Cholesterol homeostasis in pathogenesis of DR

胆固醇稳态和代谢的破坏可导致高胆固醇 在患病组织中的浓度和胆固醇晶体（CC）的形成。CC经常 在传统的组织学/免疫组织化学中被忽视的是乙醇，用于组织处理， 可以将它们溶解。这掩盖了CC的存在和潜在的致病性参与， 机制等使用扫描电子显微镜（SEM）对在没有预处理的情况下制备的组织进行观察。 在乙醇脱水步骤中，我们鉴定了CC的存在：i）在视网膜间的“脂质池”中， 色素上皮（RPE）和光感受器（PR）; ii）在糖尿病小鼠中自由循环 血液;和iii）在糖尿病人类受试者的循环单核细胞内。因为CC非常稳定 由于它们在生理学上不容易溶解并且不易于在体内溶解，因此它们成为 慢性炎症CC被先天免疫系统识别为异物 因为它们的形状、坚固性和不能溶解。此外，CC还诱导 炎症通过NLRP 3炎性体激活。这些小说初步出版 研究使我们提出了以下假设（见图1）：糖尿病- 诱导破坏i）全身胆固醇稳态，ii）血视网膜屏障功能;和 iii）SIRT 1、LXR和CYP 46 A1表达改变视网膜胆固醇稳态，导致 CC在关键部位形成：RPE、PR、单核细胞/巨噬细胞和循环中 影响内皮细胞。这种CC形成导致细胞内和细胞外补体 NLRP 3炎性体的激活和引发，用于其激活和释放高浓度的 破坏促炎细胞因子，促进DR的发展。为了验证这一假设， 我们提出以下具体目的：目的1：确定视网膜的时间变化， 胆固醇积累导致CC形成。目的2：检验糖尿病的假设- 诱导的胆固醇积累和CC形成的视网膜和 单核细胞/巨噬细胞诱导细胞内和细胞外补体活化， 引发NLRP 3炎性体和IL-1β产生。目的3：检验LXR 通过α-环糊精活化和直接螯合胆固醇可以抑制CC诱导的 糖尿病视网膜中的补体体和NLRP 3炎性体形成以及糖尿病视网膜中的 单核细胞/巨噬细胞，从而防止慢性炎症和DR的发展。 影响：该项目的成功完成将导致确定一个全新的 CC诱导的补体和NLRP 3炎性小体形成的途径， 目标是预防DR。

Disruptions in cholesterol homeostasis and metabolism can lead to high cholesterol concentrations in diseased tissue and the formation of cholesterol crystals (CC). CC are often overlooked in traditional histo/immunohistochemistry as the ethanol, used for tissue processing, can dissolve them. This masks CC presence and potential involvement in pathogenic mechanisms. Using Scanning Electron Microscopy (SEM) of tissues prepared without an ethanol dehydration step, we identified the presence of CC: i) in “lipid pools” between retinal pigment epithelium (RPE) and photoreceptors (PR); ii) circulating freely in diabetic murine blood; and iii) within circulating monocytes of diabetic human subjects. Since CC are very stable physiologically and are not easily amenable to dissolving in vivo, they become a source of chronic inflammation. CC are recognized by the innate immune system as foreign bodies because of their shape, firmness, and inability to be dissolved. Moreover, CC also induce inflammation via the NLRP3 inflammasome activation. These novel preliminary and published studies have led us to propose the following hypothesis (see schematic in Fig.1): Diabetes- induced disruption of i) systemic cholesterol homeostasis, ii) blood retinal barrier function; and iii) SIRT1, LXR and CYP46A1 expression alters retinal cholesterol homeostasis leading to CC formation within key locations: RPE, PR, monocytes/macrophages, and in the circulation affecting the endothelium. This CC formation results in intra- and extracellular complement activation and priming of the NLRP3 inflammasome for its activation and release of highly damaging pro-inflammatory cytokines, promoting development of DR. To test this hypothesis, we propose the following Specific Aims: Aim1: To determine the temporal changes in retinal cholesterol accumulation that lead to CC formation. Aim 2: To test the hypothesis that diabetes- induced cholesterol accumulation and CC formation in the retina and in monocytes/macrophages induces activation of intracellular and extracellular complement to trigger the NLRP3 inflammasome and IL-1β production. Aim 3: To test the hypothesis that LXR activation and direct sequestration of cholesterol by alpha-cyclodextrin can inhibit CC-induced complosome and NLRP3 inflammasome formation in the diabetic retina and in monocytes/macrophages cells, thus preventing chronic inflammation and development of DR. Impact: The successful completion of this project will result in identifying an entirely novel pathway in which CC-induced complosome and NLRP3 inflammasome formation that can be pharmacologically targeted to prevent DR.