Role of calcium channels in the development of diabetic retinopathy

描述（由申请人提供）：目前仍迫切需要预防糖尿病视网膜病变（DR）引起的视力丧失，这是糖尿病患者常见且重要的问题。临床前研究已经强调了视网膜氧化应激在dr发病机制中的作用。我们认为，确定糖尿病中介导致病性氧化应激的视网膜机制将对开发新的有效治疗方法具有重要意义。在神经元中，氧化应激和l型钙通道（LTCC）活性密切相关：LTCC活性的增加提高了细胞内钙含量，然后必须通过atp依赖机制（产生氧自由基）去除细胞内/细胞外钙浓度梯度。我们发现糖尿病深刻地改变了光感受器ltcc的整体活性。值得注意的是，非抗氧化和光感受器特异性治疗在体内纠正了异常的视网膜外LTCC表型，同时消除了糖尿病视网膜氧化应激。这些实验使用了一种经过验证的分析工具（MEMRI），为一个意想不到且可能非常重要的发现提供了第一个证据，该发现将异常的光感受器ltcc与糖尿病中的氧化应激联系起来。在视网膜中，LTCC有3种主要亚型（Cav）： Cav1.2（位于视网膜内），Cav1.3（位于视网膜内、光感受器和视网膜色素上皮层）和Cav1.4（仅位于光感受器）。糖尿病如何改变光感受器亚型通道尚不清楚。我们假设糖尿病诱导光感受器Cav1.3和1.4表达和活性的可重复性异常，这些变化积极促进氧化应激和糖尿病视网膜病变。为了开始评估我们的新工作假设，我们提出以下具体目标，这将充分利用我们在dr SA 1模型中混合高分辨率和体内光受体病理生理（锰增强MRI）与生物化学（活性氧，western blot分析（通道表达））和组织病理学（胰蛋白酶消化）成像的专业知识：测试糖尿病性光感受器Cav1.3和1.4 LTCC活性的改变是氧化应激和dr所必需的。提出的实验将开辟一条新的科学探究路线，有望提高我们对dr中氧化应激起源的理解。我们特定的遗传和药理学方法与一种新的成像技术相结合，使这些研究非常重要。这些实验对于确定预防和治疗DR的新分子靶点至关重要。拟议的研究具有高度创新性，因为它调查了光感受器LTCC亚型异常在糖尿病诱导的氧化应激中的先前未被怀疑但关键的作用，从而将为改变目前关于DR致病性氧化应激的起源和治疗选择的想法奠定坚实的科学基础。

DESCRIPTION (provided by applicant): There remains an urgent need to prevent vision loss from diabetic retinopathy (DR), a common and significant problem in patients with diabetes. Preclinical studies have highlighted retinal oxidative stress in the pathogenesis of DR. We reason that identifying the retinal mechanisms mediating pathogenic oxidative stress in diabetes will be important in the development of new and effective treatments. In neurons, oxidative stress and L-type calcium channels (LTCCs) activity are closely linked: increased LTCC activity elevates intracellular calcium content which must then be removed against a steep inter/extracellular calcium concentration gradient via ATP-dependent mechanisms which generate oxygen free radicals. We have discovered that diabetes profoundly changes the overall activity of photoreceptor LTCCs. Remarkably, a non-anti-oxidant and photoreceptor-specific treatment that corrected the abnormal outer retinal LTCC phenotype in vivo concurrently eliminated diabetic retinal oxidative stress. These experiments, using a validated and analytical tool (MEMRI), provide the first evidence for an unexpected and potentially very important discovery linking abnormal photoreceptor LTCCs and oxidative stress in diabetes. In the retina there are 3 major LTCC subtypes (Cav): Cav1.2 (located in inner retina), Cav1.3 (in inner retina, photoreceptor, and retinal pigment epithelium layers), and Cav1.4 (only in photoreceptors. How diabetes alters photoreceptor subtype channels are not yet known. We hypothesized that diabetes induces reproducible abnormalities in photoreceptor Cav1.3 and 1.4 expression and activity, and these changes actively contribute to oxidative stress and diabetic retinopathy. To begin to evaluate our new working hypothesis, we propose the following specific aim, which will take full advantage of our expertise in blending high resolution and analytical in vivo imaging of photoreceptor pathophysiology (manganese-enhanced MRI) with biochemistry (reactive oxygen species, western blot analysis (channel expression)) and histopathology (trypsin digest) in models of DR. SA 1: Test that diabetic alterations in photoreceptor Cav1.3, and 1.4 LTCC activity are required for oxidative stress and DR. The proposed experiments will open up a new line of scientific inquiry that is expected to improve our understanding about the origins of oxidative stress in DR. Because Cav1.3 and 1.4 subtypes are relatively insensitive to commonly used calcium channel blockers, our specific genetic and pharmacological approaches in concert with a novel imaging technique make these studies highly significant. These experiments will be crucial in the identification of new molecular targets for prevention and treatment of DR. The proposed research is highly innovative because it investigates a previously unsuspected but key role of photoreceptor LTCC subtype abnormalities in diabetes-induced oxidative stress, and will thus establish a firm scientific basis for changing current thinking regarding the origins of, and therapeutic options for, pathogenic oxidative stress in DR.