Gender Differences in Experimental Aortic Aneurysms

描述（由申请人提供）：假设：我们先前使用弹性蛋白酶灌注和血管紧张素II模型进行的腹主动脉瘤（AAA）研究表明，雌性啮齿动物的动脉瘤形成发生率和大小降低。我们最近还表明，人胎盘间充质干细胞（MSC）在AAA小鼠模型中具有保护作用。在目前的建议中，我们将研究膳食植物雌激素作为预防性治疗的作用，以及其与MSC的协同作用作为预防AAA的治疗策略。研究方法：我们将使用雄性和雌性野生型雌激素受体敲除小鼠（ER-α-/-和ER-β-/-）和ApoE-/-小鼠，建立弹性蛋白酶灌注和血管紧张素II AAA小鼠模型。将通过富含雌激素或不含雌激素的食物向雄性和雌性小鼠施用膳食植物雌激素。将用或不用雌激素引发的雌性MSC（胎盘来源、骨髓来源或脂肪来源）的各种亚组静脉内施用或通过主动脉植入AAA小鼠模型中。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-β-、KC和RANTES）和HMGB1（高迁移率族盒1;促炎性核蛋白）产生，MMP 2和MMP 9活性，丝氨酸蛋白酶蛋白质印迹法测定uPA、tPA和派-1，旁分泌因子ELISA检测血管内皮生长因子（VEGF）、肝细胞生长因子（HGF）、前列腺素E2（PGE 2）的表达，组织学检测弹性蛋白和胶原降解以及主动脉平滑肌表达，免疫细胞（巨噬细胞、CD 4 + T细胞、嗜中性粒细胞）浸润。结果如下：初步结果表明，与雄性相比，弹性蛋白酶灌注后第1、3和14天雌性小鼠中雌激素受体（ER-α）表达显著上调，AAA后与雄性相比，人类雌性主动脉组织中雌激素受体（ER-α）表达显著上调。此外，与喂食无雌激素饮食的雄性小鼠相比，喂食富含雌激素饮食的雄性小鼠显示出显著减小的主动脉直径、降低的细胞因子产生（IL-23、IL-1-β-、IL-6和IL-27）、降低的MMP 2和MMP 9表达以及降低的巨噬细胞和中性粒细胞浸润。此外，用人类雌性MSC治疗减弱AAA后的主动脉直径、促炎细胞因子产生和细胞浸润。女性MSC抑制HMGB 1和IL-17的产生比男性MSC更显著。此外，雌二醇引发的女性MSC提供了显着增加的保护，从男性AAA患者的主动脉组织血管炎症。结论：饮食雌激素治疗和间充质干细胞可以减轻AAA弹性蛋白酶灌注小鼠模型和AAA患者的人主动脉组织中的动脉瘤形成和炎症。我们建议描绘植物雌激素介导的抗炎作用，和它的串扰与性别特异性骨髓间充质干细胞的各种子集，对主动脉瘤形成的小鼠（弹性蛋白酶灌注和血管紧张素II）模型，以及主动脉瘤患者的主动脉组织和细胞。

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.