A Novel Pathogenic Pathway for Diabetic Keratopathy

项目总结/摘要 糖尿病性角膜病变是糖尿病的一种并发症，也是导致视力下降的主要原因。没有 预防或逆转糖尿病相关角膜缺损的有效药物。两个独立的纵向 临床研究已经显示出非诺贝特（一种过氧化物酶体的特异性激动剂）的强大治疗效果 过氧化物酶体激活受体-α（PPARα），糖尿病视网膜病变。我们的初步研究使用糖尿病 人类供体角膜和动物模型表明了PPARα在维持角膜神经完整性中的作用。在 在我们的初步研究中，我们已经制作了一个神经支配的三维体外人类角膜模型， 与体内角膜组织的基本解剖学和生理学相似性。利用这个新颖的模型， 阐明了PPARα在糖尿病性角膜病变中的作用。PPARα表达显著下调， 来自T1 DM和T2 DM人类供体的细胞，与PPARα水平降低一致，如 糖尿病患者的角膜我们的体内初步研究表明，非糖尿病的PPARα基因敲除 (PPARα-/-）小鼠的基底神经纤维密度降低，角膜敏感性降低，与对照组相似。 与糖尿病患者的情况相似。此外，令我们惊讶的是，老年非糖尿病的PPARα基因敲除小鼠 与年龄匹配的WT小鼠相比，自然发展出更严重的角膜溃疡。治疗 糖尿病大鼠给予非诺贝特的活性代谢物非诺贝酸， 糖尿病大鼠如Seahorse分析所示，线粒体功能在PPARα-/-视网膜中受损。基于 通过这些初步的研究，我们推测糖尿病诱导的PPARα表达下调在糖尿病的发病机制中起着重要作用。 在糖尿病性角膜病变中的关键病理作用，并代表了一种新的药物靶点。我们提议下列 研究来解决这个假设。首先，我们将在PPARα-/-小鼠和PPARα转基因小鼠中诱导糖尿病 在角膜中过表达PPARα，以确定当PPARα过表达时， 证实糖尿病引起的角膜神经密度和敏感性降低。我们还将治疗糖尿病小鼠 以确定激活的过氧化物酶体增殖物激活受体α是否阻止角膜神经纤维变性的进展。 其次，我们将确定PPARα的神经保护作用是否是通过减轻氧化应激， 炎症、线粒体功能保护和使用PPARα-/-上调神经营养因子 小鼠和PPARα转基因小鼠以及体外神经支配的3D人角膜模型。三是 将神经保护性PPARα功能转化为治疗，我们将评估局部治疗的疗效。 专利的非诺贝特滴眼液在糖尿病引起的神经纤维变性中的应用。本研究 有可能确定角膜中PPARα的新功能。这些研究有可能建立一种新的 糖尿病性角膜病变的发病机制，并导致一种新的治疗方法的发展。

PROJECT SUMMARY / ABSTRACT Diabetic keratopathy is a complication of diabetes and a major cause of vision loss. There are no effective drugs that can prevent or reverse corneal defects related to diabetes. Two independent longitudinal clinical studies have shown robust therapeutic effects of fenofibrate, a specific agonist of Peroxisome Proliferator-Activated Receptor-α (PPARα), on diabetic retinopathy. Our preliminary studies using diabetic human donor corneas and animal models suggest a role of PPARα in maintaining corneal nerve integrity. In our preliminary studies, we have fabricated an innervated 3D in vitro human corneal model that demonstrates basic anatomical and physiological similarities to the corneal tissue in vivo. Using this novel model we began unravelling PPARα’s role in diabetic keratopathy. Significant downregulation of PPARα expression was seen in cells from both T1DM and T2DM human donors, in agreement with decreased PPARα levels as shown in diabetic human corneas. Our in vivo preliminary studies have shown that non-diabetic PPARα knockout (PPARα-/-) mice have decreased densities of the sub-basal nerve fibers and reduced corneal sensitivity, similar to what is seen in diabetic humans. Furthermore, to our surprise, aged, non-diabetic PPARα knockout mice naturally developed more severe corneal ulcerations compared to that in age-matched WT mice. Treatment of diabetic rats with fenofibric acid, an active metabolite of fenofibrate, alleviates corneal nerve degeneration in diabetic rats. As shown by Seahorse analysis, mitochondrial function is impaired in PPARα-/- retina. Based on these preliminary studies, we hypothesize that diabetes-induced down-regulation of PPARα expression plays a key pathological role in diabetic keratopathy and represents a novel drug target. We propose the following studies to address the hypothesis. First, we will induce diabetes in PPARα-/- mice and PPARα transgenic mice over-expressing PPARα in the cornea, to determine if PPARα KO exacerbates while PPARα over-expression alleviates diabetes-induced decreases of corneal nerve density and sensitivity. We will also treat diabetic mice with fenofibrate to determine if activation of PPARα arrests progression of corneal nerve fiber degeneration. Second, we will determine if the neuroprotective effect of PPARα is through attenuation of oxidative stress and inflammation, protection of mitochondrial functions and up-regulation of neurotrophic factors using PPARα-/- mice and PPARα transgenic mice as well as the innervated in vitro 3D human corneal model. Third, to translate the neuroprotective PPARα function into a therapy, we will evaluate therapeutic efficacy of topical application of a proprietary fenofibrate eyedrop on diabetes-induced nerve fiber degeneration. This study has potential to identify a new function of PPARα in the cornea. These studies have potential to establish a novel pathogenic mechanism for diabetic keratopathy and to lead to the development of a novel therapy.