The Warburg Effect and Diabetic Retinopathy

项目概要/摘要 视网膜新生血管（RNV）是晚期糖尿病视网膜病变的一种使人衰弱的并发症，尽管 使用抗 VEGF 药物和激光治疗继续导致失明。人们对 RNV 为何发展的了解较少 只有在患者患有糖尿病数十年之后。尽管内皮细胞（EC）血管生成激活是一个 作为这一转变的标志，内皮细胞如何调整其新陈代谢以维持这种激活仍然是一个巨大的差距 据我们所知。我们的长期目标是确定 RNV 的生物能机制。瓦尔堡效应 是首次发现的从线粒体氧化磷酸化 (OxPhos) 到糖酵解过度的代谢转变 在癌细胞中。这种代谢转变不仅比 OxPhos 更快地产生 ATP（尽管效率较低），而且 提供细胞增殖过程中脂质、蛋白质和核苷酸合成所需的前体。最近的 瓦尔堡效应被重新发现为各种内皮相关疾病的关键因素；然而，它的作用 糖尿病视网膜病变的定义尚不明确。在此应用程序中，总体目标是定义以下角色： 糖尿病视网膜病变中的瓦尔堡效应并确定其潜在机制。在此，我们建议 需要多次打击来协同改变 EC 代谢以满足转化的生物合成需求 静态 EC 进入血管生成细胞。组织缺氧是与晚期相关的最常见危险因素 糖尿病视网膜病变。因此，我们的中心假设是糖尿病促使静止的内皮细胞产生血管（首先 击中），缺氧（第二次击中）是通过 Warburg 代谢适应进行血管生成转换所必需的 效果。 Aim1 将测试能量传感器、AMP 激活蛋白激酶持续激活的假设 (AMPK) 维持 Warburg 效应以介导 EC 血管生成激活。我们的方法是使用两次命中模型 AMPKα1 内皮特异性条件敲除 (AMPKα1End-/-) 小鼠和 AMPKα1 中糖尿病和缺氧的影响 沉默人类视网膜 EC（HREC）以实现这一目标。我们还将使用来自以下患者的玻璃体样本： 增殖性糖尿病视网膜病变，测试 RNV 的发展与 Warburg 效应之间的相关性 - 相关代谢物。 Aim2 将研究内质网 (ER) 应激在介导 Warburg 效应诱导 EC 血管生成激活。我们假设肌醇需求酶的激活 (IRE)1 是一种独特的 ER 应激传感器蛋白，具有激酶和 RNase 活性，是 Warburg 的关键介质 效应诱导的 EC 血管生成激活。我们将使用CRISPR/Cas9来测试抑制下游的效果 IRE1 激酶和 RNase 活性的信号传导对 Warburg 效应诱导的 EC 血管生成激活的影响。我们会 还使用缺氧和糖尿病的两次打击模型以及选择性药理学在体内测试我们的假设 抑制剂。总体而言，这位新研究者发起的 R01 充分利用了跨学科专业知识 生物化学家、线粒体生物学家、ER 生物学家和临床医生使用一种新型的高级二次打击模型 糖尿病视网膜病变，以获得 RNV 生物能基础的机制见解。了解角色 瓦尔堡效应将揭示治疗糖尿病视网膜病变的新靶点。

Project Summary/Abstract Retinal neovascularization (RNV) is a debilitating complication of advanced diabetic retinopathy, which despite the use of anti-VEGF and laser treatments continues to cause blindness. Less is known as to why RNV develops only after patients have had diabetes for decades. Although endothelial cell (EC) angiogenic activation is a hallmark of this transition, how ECs adapt their metabolism to sustain such activation remains a significant gap in our knowledge. Our long-term goal is to determine the bioenergetic mechanisms of RNV. The Warburg effect is a metabolic shift from mitochondrial oxidative phosphorylation (OxPhos) to hyperglycolysis that was first found in cancer cells. This metabolic shift not only produces ATP faster than OxPhos, albeit less efficiently, but also provides precursors required for lipid, protein, and nucleotide synthesis during cell proliferation. Recently the Warburg effect was rediscovered as a key contributor in various endothelial-related diseases; however, its role in diabetic retinopathy is not well-defined. In this application, the overall objective(s) are to define the role of the Warburg effect in diabetic retinopathy and to identify its underlying mechanisms. Here, we propose that multiple hits are needed to cooperatively alter EC metabolism to fulfill biosynthetic demands of transforming a quiescent EC into an angiogenic cell. Tissue hypoxia is the most common risk factor associated with advanced diabetic retinopathy. Thus, our central hypothesis is that diabetes primes quiescent ECs to be angiogenic (first hit) and that hypoxia (second hit) is necessary for angiogenic switch via the metabolic adaptation of the Warburg effect. Aim1 will test the hypothesis that persistent activation of the energy sensor, AMP-activated protein kinase (AMPK) sustains the Warburg effect to mediate EC angiogenic activation. Our approach is to use a two-hit model of diabetes and hypoxia in AMPKα1 endothelial-specific conditional knockout (AMPKα1End-/-) mice and in AMPKα1 silenced human retinal ECs (HRECs) to achieve this aim. We will also use vitreous samples from patients with proliferative diabetic retinopathy to test the correlation between the development of RNV and the Warburg effect- associated metabolites. Aim2 will investigate the role of endoplasmic reticulum (ER) stress in mediating the Warburg effect-induced EC angiogenic activation. We hypothesize that activation of Inositol-requiring enzyme (IRE)1, a unique ER-stress sensor protein with kinase and RNase activities, is a key mediator for the Warburg effect-induced EC angiogenic activation. We will use CRISPR/Cas9 to test the effect of inhibiting downstream signaling of IRE1 kinase and RNase activities on the Warburg effect-induced EC angiogenic activation. We will also test our hypothesis in vivo using a two-hit model of hypoxia and diabetes and selective pharmacological inhibitors. Overall, this new-investigator initiated R01 capitalizes on the interdisciplinary expertise of a biochemist, a mitochondrial biologist, an ER biologist, and a clinician to use a novel two-hit model of advanced diabetic retinopathy to gain mechanistic insights into the bioenergetic basis of RNV. Understanding the role of the Warburg effect will reveal novel targets in the treatment of diabetic retinopathy.