AAAs. 49, 50
Our results demonstrate that thoracic and abdominal aortas differ in the regulation of expression of several genes, most importantly AT1aR, and that effects of testosterone to increase aortic AT1aR expression were specific to abdominal aortas. Recent results demonstrated that infusion of AngII to C57BL/6 male mice resulted in hyperplasia of SMCs in the ascending aorta, but hypertrophy in other aortic regions.51
Interestingly, despite differences in AT1aR-mediated regulation of SMCs growth along the aortic length, all growth-related responses of SMCs to AngII were abolished in AT1aR deficient mice.51
In studies focused on AT1aR located to endothelial or SMCs as targets of AngII to induce aneurysms in the ascending aorta, results demonstrated that deficiency of AT1aR in endothelial cells, but not SMCs, reduced AngII-induced ascending aortic aneurysm.23
Our results demonstrate that even though testosterone promoted a region-specific increase in AT1aR mRNA abundance to abdominal aortas, all 3 AngII-induced pathologies were increased in neonatal females exposed to testosterone. Moreover, while SMC-specific deficiency of AT1aR reduced effects of testosterone to promote AngII-induced AAAs, ascending aortic aneurysms and atherosclerosis were not influenced by deficiency of AT1aR on this cell type. A lack of effect of SMC AT1aR deficiency to reduce ascending aortic aneurysms in adult females exposed to testosterone as neonates is consistent with recent results obtained from adult male mice lacking SMC AT1aR.23
In conclusion, these data demonstrate that a single dose of testosterone administered to neonatal female mice confers permanently increased susceptibility to AngII-induced AAAs, atherosclerosis and ascending aortic aneurysms in adulthood. An ability of a single dose of testosterone administered to neonatal females to promote three distinct vascular pathologies indicates that short term hormonal exposures at pivotal periods of development can markedly impact vascular disease susceptibility of adults. Moreover, mechanisms for sexual dimorphism of these vascular pathologies are distinct, since smooth muscle AT1aR deficiency reduced the ability of neonatal administration to increase AAAs, but not AngII-induced atherosclerosis or ascending aortic aneurysms in adult females. Heritability of testosterone’s effect to increase SMC AT1aR in abdominal aortic SMCs from female, but not male mice demonstrates sexual dimorphism of SMC responses to testosterone. These results demonstrate that adult AAA susceptibility can be regulated by neonatal exposures to testosterone, indicating a pivotal role for testosterone during critical periods of development as an initiator of the sexual dimorphism of AAAs.