17 β-Estradiol Impedes Aortic Root Dilation and Rupture in Male Marfan Mice

Abstract

Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with worse outcomes in male compared to female patients. In this study, we examine the effects of 17 β-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 β-estradiol, and the growth in the aortic root, along with the risk of aortic rupture, was measured. Transcriptomic profiling was used to identify enriched pathways from 17 β-estradiol treatments. Aortic smooth muscle cells were then treated with cytokines to validate functional mechanisms. We show that 17 β-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture. The Marfan transcriptome was enriched in inflammatory genes, and the addition of 17 β-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. In addition, 17 β-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro in an NF-κB dependent manner, and 17 β-estradiol decreased the formation of adventitial inflammatory foci in aortic roots in vivo. In conclusion, 17 β-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα-NF-κB signaling.

Keywords: Marfan; TNFα, NF-κB; aneurysm; estrogen.

Figure 1

17 β-estradiol attenuates aneurysm growth in male Marfan mice. (A) Aortic roots measured every two weeks from 8-week-old wild-type male (n = 3), wild-type female (n = 3), wild-type male + 17 β-estradiol (n = 3), wild-type female + 17 β-estradiol (n = 3), Marfan male (n = 10), Marfan male + 17 β-estradiol (n = 13), Marfan female (n = 5), and Marfan female + 17 β-estradiol (n = 6) mice. Data are mean +/− standard error. * denotes p < 0.05 comparing Marfan male to Marfan male + 17 β-estradiol at each time point beyond 2 weeks. (B) The ratio of aortic root diameter at eight weeks over baseline. Data are median and interquartile range. Mouse cohorts as in (A). (C) Representative aortic images from male wild-type, Marfan, and Marfan + 17 β-estradiol mice. (D) Representative H + E, trichome, and elastin sections. Mouse cohorts as in (C). Scale bar = 90 μm. (E) Western blot with quantification of aortic root lysates from male wild-type (n = 3), Marfan (n = 5), and Marfan + 17 β-estradiol (n = 5) mice probed for matrix metalloproteinases and normalized to vinculin expression. E2: 17 β-estradiol, MFS: Marfan syndrome, Sham: sham surgery, WT: Wild-type.

Figure 2

17 β-estradiol protects against aortic rupture in Marfan male mice treated with angiotensin II. (A) Survival curve of Marfan male, Marfan female, wild-type male, wild-type female, and Marfan male mice pretreated with four weeks of 17 β-estradiol following continuous infusion of angiotensin II. (B) Representative pictures of thoracic (left) and abdominal (right) aortic rupture in Marfan male mice treated with continuous infusion of angiotensin II. (C) Percentage of aortic pathologies found on gross and histological examination of aortas from angiotensin II treated Marfan males in the presence or absence of 17 β-estradiol. AII: angiotensin II, E2: 17 β-estradiol, MFS: Marfan syndrome.

Figure 3

Transcriptomic profiling indicates that 17 β-estradiol blocks TNFα-NF-κB signaling and inflammation (A) Principle component analysis plot from RNAseq performed on aortic root tissue of male wild-type (n = 5), Marfan (n = 5), Marfan treated with eight weeks of 17 β-estradiol (n = 5), and seven days of angiotensin II treated wild-type (n = 4), Marfan (n = 4), and Marfan mice treated with five weeks of 17 β-estradiol (n = 4). (B) Gene enrichment analysis of the hallmark gene set TNFα signaling via NF-κB comparing Marfan mice to a cohort of combined wild-type and Marfan mice treated with 17 β-estradiol (left). The same analysis in angiotensin II treated mice (right). (C) Dot plot of transcription factors with targets enriched in Marfan mice compared to Marfan mice with 17 β-estradiol using the TRRUST database (top). The same analysis was in angiotensin II-treated mice (bottom). (D) Normalized read counts of Mcp-1, Vcam-1, Lgasl3, Il-6, Cxcr4, and Cxcl5 gene expression from the same mouse cohorts outlined in (A). Box plots describe quartiles. Adjusted p-values from DESeq2 for pairwise comparisons are depicted in charts. AII: angiotensin II, E2: 17 β-estradiol, MFS: Marfan syndrome, WT: wild-type.

Figure 4

17 β-estradiol mitigates TNFα medicated NF-κB gene expression in murine aortic smooth muscle cells (SMCs). (A) Expression of Mcp-1, Vcam-1, Il-6, and C3 in n = 4 independent Marfan male aortic SMC lines pre-treated with 17 β-estradiol or vehicle followed by the addition of TNFα or vehicle. (B) Expression of genes as in (A). following pre-treatment with PDTC or vehicle followed by the addition of TNFα or vehicle. Bar charts display mean +/− standard error. Relative expression is normalized to the cytokine or drug-alone treated group. E2: 17 β-estradiol, PDTC: Pyrrolidine Dithiocarbamate.

Figure 5

17 β-estradiol prevents inflammatory foci in the adventitia of Marfan mice. (A) Top enriched biological process ontology terms from the RNA-seq genes upregulated in Marfan compared to 17 β-estradiol treated Marfan male mice. (B) RNA-seq normalized read counts of CD45 gene expression from male wild-type, Marfan, and 17 β-estradiol-treated Marfan mice. The box plot describes quartiles. Adjusted p-values from DESeq2 for pairwise comparisons are depicted above the chart. (C) Representative images of immunofluorescence staining of aortic roots from the same cohorts described in (B). Tissue is probed for CD45, αSMA, and DAPI nuclear stain. White arrows indicate CD45⁺ inflammatory foci. Scale bars are included below each panel. The far right panel is a higher magnification of overlay. (D) Mean fluorescent intensity of CD45⁺ stain from n = 5 mice in each cohort described in (D). p-values for pairwise comparisons are shown above the chart. E2: 17 β-estradiol, MFS: Marfan syndrome, WT: wild-type.