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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC May 25, 2013.
Published in final edited form as:
PMCID: PMC3518797
NIHMSID: NIHMS386006
Transient Exposure of Neonatal Female Mice to Testosterone Abrogates the Sexual Dimorphism of Abdominal Aortic Aneurysms
Xuan Zhang,1 Sean E. Thatcher,2 Debra L. Rateri,3 Dennis Bruemmer,2,3 Richard Charnigo,4 Alan Daugherty,1,2,3 and Lisa A. Cassis1,2
1Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA
2Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, USA
3Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA
4Department of Statistics, University of Kentucky, Lexington, KY, USA
Corresponding author: Lisa Cassis, Ph.D., Professor and Chair, Graduate Center for Nutritional Sciences, University of Kentucky, Room 521b Wethington Building, 900 S. Limestone, Lexington, KY 40536-0200, Tel: (859)-323-4933 ext 81400, Fax: (859)-257-3646, lcassis/at/uky.edu
Rationale
Abdominal aortic aneurysms (AAAs) exhibit marked sexual dimorphism with higher prevalences in men. Similarly, AAAs induced by angiotensin II (AngII) infusion into mice exhibit a higher prevalence in males. Testosterone promotes AAA pathology in adult male mice through regulation of angiotensin type 1A receptors (AT1aR) in abdominal aortas. However, mechanisms for sexual dimorphism of regional aortic angiotensin receptor expression and AAA formation are unknown.
Objective
To define the role of developmental testosterone exposures in sexual dimorphism of AAAs, we determined if exposure of neonatal female mice to testosterone confers adult susceptibility to AngII-induced AAAs.
Methods and Results
One day old female hypercholesterolemic mice were administered a single dose of either vehicle or testosterone. Neonatal testosterone administration increased abdominal aortic AT1aR mRNA abundance and promoted a striking increase in AngII-induced AAAs in adult females exhibiting low serum testosterone concentrations. AngII-induced atherosclerosis and ascending aortic aneurysms were also increased by testosterone administration to neonatal females. In contrast, neonatal testosterone administration in males had no effect on AngII-induced vascular pathologies. Deficiency of AT1aR in smooth muscle cells (SMCs) reduced effects of neonatal testosterone to promote AAAs in adult females, but did not alter atherosclerosis or ascending aortic aneurysms. Testosterone increased AT1aR mRNA abundance and hydrogen peroxide generation in cultured abdominal aortic SMCs. Increased AT1aR mRNA abundance was maintained during progressive passaging of female SMCs.
Conclusions
These data reveal an unrecognized role of transient sex hormone exposures during neonatal development as long-lasting mediators of regional aortic AT1aR expression and sexual dimorphism of AAAs.
Keywords: aneurysm, sexual dimorphism, testosterone, vascular smooth muscle, angiotensin
Abdominal aortic aneurysms (AAA) are permanent dilations of the abdominal aorta with an 85% chance of death after rupture.1-3 Recent estimates of AAA prevalence are 1.1 million people in the US aged 50 to 84.4 As the population lives longer, it is anticipated that AAA prevalence will increase, which is of concern since there is no proven medical therapy to change aneurysm growth or rupture. A variety of risk factors influence AAA formation and progression including age,5, 6 smoking,7-9 obesity,10, 11 and gender.12, 13 Several studies have demonstrated that male sex is a prominent risk factor for human AAAs,6, 14, 15 with recent results from the Tromsǿ study estimating that males are 2.66 times as likely as females to develop AAAs.15 Despite a strong influence of male sex on AAA formation, mechanisms contributing to increased AAA formation in males are unclear.
Angiotensin II (AngII) infusion into hypercholesterolemic mice increased atherosclerosis and induced formation of aneurysms in both the ascending and suprarenal aortas of male mice.16-18 Notably, the renin-angiotensin system has been clinically implicated in the etiology of atherosclerosis, ascending aortic aneurysms, and AAAs.19, 20 In mice, these AngII-induced vascular pathologies are mediated through angiotensin type 1a receptors (AT1aR).21-23 While each of these vascular pathologies are induced by infusion of AngII, only AAAs exhibit marked sexual dimorphism with a higher prevalence (4-fold) in male compared to female mice.16, 17, 24, 25 Testosterone was determined to be a primary mediator of higher AAA prevalence in adult AngII-infused male mice.25, 26 As a mechanistic target of testosterone to promote AngII-induced AAAs, AT1aR mRNA abundance was greater in abdominal compared to thoracic aortas of male, but not female mice. This regional difference was abolished by orchiectomy and restored in castrated males by androgen administration.26 However, the mechanistic contributions of the renin-angiotensin system to the sexual dimorphism of AAA formation have not been defined.
Sexual dimorphism has been studied extensively in several organ systems. For example, sexual dimorphism of the mammalian brain has been examined by exposing females to testosterone during the neonatal period.27 This androgenization imposes upon females transient exposures to testosterone shortly after birth, and mimics developmental effects of androgen in males during the neonatal period.28, 29 Sex hormone differences between adult males and females have been implicated as mediators of sexual dimorphism of several cardiovascular diseases. However, the role of sex hormone exposures during neonatal development on vascular diseases that display pronounced sexual dimorphism, such as AAAs, has not been defined. Moreover, mechanisms for region-specific regulation of AT1aR expression by testosterone in different aortic regions are unknown, but may relate to embryonic origins of vascular wall cells and epigenetic influences of sex hormone exposures during critical periods of development. In this study, we sought to quantify effects of transient exposures of neonatal females to testosterone on regional expression of aortic AT1aR and adult susceptibility to AngII-induced vascular diseases. Remarkably, we found that exposure of 1 day old female mice to a single dose of testosterone increased AT1aR mRNA abundance that was restricted to abdominal aortas of adult females. In addition, females exposed to testosterone as neonates exhibited a striking increase in adult susceptibility to AngII-induced AAAs, atherosclerosis and ascending aortic aneurysms. Deficiency of AT1aR in smooth muscle cells (SMCs) reduced effects of neonatal testosterone to promote AngII-induced AAAs in adult females, but had no significant impact on the effects of neonatal testosterone in promoting atherosclerosis or ascending aortic aneurysms. Testosterone promoted AT1aR mRNA abundance in SMCs from abdominal, but not thoracic aortas of male and female mice. Notably, effects of testosterone to promote AT1aR mRNA abundance were heritable in passaged abdominal aortic SMCs from female, but not male mice. Our findings indicate that transient exposure of neonatal female mice to testosterone abrogates the sexual dimorphism of AngII-induced AAAs.
Mice
Female ApoE-/- and LDLR-/- mice (both N10 C57BL/6 background) were bred to males in house. Within 24 hours of birth, female mice were injected once with either vehicle (corn oil) or testosterone propionate (100 or 400 μg/mouse, s.c., Sigma Aldrich, St. Louis, MO). Female ApoE-/- and LDLR-/- mice administered vehicle or testosterone propionate (400 μg/mouse) as neonates were maintained on standard diet and terminated at 5 months of age for aortic gene analysis and aortic vascular morphology measurements without AngII infusion. In separate studies, 3 month old female mice (ApoE-/-, LDLR-/-, AT1aRfl/fl, AT1aRSM22 KO on an LDLR-/-background) administered vehicle or testosterone (400 μg/mouse) as neonates were infused with either saline or AngII (500, 750 or 1,000 ng/kg/min, Bachem, Torrance, CA) using osmotic pumps (Alzet, model 1004, Durect Co., Cupertino, CA) for 4 weeks. For studies in male mice, 1 day old male ApoE-/- or LDLR-/- mice were injected with either vehicle or testosterone propionate (400 μg/mouse) and then infused with AngII (500 or 750 ng/kg/min for 4 weeks) at 3 months of age. In separate studies, female LDLR-/- mice were injected with a lower dose of testosterone (100 μg/mouse) within 24 hours of birth, and then infused with AngII (1,000 ng/kg/min for 28 days) at 2 months of age. ApoE-/- mice were maintained on standard diet; LDLR-/- mice were fed a high fat diet containing 21% milk fat and 0.2% cholesterol (TD88137, Harlan Teklad, Indianapolis, IN) 1 week prior to pump implantation and throughout the study. All experiments were performed in accordance with the University of Kentucky Institutional Animal Care and Use Committee.
Generation of AT1aRSM22 KO mice
As described previously, AT1aRfl/fl were generated by InGenious Targeting Laboratory (Stony Brook, NY) directly in ES cells of C57BL/6 mice and subsequently crossed to LDLR-/- mice.1 SM22-Cre mice (The Jackson Laboratory, Tg(Tagln-cre)1Her/J, #004746, Bar Harbor, ME) were bred to LDLR-/- mice and were identified to be the equivalent of N10. Details regarding breeding of experimental mice are provided in the Online Supplement.
Quantification of atherosclerosis, AAA and ascending aortic aneurysms
Atherosclerosis was quantified in the aortic arch and sinus, details are provided in the Online Supplement. AAAs were defined as > 50% dilation of lumen diameters from saline-infused mice. The extent of ascending aortic dilation as an index of ascending aortic aneurysms was quantified by measuring aortic arch intimal areas (to 3 mm distal to the subclavian branch) and ascending aortic diameters.18 The incidences of ulceration in ascending aortas were assessed by observers who were blinded to the experimental design. Details regarding quantification of vascular pathologies are provided in the Online Supplement.
All other procedures used in this study were described previously, although an expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Abdominal aortic AT1aR mRNA abundance was increased in adult females administered testosterone as neonates
The embryonic origins of cells can influence site-specific gene expression patterns in different aortic regions.30, 31 To define potential gene targets in aortic regions that are influenced by transient exposures to testosterone during development, we administered a single dose of either testosterone or vehicle to neonatal LDLR-/- mice within the first 24 hours after birth. This dosing regimen was chosen to mimic testosterone surges in neonatal males.28, 29 Targeted PCR arrays in aortas from adult females examined 40 genes that were implicated previously in both AngII-induced AAAs in mice 32 and human AAAs (Online Table I). Of the 40 genes examined, 16 (40%) were differentially expressed between thoracic and abdominal aortas of adult female LDLR-/- mice administered vehicle as neonates (Online Table II). The majority of these genes continued to be expressed at different levels in thoracic compared to abdominal aortas of adult female LDLR-/- mice administered testosterone as neonates (Online Table III). In aortas from adult females administered testosterone as neonates, 8 genes (20%) exhibited differences compared to vehicle in abdominal (5 genes) or thoracic (3 genes) aortas (Online Table IV). Notably, AT1aR mRNA abundance was increased in abdominal, but not in thoracic aortas from adult females administered testosterone as neonates. Since this receptor mediates AngII-induced atherosclerosis, ascending aortic aneurysms and AAA formation in adult male mice,21-23 we used RT-PCR to confirm region-specific increases in AT1aR mRNA abundance in abdominal aortas from adult ApoE-/- and LDLR-/- female mice that were administered vehicle or testosterone within 24 hours after birth. As reported previously,26 AT1aR mRNA abundance was not significantly different between thoracic and abdominal aortas of adult female ApoE-/- or LDLR-/- mice administered vehicle as neonates (Figure 1A and B, respectively). In contrast, administration of testosterone to neonatal females significantly increased AT1aR mRNA abundance in abdominal, but not in thoracic aortas of adult female mice of each strain (Figure 1A and B). Thus, transient exposures of neonatal female mice to testosterone imparted a long-lasting increase in AT1aR mRNA abundance specifically in abdominal aortas of adult females. Unfortunately, we were unable to quantify AT1aR protein expression in aortas due to a lack of antibodies exhibiting specificity to AT1aR.23
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Administration of testosterone to neonatal females increased AT1aR mRNA abundance in abdominal aortas from adult female ApoE-/- and LDLR-/- mice, but did not change aortic vascular morphology or contractile function in adult females. (A,B) AT1aR mRNA (more ...)
To determine whether neonatal exposures to testosterone changed aortic structure and/or function, we quantified morphology (medial thickness, Figure 1C and D) of selected regions of aortas from adult LDLR-/- females that were administered either testosterone or vehicle as neonates. Medial thickness was greatest in ascending aortas from adult females administered either testosterone or vehicle compared to other aortic regions (Figure 1D). However, medial thickness was not significantly influenced by neonatal administration of testosterone. We also quantified contractile responses of different aortic regions to 5-hydroxytryptamine (5-HT) or AngII (Figure 1E and F). Contractile responses to 5-HT were greatest in suprarenal aortic segments from mice in each group (Figure 1E), while responses to AngII were greatest in infrarenal aortas (Figure 1F). However, there were no significant differences in contractile responses to either agonist in aortic ring segments extracted from adult females administered testosterone within 24 hours after birth compared to vehicle controls. While it may appear surprising that the contractile response to AngII was not influenced by neonatal exposures to testosterone given the ability of the hormone to increase AT1aR mRNA abundance, previous investigators inferred that AT1bR, rather than AT1aR mediate aortic smooth muscle contractions.33 In addition, mRNA abundance of AT1bR was greater in abdominal compared to thoracic aortas of adult female mice administered either vehicle or testosterone as neonates, with no differences in expression of this angiotensin receptor subtype between females administered vehicle or testosterone as neonates (Online Tables I-III). Elevated expression of AT1bR in abdominal aortas is consistent with increased contractile responses to AngII in this aortic region (Figure 1F). These results demonstrate that administration of testosterone to neonatal females had no overt effects on aortic morphology or contractile function.
AngII-induced AAAs were strikingly increased in adult females administered testosterone as neonates
We next investigated if elevated AT1aR mRNA abundance in abdominal aortas of adult females administered testosterone as neonates translated into increased susceptibility to AngII-induced AAAs. Adult ApoE-/- females administered vehicle or testosterone as neonates were infused with either saline or AngII (1,000 ng/kg/min) for 28 days. In agreement with previous reports,34, 35 neonatal administration of testosterone resulted in a modest but significant increase in body weights of adult females (Online Table V for AngII-infused groups). However, there was no significant effect of testosterone on systolic blood pressure, blood monocyte counts, serum cholesterol concentrations (Online Table V), or lipoprotein cholesterol distribution (Online Figure I) in adult females infused with AngII at different rates. Notably, serum testosterone concentrations were not significantly elevated in adult female mice administered testosterone as neonates (Online Table V), and were 7-fold lower than those observed previously in adult male ApoE-/- mice.25
In saline-infused adult female ApoE-/- mice, neonatal administration of testosterone had no significant effect on maximal aortic diameters of suprarenal abdominal aortas (Figure 2B). AAA incidence, which included mice that died from aneurysmal rupture, was increased dose-dependently by AngII in female mice administered vehicle as neonates (Figure 2A). However, it did not rise above 21% at the highest infusion rate (1,000 ng/kg/min) of AngII. Infusion of AngII (1,000 ng/kg/min) to adult females administered vehicle as neonates resulted in modest but significant increases in external diameters of abdominal aortas (Figure 2B), indicative of aneurysm formation in a small percentage of mice (Figure 2A and 2C). In contrast, AAA incidence was strikingly elevated with increasing infusion rates of AngII in adult females administered testosterone as neonates compared to vehicle-administered controls (Figure 2A), with a 3-fold increase in AAA incidence at infusions of 1,000 ng/kg/min of AngII (21% vs. 64%; P<0.05). In addition, adult female mice exposed to testosterone as neonates exhibited striking increases in external diameters of abdominal aortas with pronounced aneurysmal pathology similar to that observed previously in adult males (Figure 2A-C).24, 25 An increase in mortality from aneurysmal rupture contributed to higher AAA incidences in females administered testosterone as neonates (rupture: vehicle, 7%; testosterone, 20%). These results demonstrate that transient exposure of neonatal females to testosterone abrogates the sexual dimorphism of AngII-induced AAAs in adult females that exhibited low concentrations of serum testosterone.
Figure 2
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Administration of testosterone to neonatal females significantly increased AngII-induced AAAs in adult female ApoE-/- mice. (A) AAA incidences in mice infused with 500, 750 or 1000 ng/kg/min of AngII for 28 days (N = 10-25/group). *P<0.05 compared (more ...)
We also examined effects of the same dose of testosterone administered to 1 day old male ApoE-/- mice on susceptibility to AngII-induced vascular diseases as adults. In contrast to findings in females, AAA incidence and maximal external diameters were not altered in adult male mice administered testosterone as neonates (Online Figure II, A and B). These results suggest that in male mice, with the natural occurrence of neonatal androgen surges, the dose of testosterone administered in this study did not impose supraphysiological effects on AngII-induced AAAs.
SMC deficiency of AT1aR blunted the effects of neonatal testosterone exposure to promote AngII-induced AAAs
AT1aRs are expressed in several cell types within the vascular wall. Previous studies examining temporal changes in aneurysmal pathology with AngII infusion demonstrated elastin degradation within the medial layer of abdominal aortas early in AngII-induced AAAs.36 These results suggested that testosterone exposures in neonatal females may regulate AT1aR mRNA abundance within SMCs of the abdominal aorta to promote AngII-induced AAA formation. Therefore, we investigated if SMC-specific AT1aR deficiency reduced the effect of neonatal testosterone in promoting AngII-induced AAAs. As described previously,23 we used Cre-LoxP technology to generate mice with deficiency of AT1aR in SMCs. Mice were engineered with loxP sites flanking exon 3 of AT1aR which includes the entire coding region (Online Figure III, A). AT1aRfl/fl females on an LDLR-/- background were mated to male mice expressing Cre recombinase under the control of an SM22 promoter to generate smooth muscle cell-specific AT1aR deficient mice (AT1aRSM22 KO) and wild type littermates (AT1aRfl/fl).23, 37, 38 Depletion of AT1aR from SMCs was verified by PCR of genomic DNA from aortas (Online Figure III, B). Female AT1aRfl/fl control and AT1aRSM22 KO mice were administered a single dose of vehicle or testosterone within 24 hours of birth, and then infused with AngII at 2 months of age. Notably, neonatal administration of testosterone, as well as SMC-specific AT1aR deficiency, had no significant effect on baseline systolic blood pressures, AngII-induced hypertension,23, 39 plasma aldosterone concentrations, serum cholesterol concentrations (Table 1) or serum lipoprotein cholesterol distributions (Online Figure IV). Moreover, serum testosterone concentrations did not significantly differ across study groups (Table 1).
Table 1
Table 1
Characteristics of adult female AT1aRfl/fl and AT1aRSM22 KO administered either vehicle or testosterone as neonates.
Similar to findings from adult ApoE-/- females exposed to testosterone within 24 hours of birth, adult AT1aRfl/fl LDLR-/- females administered testosterone as neonates exhibited a striking increase in the incidence (Figure 3A) and size (Figure 3B and 3C) of AngII-induced AAAs. Interestingly, SMC specific AT1aR deficiency significantly reduced, but did not ablate effects of neonatal testosterone to promote AngII-induced AAAs (Figure 3A-C). Notably, even though AAA incidence was significantly decreased in AT1aRSM22 KO females administered testosterone as neonates, females of each genotype that exhibited an AAA developed aneurysms of similar sizes (Figure 3D). Moreover, we examined tissue characteristics of AAAs of equivalent sizes that formed in adult female mice of each genotype exposed to testosterone as neonates. In both genotypes, AAAs exhibited typical characteristics of pronounced adventitial thrombus, elastin degradation within the aortic media and pronounced lumen dilation (Figure 3E). These data demonstrate that exposure of neonatal females to testosterone results in a long-lasting increase in adult susceptibility of females to AngII-induced AAAs partially through smooth muscle cell AT1aR.
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SMC-specific AT1aR deficiency reduced AngII-induced AAAs in adult female mice administered testosterone as neonates. AAA incidence (A) and maximal external diameters (B) of abdominal aortas in female mice infused with AngII (1000 ng/kg/min) for 28 days. (more ...)
AngII-induced atherosclerosis and ascending aortic aneurysms were increased in adult females administered testosterone as neonates, but SMC deficiency of AT1aR had no effect
We also quantified ascending aortic aneurysms18 and atherosclerosis17 as additional vascular pathologies induced by infusion of AngII that do not display sexual dimorphism between adult males and females.25, 26 In females administered vehicle as neonates, there was no significant effect of SMC AT1aR deficiency on AngII-induced atherosclerosis in aortic sinuses (Figure 4A) or arches (Online Figure V, A). Surprisingly, testosterone administration to neonatal female mice resulted in pronounced increases in AngII-induced atherosclerosis in AT1aRfl/fl and AT1aRSM22 KO adult female mice (Figure 4A and Online Figure V, A). However, deficiency of AT1aR in SMCs did not significantly influence the ability of neonatal testosterone administration to promote AngII-induced atherosclerosis (Figure 4A-C). Lesional phagocytes, as defined by quantification of CD68 immunostaining in aortic sinus sections from adult females, were increased significantly by neonatal administration of testosterone in lesions from AT1aRfl/fl and AT1aRSM22 female mice (Figure 4B). Moreover, cellular compositions (phagocytes, SMCs) of atherosclerotic lesions in aortic sinus sections were similar between AT1aRfl/fl and AT1aRSM22 female mice administered testosterone as neonates (Figure 4C).
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Testosterone administration to neonatal females increased AngII-induced atherosclerosis and ascending aortic aneurysms in adult AT1aRfl/fl and AT1aRSM22 KO female LDLR-/- mice. (A) Quantification of atherosclerotic lesions stained with Oil Red O in aortic (more ...)
In addition to promoting atherosclerosis, neonatal testosterone administration to female LDLR-/- mice augmented AngII-induced increases in ascending aortic diameters (Figure 4D) and ascending aortic intimal areas (Online Figure V, B), indicative of ascending aortic aneurysms.18 Cross sections from ascending aortas of female mice exposed to testosterone as neonates and then infused with AngII as adults exhibited significant medial thickening (Figure 4E and F) and aortic ulceration (Online Figure V, C and D). Macrophage immunostaining was prominent in media and adventitia of aortic arch sections from AngII-infused adult female mice administered testosterone as neonates (Figure 4E), similar to results previously observed in ascending aortas of adult male mice.18 SMC deficiency of AT1aR did not significantly influence the ability of neonatal testosterone administration to promote AngII-induced aneurysms in the ascending aorta (Figure 4D-F). Thus, SMC AT1aR deficiency resulted in a selective reduction in AngII-induced AAAs, but not atherosclerosis or ascending aortic aneurysms, in female mice administered testosterone as neonates.
Neonatal female mice were more sensitive compared to neonatal males to testosterone exposures to promote AngII-induced vascular pathologies
Our approach has been to attempt to mimic neonatal surges of testosterone in male mice shortly after birth28, 29 and superimpose similar systemic concentrations of testosterone on neonatal females. It is technically challenging to measure serum testosterone concentrations in 1 day old male and female mice to confirm whether we had indeed achieved similar circulating testosterone concentrations between males and females. Since administration of testosterone to neonatal ApoE-/- males had no significant effect on adult male susceptibility to AngII-induced AAAs, it is likely that the dose of testosterone was not supra-physiologic in addition to endogenous neonatal surges of testosterone. However, as an alternative approach, we determined if administration of a 4-fold lower dose of testosterone (100 μg) to 1 day old female LDLR-/- mice was sufficient to confer increased adult susceptibility to AngII-induced AAAs. We also administered a higher dose (400 μg) of testosterone to neonatal LDLR-/- male mice and then defined adult sensitivity to AngII-induced AAAs. AAA incidence (from 30 to 64%, Figure 5A) and size (Figure 5B and 5C) increased in adult female LDLR administered the lower dose of testosterone as neonates. Atherosclerosis was increased significantly in aortic sinus sections from adult AngII-infused females administered the lower dose of testosterone as neonates (Figure 5D). In addition, aortic arch areas (vehicle: 17.6 ± 0.8; testosterone: 20.4 ± 0.8 mm2; P<0.05) and ulceration of the aortic arch (vehicle: 20%; testosterone: 57%) were significantly increased in females administered the lower dose of testosterone as neonates. In contrast, male LDLR-/- mice exhibited no response to a higher dose (400 μg) of neonatal testosterone exposure in the development of AngII-induced AAAs, atherosclerosis and ascending aortic aneurysms (Online Figure VI, A-C). These results demonstrate pronounced sensitivity of neonatal females to testosterone to promote a long-lasting increased susceptibility to AngII-induced vascular diseases.
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Administration of a 4-fold lower dose (100 μg/mouse) of testosterone to neonatal females significantly increased AngII-induced AAAs in adult female LDLR-/- mice. (A) AAA incidences (N =10-14 mice/group). (B) Representative aortas from adult female (more ...)
Testosterone-induced increases in AT1aR mRNA abundance in SMCs were region-specific and sexually dimorphic
Neonatal testosterone exposures exerted a region-specific effect to promote a long-lasting increase in AT1aR mRNA abundance in abdominal aortic SMCs to increase susceptibility to AngII-induced AAAs in adult females. To define mechanisms for these effects, we quantified androgen receptor mRNA abundance in thoracic and abdominal aortas of adult male and female ApoE-/- mice. Interestingly, similar to regional differences in AT1aR expression (Figure 1A and 1B), androgen receptor mRNA abundance was expressed at a significantly higher abundance in abdominal compared to thoracic aortas of male and female mice (Online Figure VII). To determine if regional differences in androgen receptor expression abundance arose from SMC cells within different aortic regions, we cultured SMCs from thoracic and abdominal aortic regions of adult male and female mice. Notably, androgen receptor mRNA and protein expression were significantly increased in SMCs cultured from abdominal compared to thoracic aortas from male and female mice (Figure 6A and 6B). Furthermore, testosterone significantly increased AT1aR mRNA abundance in primary cultures of SMCs from abdominal, but not thoracic aortas of male and female mice (Figure 6C and 6D). These results suggest that regional differences in androgen receptor expression contribute to site-specific increases in abdominal aortic SMC AT1aR expression by testosterone.
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Regional specificity and sexually dimorphic heritability of testosterone-induced increased in AT1aR expression in SMCs. (A) Androgen receptor mRNA abundance in primary cultured SMCs from thoracic and abdominal aortas of male and female mice. *, P<0.01 (more ...)
Since heritability is a characteristic of epigenetic gene regulation, we examined heritability of testosterone-induced increases in AT1aR mRNA abundance in passaged SMCs from abdominal aortas of male and female mice. After incubation of abdominal aortic SMCs with vehicle or testosterone for 24 hours, cells from both male and female mice were passaged to proliferate for one additional generation. Elevated AT1aR mRNA abundance persisted in passaged abdominal SMCs from female, but not male mice (Figure 6E). These results demonstrate sexual dimorphism of testosterone-induced regulation of AT1aR expression in abdominal SMCs.
To determine if testosterone-induced increases in AT1aR mRNA abundance in abdominal aortic SMCs result in increased functional responses to AngII, we examined AngII-induced hydrogen peroxide (H2O2) generation in testosterone-treated SMCs cultured from thoracic and abdominal aortas of female mice. Incubation of thoracic and abdominal aortic SMCs with AngII significantly increased H2O2 generation, which was abolished by losartan (Figure 6F and 6G). In abdominal, but not thoracic aortic SMCs previously exposed to testosterone, AngII-induced H2O2 generation was increased compared to vehicle (DMSO). These results demonstrate that testosterone-induced increases in AT1aR mRNA abundance specifically in abdominal aortic SMCs result in heightened regional-specific functional responses to AngII.
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.
Novelty and Significance
What is Known?
  • Abdominal aortic aneurysms (AAAs) in humans and AngII-induced AAA in mice exhibit pronounced sexual dimorphism with greater prevalence in males than in females.
  • In adult mice, testosterone increases angiotensin type 1a receptor (AT1aR) in abdominal aortas where the AAAs form. It also increases the incidence of AAAs in male mice.
What New Information Does this Article Contribute?
  • Exposure of female mice to a single dose of testosterone within 24 h of birth, to mimic testosterone surges in neonatal males, has a long lasting effect in increasing abdominal aortic AT1aR expression and AngII-induced AAAs in adult females. It also increases AngII-induced atherosclerosis and ascending aortic aneurysms.
  • Smooth muscle cell (SMC)-specific deficiency of AT1aR results in a selective reduction in the neonatal effects of testosterone in promoting AngII-induced AAAs, but it has no effect on atherosclerosis or ascending aortic aneurysms.
  • Heritability of testosterone-induced increases in AT1aR expression in abdominal aortic SMCs from female, but not male, mice suggests that epigenetic mechanisms contribute to sexual dimorphism of testosterone responses.
AAAs exhibit pronounced sexual dimorphism with much higher prevalence in males. Thus, it is important to identify mechanisms underlying sexual dimorphism of AAAs in order to understand the pathogenesis and develop more effective strategies for treating this life-threatening disease. We report here that transient exposures of female mice to testosterone shortly after birth induces a long-lasting increase in AT1aR mRNA abundance in abdominal aortas and a striking increase in susceptibility to AAA during adulthood, Transient testosterone exposures in neonatal females promoted atherosclerosis and ascending aortic aneurysms. Our results demonstrate that deletion of AT1aR alleles in SMCs resulted in a selective decrease in AAAs, but not atherosclerosis or ascending aortic aneurysms induced by exposures of neonatal females to testosterone. Finally, testosterone exerted a region-specific increase in abdominal aortic SMC AT1aR expression that was heritable in passaged cells from female, but not male mice. Regional differences in androgen receptor expression with increased localization to abdominal aortic SMCs may have contributed to site-specific effects of testosterone.
Supplementary Material
Acknowledgments
We thank Jessica Moorleghen for technical assistance with aortic contractility measurement, breeding and administration of testosterone to neonatal female mice, Anju Balakrishnan for assistance with mouse breeding, Deborah Howatt for tissue pathology and FPLC, and Victoria English for technical assistance with biochemical assays.
Sources of Funding
This study was funded by research grants (HL08100, HL107326, LAC, AD) from the National Institutes of Health. Morphologic assessment of aortic segments was performed through a pathology core supported by an NIH NCRR Center of Biomedical Research Excellence (P20RR021954; LAC).
Non-standard Abbreviations and Acronyms
AngIIAngiotensin II
AAAsAbdominal aortic aneurysms
AT1aRAngiotensin type 1a receptor
ApoEApolipoprotein E
LDLRLow density lipoprotein receptor

Footnotes
Disclosures
None.
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