Gender Differences in Experimental Aortic Aneurysms

描述(申请人提供)：假设：我们之前使用弹性酶灌流和血管紧张素II建立腹主动脉瘤(AAA)模型的研究表明，雌性啮齿动物的动脉瘤发生率和动脉瘤形成的大小都有所降低。我们最近还表明，人胎盘间充质干细胞(MSCs)在AAA小鼠模型中具有保护作用。在目前的建议中，我们将研究饮食中植物雌激素作为预防治疗的作用，以及它与骨髓间充质干细胞作为预防AAA的治疗策略的协同作用。方法：我们将使用弹性酶灌流和血管紧张素II小鼠模型，使用雌雄不同的野生型雌激素受体基因敲除小鼠(ER-α-/-和ER-？？/-)和载脂蛋白E-/-小鼠。饮食中的植物雌激素将通过富含雌激素或不含雌激素的食物给予雄性和雌性小鼠。不同亚群的雌性间充质干细胞(胎盘、骨髓或脂肪来源)，无论有没有雌激素，都将通过静脉或主动脉植入的方式在AAA小鼠模型中进行注射。分别于第3、7、14天(弹性酶灌流模型)和28天(血管紧张素II模型)测量主动脉内径。采用酶联免疫吸附试验检测促炎症细胞因子(IL-17、肿瘤坏死因子-α-、单核细胞趋化蛋白-1、白细胞介素1-β、KC和RANTES)和高迁移率族蛋白1(高迁移率族蛋白1；促炎症核蛋白)的产生；用免疫印迹法检测丝氨酸蛋白酶(uPA、tPA和PAI-1)；结果：初步结果显示，雌鼠在弹性酶灌流后第1、3和14天雌激素受体(ER-α)的表达显著高于雄鼠，而在腹主动脉注射后雌鼠的主动脉组织中雌激素受体(ER-ER)的表达也显著高于雄鼠。此外，与饲喂无雌激素饲料的雄性小鼠相比，饲喂富含雌激素的饲料的雄性小鼠的主动脉内径显著减小，细胞因子(IL-23、IL-1、IL-6和IL-27)的产生减少，MMP2和9的表达减少，巨噬细胞和中性粒细胞的浸润减少。此外，人的女性MSCs治疗可以减少AAA后的主动脉内径、促炎细胞因子的产生和细胞的浸润。雌性MSCs对HMGB1和IL-17产生的抑制作用明显高于雄性MSCs。此外，雌激素刺激的女性MSCs显著增强了对男性AAA患者主动脉组织中血管炎症的保护作用。结论：饮食雌激素治疗和间充质干细胞可以减轻弹性酶灌流的AAA小鼠模型和AAA患者的人主动脉组织中动脉瘤的形成和炎症。我们建议描述植物雌激素介导的抗炎作用，以及它与性别特异性MSCs的不同亚群在小鼠(弹性酶灌流和血管紧张素II)模型以及AAA患者的主动脉组织和细胞中对主动脉瘤形成的影响。

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.