Neuroangiogenesis in Deep Infiltrating Endometriosis

描述（由申请人提供）：本提案的科学目标是测试雌激素和白细胞介素（IL）-1的作用，已知其可加重炎症，对与深部浸润性子宫内膜异位症（DIE）相关的盆腔疼痛的特别严重原因。子宫内膜异位症影响多达10%的育龄妇女，在美国每年直接和间接的医疗费用超过220亿美元。DIE，其中异位植入物穿透>5 mm进入腹膜下空间，代表疾病的最痛苦的表现。据报道，约95%患有这种类型的子宫内膜异位症的女性经历中度至重度疼痛，通常表现为痛经（疼痛期），排便困难（排便疼痛）和性交困难（性交疼痛）的症状。DIE病变充满了密集的神经和血管网络，我们假设通过一个综合的发展计划，我们创造了“神经血管生成”。我们已经确定了一种多功能蛋白质，脑源性神经营养因子（BDNF），这是一个候选人的神经血管生成介质在DIE，我们将详细调查下，这个探索性的R21补助金的支持。BDNF对神经元和内皮细胞都有促有丝分裂作用，并且与无疾病的对照组相比，它在患有子宫内膜异位症的妇女的异位和在位子宫内膜中过表达。此外，其同源受体酪氨酸激酶B（Trk B）的mRNA和蛋白在子宫内膜异位症病例中均上调。因此，BDNF表现出预期的神经血管生成介质的表达模式和生化特征。我们推测这种蛋白质有助于DIE病变生长、血管形成和疼痛。将收集术前疼痛和生活质量工具，以与生化数据进行比较。我们将使用原位分子组织化学分析来验证脑源性神经营养因子mRNA和蛋白质在脑缺血性脑病活检组织中的细胞分布。来自没有子宫内膜异位症或疼痛的妇女的正常子宫内膜将作为对照。将通过蛋白质印迹法评价BDNF的同种型。上述描述性研究将伴随机械实验，使用我们开发和表征的细胞培养模型。对照和子宫内膜异位症细胞培养物将用于评估雌激素和IL- 1对体外BDNF mRNA和蛋白调节的影响，包括BDNF原的细胞内加工成其成熟的生物活性形式。我们的初步数据表明，雌激素和IL-1 β刺激BDNF的产生。剂量-反应和时程实验，沿着药理学抑制剂和siRNA敲除ER？ER？GPER或IL-1 I型受体（使用scrambled siRNA作为对照），将确定神经血管生成机制特异性药物靶点，以便将来可以合理设计有效的治疗子宫内膜异位症相关疼痛的药物。两个新的雌激素受体拮抗剂，合成和我们的合作者提供的，也将进行评估。在R21期结束时，将准备R 01申请，以解决BDNF诱导的神经和毛细血管增殖和迁移的相关生物活性及其与盆腔疼痛症状的相关性。

DESCRIPTION (provided by applicant): The scientific goal of this proposal is to test the roles of estrogen and interleukin (IL)-1�potent autacoids known to exacerbate inflammation, on a particularly severe cause of pelvic pain associated with deep infiltrating endometriosis (DIE). Endometriosis affects as many as 10% of reproductive age women and accounts for >$22 billion annually in direct and indirect medical costs here in the USA. DIE, wherein ectopic implants penetrate >5 mm into the subperitoneal space, represents the most painful manifestation of the disease. It is reported that ~95% of women with this type of endometriosis experience moderate to severe pain, commonly presenting with symptoms of dysmenorrhea (painful periods), dyschezia (painful bowel movements) and dyspareunia (painful intercourse). DIE lesions are imbued with dense nerve and vascular networks that we postulate arise via an integrated developmental program we have coined "neuroangiogenesis". We have identified one multifunctional protein, brain-derived neurotrophic factor (BDNF), that is a candidate neuroangiogenesis mediator in DIE, which we will investigate in detail under the aegis of this exploratory R21 grant. BDNF is mitogenic for both neurons and endothelial cells and it is overexpressed in ectopic and eutopic endometrium of women with endometriosis compared to controls without the disease. Moreover, both mRNA and protein of its cognate receptor, tyrosine kinase B (TrkB), are upregulated in endometriosis cases. Thus, BDNF exhibits the expression pattern and biochemical characteristics expected of a neuroangiogenesis mediator. We postulate that this protein contributes to DIE lesion growth, vascularization and pain. Preoperative pain and quality of life instruments will be collected for comparison with the biochemcial data. We will validate the cellular distribution of BDNF mRNA and protein in DIE biopsies using in situ molecular histochemical assays. Normal endometrium from women without endometriosis or pain will serve as a control. Isoforms of BDNF will be evaluated by Western blotting. The aforementioned descriptive studies will be accompanied by mechanistic experiments, using cell culture models that we developed and characterized. Control and endometriosis cell cultures will be used to evaluate the effects of estrogens and IL- 1�n BDNF mRNA and protein regulation in vitro, including the intracellular processing of pro-BDNF to its mature, bioactive form. Our preliminary data indicate that estrogens and IL-1�timulate BDNF production. Dose-response and time-course experiments, along with pharmacological inhibitors and siRNA knock down of ER�ER�GPER or IL-1 type I receptor (using scrambled siRNA as a control) will identify mechanism-specific drug targets for neuroangiogenesis, so that effective therapeutics can be rationally designed for endometriosis- associated pain in the future. Two novel estrogen receptor antagonists, synthesized and provided by our collaborators, also will be evaluated. At the completion of the R21 period, an R01 application will be prepared to address the relevant biological activities of BDNF-induced nerve and capillary proliferation and migration and their correlation with pelvic pain symptoms.