Gender Differences in Experimental Aortic Aneurysms

假设：我们之前使用弹性蛋白酶灌注和血管紧张素II模型对腹主动脉瘤（AAA）进行的研究表明，雌性啮齿动物动脉瘤形成的发生率和大小都有所减少。我们最近也表明，人胎盘间充质干细胞（MSCs）在AAA小鼠模型中具有保护作用。在目前的提议中，我们将研究饮食植物雌激素作为预防治疗的作用，以及它与MSCs的协同作用作为预防AAA的治疗策略。方法：我们将使用雄性和雌性野生型AAA小鼠，雌激素受体敲除小鼠（ER-α- /-和ER-ß-/-）和ApoE-/-小鼠进行弹性酶灌注和血管紧张素II小鼠模型。将通过富含雌激素或不含雌激素的食物给雄性和雌性小鼠喂食植物雌激素。在AAA小鼠模型中，通过静脉注射或主动脉植入方式，将不同亚群的雌性间充质干细胞（胎盘、骨髓或脂肪来源）注入或不注入雌激素。在第3,7和14天（弹性蛋白酶灌注模型）和第28天（血管紧张素II模型）测量主动脉直径。采集主动脉组织，分析促炎细胞因子（IL-17、TNF-α-、MCP-1、IL-1-ß-、KC和RANTES）和HMGB1(高迁移率组1；ELISA检测促炎核蛋白（促炎核蛋白）产生，酶谱检测MMP2和MMP9活性，western blot检测丝氨酸蛋白酶（uPA、tPA和PAI-1）， ELISA检测旁分泌因子（VEGF、HGF、PGE2），组织学检测弹性蛋白和胶原降解及主动脉平滑肌表达，流式细胞仪检测免疫细胞（巨噬细胞、CD4+ T细胞、中性粒细胞）浸润。结果:初步结果表明，雌性小鼠在弹性酶灌注后的第1、3和14天，雌激素受体（ER-α）的表达明显高于雄性小鼠，人类雌性小鼠的主动脉组织中雌激素受体（ER-α）的表达也明显高于AAA后的雄性小鼠。此外，喂食富含雌激素的雄性小鼠的主动脉直径显著减少，细胞因子（IL-23、IL-1-ß-、IL-6和IL-27）的产生显著减少。MMP2和9表达减少，巨噬细胞和中性粒细胞浸润减少。此外，用人女性间充质干细胞治疗可以减少AAA后主动脉直径、促炎细胞因子的产生和细胞浸润。女性间充质干细胞比男性间充质干细胞更明显地抑制HMGB1和IL-17的产生。此外，雌性间充质干细胞的雌二醇启动对男性AAA患者主动脉组织血管炎症的保护作用显著增强。结论：膳食雌激素治疗和间充质干细胞治疗可减轻AAA小鼠弹性酶灌注模型和AAA患者主动脉组织的动脉瘤形成和炎症。我们建议描述植物雌激素介导的抗炎作用，以及它与不同亚群的性别特异性间充质干细胞的串扰，在小鼠（弹性酶灌注和血管紧张素II）模型以及AAA患者的主动脉组织和细胞中形成主动脉瘤。

DESCRIPTION (provided by applicant): Hypothesis: Our previous studies using elastase-perfusion and angiotensin II models of abdominal aortic aneurysms (AAA) have shown that female rodents have a decreased incidence and size of aneurysm formation. We have also recently shown that human placental mesenchymal stem cells (MSCs) are protective in a mouse model of AAA. In the current proposal, we will investigate the role of dietary phytoestrogen as a preventive therapy, and its synergistic effects with MSCs as a treatment strategy to protect against AAA. Methods: We will use an elastase-perfusion and angiotensin II murine model of AAA using male and female wild-type, estrogen receptor knockout mice (ER-α--/- and ER-ß-/-) and ApoE-/- mice. Dietary phytoestrogen will be administered to male and female mice via estrogen-rich or estrogen-free chow. Various subsets of female MSCs (placenta-, bone marrow- or adipose-derived) primed with or without estrogen will be administered intravenously or by aortic implantation in mice models of AAA. Aortic diameter will be measured on day 3, 7 and 14 (elastase perfusion model) and day 28 (angiotensin II model). Aortic tissue will be harvested to analyze pro-inflammatory cytokine (IL-17, TNF-α-, MCP-1, IL-1-ß-, KC and RANTES) and HMGB1 (high mobility group box 1; a pro-inflammatory nuclear protein) production by ELISA, MMP2 and MMP9 activity by zymography, serine proteases (uPA, tPA and PAI-1) by western blots, paracrine factors (VEGF, HGF, PGE2) by ELISA, elastin and collagen degradation as well as aortic smooth muscle expression by histology, and immune cell (macrophages, CD4+ T cells, neutrophils) infiltration by flow cytometry. Results: Preliminary results demonstrate a significant upregulation of estrogen receptor (ER-α) expression in female mice on days 1, 3 and 14 after elastase-perfusion compared to males, as well as in aortic tissue from human females compared to males after AAA. Also, male mice fed a diet rich in estrogen display a significantly decreased aortic diameter, decreased cytokine production (IL-23, IL-1-ß-, IL-6 and IL-27), decreased MMP2 and 9 expression and decreased macrophage and neutrophil infiltration compared to male mice fed an estrogen free diet. Furthermore, treatment with human female MSCs attenuates aortic diameter, pro-inflammatory cytokine production and cell infiltration after AAA. Female MSCs inhibit HMGB1 and IL-17 production more significantly than male MSCs. Also, estradiol-priming of female MSCs offer significantly increased protection from vascular inflammation in aortic tissue from male AAA patients. Conclusions: Dietary estrogen therapy and mesenchymal stem cells can attenuate aneurysm formation and inflammation in the elastase-perfusion murine model of AAA and in human aortic tissue from AAA patients. We propose to delineate the phytoestrogen mediated anti-inflammatory effects, and its crosstalk with various subsets of gender-specific MSCs, on aortic aneurysm formation in the murine (elastase-perfusion and angiotensin II) models as well aortic tissue and cells from AAA patients.