GENETIC MOUSE MODEL OF PREECLAMPSIA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。试点项目II：子痫前期小鼠遗传模型主要研究者：赖仲斌，M.D, Ph.D. A.具体目标在本提案中，具体目标旨在解决以下问题：具体目标#1。IL-10敲除小鼠（IL-10 KO）是否会表现出妊娠结局受损（胎儿吸收、早产、先兆子痫或宫内生长迟缓），以响应孕龄依赖性治疗，如脂多糖（LPS）、肽聚糖和缺氧？具体目标2。子痫前期相关的促炎细胞因子网络、白细胞侵入胎盘、高血压和蛋白尿等特征是否需要母体炎症程序？具体目标#3。子痫前期患者血清是否会导致IL-10妊娠小鼠胎盘异常（细胞凋亡、滋养细胞侵袭蜕膜能力差和白细胞侵袭）和高血压？B.研究和结果本报告涵盖的时间为7/04至5/05。我们实验室有兴趣建立妊娠相关高血压疾病子痫前期小鼠模型。我们发表的观察结果表明，IL-10在正常和异常妊娠结局中的时间作用。下面，我们展示了我们未发表的初步结果，以强调IL-10和子宫NK细胞在胎儿存活中的体内作用。此外，我们提供的数据表明，IL-10能够抑制COX-2的表达和前列腺素的释放。我们还证明，子痫前期妇女的母体血清可导致滋养细胞死亡。子宫NK细胞介导il -10缺失小鼠炎症诱导的胎儿死亡（Nature Immunology）胎儿死亡是妊娠早期常见的并发症，局部免疫反应失调可能是其病因。在这种情况下，证明细胞因子如IL-10缺乏、炎症、子宫NK （uNK）细胞毒性增强和妊娠丢失之间的一种新的调节关系是很重要的。我们在这里表明，IL-10-/-小鼠的交配导致胎儿吸收或宫内生长限制（IUGR），以响应极低剂量的脂多糖（LPS）。即使LPS剂量高出10倍，野生型小鼠的妊娠也正常（图1）。IL-10-/-小鼠的胎儿吸收与子宫自然杀伤细胞（uNK）细胞毒激活和侵入胎盘的显著增加有关。在lps处理的IL-10-/-动物中，uNK细胞耗尽或IL-10给药可挽救妊娠（图2）。我们的研究结果确定了涉及IL-10缺乏和炎症的胎儿死亡或IUGR的双重打击机制。我们的结果可能为人类不良妊娠结局提供见解。有证据表明，子痫前期妇女的母体血清诱导滋养细胞死亡子宫胎盘循环不足和内皮功能障碍是自然流产和子痫前期的标志性特征（13,14）。据报道，炎症引起的胎盘变性可导致细胞毒性细胞因子、核酸片段化和血管紧张素渗漏到外周循环。因此，努力应该集中在使用这些观察结果来检测子痫前期和血压正常的患者血清样本中的细胞毒性因子。如果成功，这些发现将对建立基于血清中存在的因素来预测妊娠相关疾病的体外检测具有重要意义。为了启动这些研究，我们对暴露于正常血压孕妇和子痫前期妇女血清样本的人上皮外滋养细胞TCL1进行了细胞凋亡和丙酸排除试验。我们的目的是证明子痫前期血清中含有诱导细胞死亡的可溶性成分。为了描述子痫前期妇女血清中因子的潜在细胞毒性作用，我们用20%的子痫前期患者（n=15）和正常妊娠妇女（n=8）的血清样本处理细胞外滋养细胞（TCL-1细胞系）。此外，细胞生长在10% FCS或10% FCS +放线菌素D (20 ？G /ml)分别用于正常生长和诱导细胞凋亡。用碘化丙啶排除法和caspase 3活化法测定这些处理细胞的细胞损伤和凋亡。代表性实验如图3和图4所示。在图3中，我们显示子痫前期血清，而不是正常妊娠血清，诱导碘化丙啶摄取早在治疗两小时。实验细节在图例中提供。该活性对热敏感，可滴定至血清的5%（数据未显示）。同样，我们发现，通过caspase 3阳性人群的FACS分析，子痫前期患者的血清而非正常妊娠妇女的血清会导致TCL-1细胞显著凋亡（图4）。值得注意的是，子痫前期患者的血清样本存在个体间差异，强调了子痫前期的异质性（数据未显示）。在这个节骨眼上，我们只使用了有限数量的样品。我们打算用这种方法筛选大量来自子痫前期患者和正常妊娠个体的样本，长期目标是鉴定母体血清中的细胞毒性分子，并评估子痫前期血清在妊娠IL-10 KO小鼠中的体内活性。1)我们的数据清楚地表明IL-10-/-小鼠在对炎症信号的反应中会出现妊娠并发症，我们计划利用这一模型研究与子痫前期相关的病理生理参数。我们计划使用缺氧条件，LPS和多I-C作为子痫前期可能的致病因素。此外，我们将试图建立子痫前期症状与子宫NK细胞和滋养细胞侵袭的细胞毒性活性之间的联系。2)我们的研究结果表明，滋养细胞毒性活性存在于子痫前期患者的血清样本中。利用IL-10-/-妊娠小鼠，我们将尝试评估血清活性的体内效应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PILOT PROJECT II: Genetic Mouse Model of Preeclampsia Principal Investigator: Zhongbin Lai, M.D., Ph.D. A. Specific Aims In this proposal, the specific aims are designed to address the following questions: Specific Aim #1. Will IL-10 knockout mice (IL-10 KO) exhibit compromised pregnancy outcome (fetal resorption, premature birth, preeclampsia, or intrauterine growth retardation) in response to gestational age-dependent treatment with inflammatory signals such as lipopolysaccharide (LPS), peptidoglycan, and hypoxia? Specific Aim #2. Is the maternal inflammatory programming required for preeclampsia-associated signature features of pro-inflammatory cytokine network, leukocyte invasion of the placenta, hypertension, and proteinurea? Specific Aim #3. Will administration of serum from preeclampsia patients cause placental abnormalities (apoptosis, poor trophoblast invasion of the decidua, and leukocyte invasion) and hypertension in pregnant IL-10 KO mice? B. Studies and Results This report covers the period from 7/04 to 5/05. Our lab is interested in establishing a mouse model for pregnancy-associated hypertension disease preeclampsia. Our published observations point to a temporal role for IL-10 in normal and abnormal pregnancy outcomes. Below, we present our unpublished preliminary results to highlight the in vivo role of IL-10 and uterine NK cells in fetal survival. In addition, we present data to suggest that IL-10 is capable of inhibiting COX-2 expression and prostaglandin release. We also demonstrate that maternal serum from preeclamptic women causes cell death in trophoblasts. Uterine NK cells mediate inflammation-induced fetal demise in IL-10-null mice (Manuscript Suibmitted to Nature Immunology) Fetal demise is a common complication of early pregnancy, and dysregulated local immune responses may underlie its etiology. In this context, it is important to demonstrate a novel regulatory relationship between deficiency in cytokines such as IL-10, inflammation, enhanced uterine NK (uNK) cell cytotoxicity, and pregnancy loss. We show here that mating of IL-10-/- mice resulted in fetal resorption or intrauterine growth restriction (IUGR) in response to very low doses of lipopolysaccharide (LPS). Pregnancy in wild type mice was normal even at 10 fold higher LPS doses (Figure 1). Fetal resorption in IL-10-/- mice was associated with significant increase in uterine natural killer (uNK) cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells or IL-10 administration rescued pregnancy in LPS-treated IL-10-/-animals (Figure 2). Our results identify a two-hit mechanism of fetal demise or IUGR involving IL-10 deficiency and inflammation. Our results may provide insights into adverse pregnancy outcomes in humans. Evidence that maternal serum from preeclamptic women induces cell death in trophoblast cells Insufficient uteroplacental circulation and endothelial dysfunction are the hallmark features of spontaneous abortions and preeclampsia (13, 14). It has been reported that placental degeneration due to inflammation may result in leakage of cytotoxic cytokines, fragmented nucleic acids, and angiotensinogens into the perpheral circulation. Thus, efforts should be focused on using such observations to detect cytototoxic factors in serum samples from preeclamptic and normotensive patients. If successful, such findings will be of great significance in establishing in vitro assays to predict pregnancy-associated disorders based on the factors present in the sera. To initiate these studies, we have performed apoptosis and propidium exclusion assays on the human extravillous trophoblast cell line TCL1 exposed to serum samples from normotensive pregnant and preeclamptic women. Our aim was to show that preeclamptic serum contains soluble components that induce cell death. In order to delineate the potential cytotoxic effect of factors in the serum of pre-eclamptic women, extravillous trophoblasts (TCL-1 cell line) were treated with 20% serum samples from preeclamptic patients (n=15) as well as from women experiencing normal pregnancy (n=8). In addition, cells were grown in 10% FCS or 10% FCS + actnomycin D (20 ?g/ml) for normal growth or to induce apoptosis, respectively. Cell injury and apoptosis in these treated cells were measured using propidium iodide exclusion and caspase 3 activation assays. Representative experiments are shown in Figures 3 and 4. In Figure 3, we show that preeclamptic serum, not normal pregnancy serum, induces uptake of propidium iodide as early as two hrs of treatment. The experimental details are provided in the figure legend. This activity is heat sensitive and can be titrated down to 5% of serum (data not shown). Similarly, we find that serum from patients with preeclampsia, but not serum from women with normal pregnancy causes significant apoptosis in TCL-1 cells as measured by FACS analysis of caspase 3-positive population (Figure 4). It is noteworthy that there were interindividual differences in serum samples from preeclamptic patients, underscoring the heterogeneous nature of preeclampsia (data not shown). At this juncture, we have used only a limited number of samples. We intend to use this approach to screen a sizeable number of samples from both preeclamptic patients and normal pregnancy individuals The long term goal is to identify the cytotoxic molecule(s) in maternal serum and to evaluate preeclampsia serum for its in vivo activity in pregnant IL-10 KO mice. Future Directions 1) Our data clearly suggest that IL-10-/- mice experience pregnancy complications in response to inflammatory signals, we plan to exploit this model to study pathophysiologic parameters associated with preeclampsia. We plan to use hypoxic conditions, LPS, and poly I-C as the possible causative factors for preeclampsia. Furthermore, we will attempt to establish a link between preeclamptic symptoms and the cytotoxic activity of uterine NK cells and trophoblast invasion. 2) Our results suggest that a trophoblast cytotoxic activity is present in serum samples from preeclampsia patients. Using IL-10-/- pregnant mice, we will attempt to evaluate the in vivo effect of the serum activity.