Prolonged Inhibition of Pathologic Neovascularization by Catalytic Antioxidants

描述(申请人提供)：在某些形式的糖尿病视网膜病变(DR)和黄斑变性(AMD)中，失明是由于新生血管的病理发育造成的，这些血管不完整、脆弱和疏松。这些新生血管疾病的进展被认为是通过产生有毒分子，即活性氧物种(ROS)来实现的。这些疾病对患者造成毁灭性的影响，美国每年花费超过500亿美元，目前还没有针对这些疾病的长期成功治疗方法。我们的长期目标是开发一种治疗方法，以保护视网膜细胞的健康和功能，从而延长视力，提高DR或AMD患者的生活质量。由于ROS的过度升高发生在大多数其他视网膜病变的“上游”，它代表着一个常见的结节，可以作为抗氧化剂和其他分子的靶点，这些分子增加了“第二阶段”抗氧化酶的表达。我们已发表的初步数据表明，氧化铈纳米颗粒可以催化破坏ROS，通过调节包括血管内皮生长因子(VEGFR)在内的许多视网膜基因的表达，阻止病理性脉络膜和视网膜新生血管病变的发展，并导致极低密度脂蛋白受体缺失视网膜中现有病理性新生血管的消退。我们的中心假设是，氧化铈纳米颗粒由于其催化抗氧化活性和在视网膜中的长期滞留，将持续清除ROS，并在长达12个月的时间内抑制病理性新生血管。具体目标1将确定纳米钙在视网膜中的持续时间以及它们在多大程度上保持抗新生血管的活性。电感耦合等离子体质谱将在十亿分之几的水平上定量测定Ce。眼底检查、视网膜电描记术和光学相干断层扫描将用于对同一动物进行纵向研究，以评估新生血管、视网膜功能和外核层的厚度。纳米陶瓷对氧化应激、炎症和新生血管相关特定基因的影响将使用共聚焦显微镜、蛋白质印迹和聚合酶链式反应阵列进行分析。具体目标2将证明纳米氧化钙通过减少氧化应激对光感受器和视网膜色素上皮(RPE)细胞的影响来保护视网膜。将对指示光感受器-和/或RPE-氧化应激的基因活性、蛋白质和结构进行评估。具体目标3将测试这样一种假设，即纳米氧化钙和萝卜硫素的组合使用将在Vldlr视网膜中产生相加或协同效应。萝卜硫素是第二相抗氧化酶的诱导剂。预期结果--拟议的工作有望证明纳米氧化钙抑制视网膜病理性新生血管的寿命、效力和机制。预计这一结果将产生重要的积极影响，因为纳米陶瓷长期有效性的证明很可能支持它们的治疗用途，已识别基因的活性变化应该会为治疗DR、AMD和其他涉及氧化应激的疾病提供更多重要的靶点。

DESCRIPTION (provided by applicant): In some forms of Diabetic Retinopathy (DR) and Macular Degeneration (AMD), blindness results from the pathologic development of new blood vessels which are incomplete, weak and porous. The progression of these neovascular diseases is thought to occur through the production of toxic molecules, Reactive Oxygen Species (ROS). There are no long term successful therapies for such diseases which have devastating effects on patients and cost the USA over $50 billion/yr. Our long term goal is to develop a therapeutic treatment to protect the health and function of retinal cells and thereby prolong vision and improve the quality of life for patients with DR or AMD. Because the excessive rise in ROS occurs "upstream" of most other retinal pathologies, it represents a common node which can be targeted by antioxidants and other molecules which increase the expression of "Phase II" antioxidant enzymes. Our published and preliminary data show that cerium oxide nanoparticles, which catalytically destroy ROS, can prevent development of pathologic choroidal and retinal neovascular lesions and cause the regression of existing pathologic neovessels in the Very Low Density Lipoprotein Receptor null retina by modulating the expression of many retinal genes including Vascular Endothelial Growth Factor (VEGF). Our central hypothesis is that cerium oxide nanoparticles, because of their catalytic antioxidant activity and long term retention in the retina, will continuously scavenge ROS and inhibit pathologic neovascularization over prolonged times -up to 12 months. Specific aim 1 will determine duration of nanoceria in the retina and the extent to which they retain activity against neovascularization. Inductively coupled plasma mass spectrometry will quantitate cerium at the parts per billion levels. Fundoscopy, electroretinography and optical coherence tomography will be used for longitudinal studies on the same animal to evaluate neovascularization, retinal function and thickness of the outer nuclear layer. Nanoceria effects on specific genes involved in oxidative stress, inflammation and neovascularization will be analyzed using confocal microscopy, Western blots and PCR arrays. Specific Aim 2 will demonstrate that nanoceria provide protection to the retina by reducing the effects of oxidative stress on photoreceptors and Retinal Pigment Epithelial (RPE) cells. Gene activity, proteins and structures indicative of photoreceptor- and/or RPE- oxidative stress will be evaluated. Specific Aim 3 will test the hypothesis that the combinatorial use of nanoceria and sulforaphane, an inducer of Phase II antioxidant enzymes, will result in an additive or synergistic effects in the Vldlr retina. Expected outcomes - the work proposed is expected to demonstrate the longevity, potency and mechanisms by which nanoceria inhibit pathologic neovascularization in the retina. The results are expected to have an important positive impact because the demonstration of the long term effectiveness of the nanoceria will most likely support their therapeutic use and changes in activity of identified genes should provide additional targets important for treating DR, AMD and other diseases which involve oxidative stress.