Sex chromosome complement defines diffuse versus focal angiotensin II-induced aortic pathology

Objective-Aortic pathologies exhibit sexual dimorphism, with aneurysms in the ascending, thoracic and abdominal aorta (AAA) exhibiting higher prevalence in males. Despite lower incidence of aortic vascular disease in women, aneurysms progress rapidly. Mechanisms for these sex differences are unclear. We defined the role of sex chromosome complement and testosterone in regional development and progression of angiotensin II (AngII)-induced vascular pathologies. Approach and Results-We used transgenic male mice expressing Sry on an autosome to create low density lipoprotein receptor (Ldlr) deficient male mice with an XY or XX sex chromosome complement. Subjects were then sham operated or orcheictomized. Transcriptional profiling on abdominal aortas from XY or XX males demonstrated1746 genes influenced by sex chromosomes, sex hormones, or an interaction. A second cohort of animals was then infused with AngII for 28 days. Diffuse aortic aneurysm pathology developed in XY AngII-infused males, while XX males developed discrete AAAs. Castration reduced all AngII-induced aortic pathologies in XY and XX males. Thoracic aortas from AngII-infused XY males, but not XX males exhibited adventitial thickening. We infused male XY and XX mice with saline or AngII and quantified mRNA abundance of key genes in thoracic versus abdominal aortas. Regional differences in mRNA abundance existed before AngII infusions, which were differentially influenced by AngII between genotypes. Prolonged AngII infusions resulted in AAA aortic wall thickening in XY males with diffuse aortic pathology, while XX males had dilated focal AAAs. Conclusions-An XY sex chromosome complement mediates diffuse aortic pathology, while an XX sex chromosome complement contributes to discrete AngII-induced AAAs. All studies using mice were approved by the institutional Animal Care and Use Committee at the University of Kentucky and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH. Male transgenic mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were backcrossed 10 times on a C57BL/6J background (Stock#010905, The Jackson Laboratories, Bar Harbor MA) and bred to low density liproprotein receptor deficient (Ldlr-/-) females (Stock# 002207, The Jackson Laboratories, Bar Harbor MA) to generate Ldlr-/- male mice with an XY or an XX sex chromosome complement. DNA was extracted from ear or tail clips and subjected to PCR using a commercial PCR mix (Promega 2X Master Mix, cat#m7123, Fitchburg WI) and specific primers. Mice were fed a western diet (TD88137, Harlan Teklad, Indianapolis, IN) for the duration of the experiment and randomly assigned to sham and orchiectomized (ORC) groups. Two weeks later, anesthetized mice were implanted subcutaneously with osmotic micropumps (Alzet model 1004, Durect Co., Cupertino, CA) for infusion of AngII for 28 days (1,000 ng/kg/min, Bachem, Torrance, CA). At the end of the experiment, mice were euthanatized under anesthesia (ketamine/xylazine, 100:10 mg/kg, i.p.) and blood was collected via cardiac puncture. Orchiectomy. Male (8-12 weeks of age) XX and XY Ldlr-/- mice were orchiectomized under isoflurane anesthesia (2-3%) and given pre and post-operative analgesic (24 hr. after surgery; flunixin; 2.5 mg/kg). Mice were shaved in the scrotum region and a depilatory cream (Nair, Inc.) applied to the area to remove hair, followed by sterilizing with povidone-iodine/ethanol (three times). A small incision was made in this region. Vas deferens were collapsed using a hemostat and the testes were removed. The vascular supply was ligated by cauterization using a high-temperature fine-tip loop cauterizer and the hemostat was released. The wound site was monitored for bleeding, and the skin was closed by wound clips (Autoclip stapler). The site was then treated with povidone-iodine, and mice were allowed to recover. The testes were manipulated but left intact in anesthetized sham-operated mice. All mice were allowed to recover from surgery and to clear their endogenous testicular hormones for two weeks. After two weeks, mice were placed on Western diet (TD88137, Harlan Teklad, Indianapolis, IN) for one week. RNA from abdominal aortas was extracted using the RNeasy fibrous tissue mini kit (Qiagen, cat # 74704). Harvested abdominal aortic RNA quality and quantity were measured by Agilent 2100 Bioanalyzer using RNA 6000 Nano Labchip kits (Agilent Technologies, Cat # 5067-151). One RNA sample (subject #9) was excluded due to poor quality. The remaining 17 samples had excellent quality and did not differ significantly by treatment group (RNA Integrity Number [RIN]: 9.64 ± 0.03; RNA concentration (ng/ul): 55.7 ± 7.4; 28s:18s ratio: 3.00 ± 0.07; all p-values for main effects of gene and surgery, as well as of interaction terms were n.s. [p > 0.1]). Extracted RNA was labeled and hybridized to Affymetrix Mouse Transcriptome Assay 1.0 arrays (one array per subject; N = 17). Signal intensities were calculated using the RMA algorithm (Bolstad et al., 2003) at the transcript level using Genomics Suite software (Partek, St Louis). Data were transferred to flat files in Excel and associated with vendor-provided annotation data. Pre-statistical filtering retained unique, annotated probe sets with adequate signal intensity (signal intensity ≥ 4.2 on at least 3 arrays in the study). Filtered signal intensities were analyzed by two-way ANOVA to identify significant main effects of genotype (XX versus XY), surgery (Sham vs ORC), as well as interaction. The False Discovery Rate (FDR) procedure (Hochberg and Benjamini, 1990) was used to estimate the error of multiple testing.