Effect of Hyperglycemia on Embryonic Heart Development

描述（由申请人提供）：人群中糖尿病的发病率正在上升;母体糖尿病影响7%的妊娠，导致胎儿心脏和瓣膜畸形的风险增加3-5倍。由于母体血糖水平升高，通过胎盘促进母胎扩散导致胎儿葡萄糖水平升高，导致慢性高血糖或脉冲性高血糖胚胎血糖水平。高血压调节内皮细胞（EC）对血流动力学条件变化的反应能力，这在发育过程中至关重要。本研究的目的是确定高血糖对早期胚胎心脏发育的作用及其对心脏瓣膜形成的影响。我们假设胚胎中的高血糖症将导致氧化应激和蛋白质硝化增加，导致EC功能障碍和TGF-β和Wnt信号通路的改变，这可能最终导致心脏畸形，包括瓣膜畸形和流出道缺陷。改变的葡萄糖的影响将通过以下方式确定：确定慢性高血糖症或脉冲性高血糖症将改变（1）胚胎心脏流出道中的EC和（2）胚胎EC转化为间充质细胞的能力（心脏发育的关键步骤）的程度。将使用qPCR、免疫组织化学和免疫荧光（IF）在鸡胚心脏的流出道中测定高血压诱导的体内基因表达变化。从这些流出道分离的EC将用于使用qPCR和IF研究胚胎EC对高血糖症的体外反应。将通过测量鸡心脏中硝基酪氨酸的量来研究蛋白质硝化，并且将通过测量WNT 3、BMP 10、<$-连环蛋白和E-钙粘蛋白中的基因表达变化来确定信号传导途径中的所得变化。初步数据表明，血糖水平的脉冲和慢性增加导致发育迟缓，细胞增殖减少和死亡率增加。光学相干断层扫描测量表明，在高血糖反应的流出道的结构的变化，而微阵列的结果表明，在关键的发育细胞信号通路的TGF-β和Wnt的变化。已组建了一个心脏发育、内皮细胞功能、鸡胚、临床糖尿病和蛋白质硝化方面的专家团队来培训申请人。这项研究的结果将确定心脏发育早期高血糖改变EC激活的机制，并将指导未来的研究，以预防母体糖尿病导致的胎儿心脏和瓣膜畸形。 公共卫生相关性：全球糖尿病发病率正在上升，影响7%的孕妇。葡萄糖的波动影响心脏发育，特别是母体糖尿病与心脏畸形风险增加3-5倍相关。需要对葡萄糖对心脏畸形发展的影响进行研究。

DESCRIPTION (provided by applicant): The incidence of diabetes is rising in the population; maternal diabetes affects 7% of pregnancies resulting in a 3-5 fold increased risk for fetal cardiac and valve malformations. As a result of elevated maternal blood glucose levels, facilitated maternofetal diffusion through the placenta causes elevated fetal glucose levels leading to chronic hyperglycemic or pulsed hyperglycemic embryonic blood glucose levels. Hyperglycemia modulates the ability of endocardial cells (ECs) to respond to changes in hemodynamic conditions which is critical during development. The objective of this study is to determine the role of hyperglycemia on early embryonic heart development and its effect on heart valve formation. We hypothesize that hyperglycemia in the embryo will result in increased oxidative stress and protein nitration leading to EC dysfunction and alterations of the TGF-¿ and Wnt signaling pathways, which could ultimately lead to cardiac malformations including valve malformation and outflow tract defects. The effect of altered glucose will be established by: determining the degree to which chronic hyperglycemia or pulsed hyperglycemia will alter (1) ECs in the outflow tract of the embryonic heart and (2) the ability of embryonic ECs to transform into mesenchymal cells, a critical step in heart development. Hyperglycemia induced in vivo gene expression alterations will be determined in the outflow tracts of embryonic chick hearts using qPCR, immunohistochemistry, and immunofluorescence (IF). ECs isolated from these outflow tracts will be used to study the in vitro response of the embryonic ECs to hyperglycemia using qPCR, and IF. Protein nitration will be studied by measuring the amount of nitrotryosine in the chick hearts, and the resulting changes in signaling pathways will be determined by measuring gene expression changes in WNT3, BMP10, ¿-Catenin, and E-Cadherin. Preliminary data indicates that pulsed and chronic increases in blood glucose levels lead to developmental delays, decreased cell proliferation and increased mortality. Optical coherence tomography measurements demonstrated a change in the structure of the outflow tracts in response to hyperglycemia, while microarray results indicated a change in the critical developmental cell signaling pathways of TGF-¿ and Wnt. A team of experts in heart development, endothelial cells functions, chick embryos, clinical diabetes, and protein nitration has been assembled to train the applicant. The results of the proposed study will determine the mechanisms by which hyperglycemia in early heart development alters EC activation and will guide future research to prevent the fetal cardiac and valve malformations which result from maternal diabetes. PUBLIC HEALTH RELEVANCE: The worldwide incidence of diabetes is rising worldwide, affecting 7% of pregnancies. Fluctuations in glucose affects cardiac development, specifically maternal diabetes is associated with a 3-5 fold increased risk for cardiac malformations. Research needs to be conducted on the effect of glucose on the development of cardiac malformations.