Downregulation of Emilin1 and Thbs2 Contributes to Aortic Structural Stiffness of the XX Female Four Core Genotype Mice

Abstract

Background: The aorta comprises endothelial cells in the tunica intima, and smooth muscle cells in the tunica intima intertwine with elastin fibers and fibroblast cells. A network of extracellular matrix (ECM) proteins, including collagen, integrins, and adhesion proteins, provide rigidity and support the vessel. Protein deficiencies within the ECM compromise the structural integrity, which can foster an array of vascular pathologies, including aneurysms and atherosclerosis. However, ECM disorganization can occur due to vascular remodeling in mice, which is dependent on age and sex. Sex differences between females and males can be hormonal (estradiol versus testosterone) or chromosomal (XX versus XY). Studies have shown that females, compared to males, exhibit accelerated arterial stiffness with aging. The decline in sex hormones promotes arterial stiffening in male and female mice. However, whether the driving factor in arterial stiffening is sex chromosome complement (XX versus XY), sex hormone-dependent, and their interaction is unknown. Methods: To determine the contribution of sex hormones and sex chromosomes or their interaction to arterial stiffening, we used the Four-Core genotype (FCG) mouse model. FCG mice comprise females and males with either XX or XY sex chromosome complement. First, we performed vaginal swabs in female mice to determine estrous cycles. After euthanasia, we isolated male and female mice descending thoracic to the abdominal aorta. Aortic length, thickness, and diameter were assessed. Next, we used a tissue puller to assess the uniaxial tension of the aorta. Stress-strain and force-displacement curves of the aorta were automatically generated in control, collagenase, and elastase digestion conditions. Aortic histology by Masson’s Trichome (MTC) was used to determine collagen, and Verhoeff-Van Gieson (VVG) was used to determine elastin strands. PCR microarray assessed aorta ECM and adhesive genes, followed by RT-qPCR validation. Results: The estrous cycling did not significantly impact aorta mechanical testing. Stress-strain tests showed an increased leftward shift in the following order: XXF, XYF, XXM, and XYM, respectively, recapitulated by the tensile strength assessed by force-displacement tests. Aortas were enzymatically digested with collagenase to determine collagen contribution to tensile strength. Collagenase digestion indicated a rightward shift in XXF compared to XYF (P<0.0007) and removed any significant differences in XX compared to XYF, XXM, and XYM. Elastase digestion showed a rightward shift in XXF compared to XYF, while no significant difference was detected in the male mice. PCR microarray indicated significant changes in Adamts, Emilin1, Icam, Thbs2, Ctnna1, Ecm1, and Fbln1 genes, while RT-qPCR validation showed a significant decrease in Emilin1, Icam, Thbs2, Ctnna1, Ecm1, and Fbln1 genes in XX mice. Aortic histology data indicated increased Masson’s trichrome staining in XX than XY female mice that was removed by collagenase digestion. Verhoeff-Van Giesen staining showed linearized elastin structures in elastase-digested aortas but not in control and collagenase groups. Conclusion: We show that gonadal intact female FCG mice with XX sex chromosomes complement have increased intrinsic stiffness that was not impacted by the estrous cycle. PCR microarray and RT-qPCR indicated that the Emilin1 gene plays a role in aortic stiffness. New and Noteworthy: For the first time, segregating mice by sex hormones and sex chromosome complement shows increased aortic structural stiffening in XX female mice, partly driven by Emilin1 and Thbs2 genes. Previous studies have shown that Emilin1 null mice are hypertensive and have increased myogenic tone and vascular abnormalities. Additionally, we show a significant decrease in Emilin1 and Thbs2 genes in stretched primary isolated cells from XX female mice. The sex-specific change suggests a unique role of Emilin1 and Thbs2 genes in XX female mice in regulating the aorta’s integrity.