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-1? 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.

Public Health Relevance

Cholesterol crystals (CC), which are formed by increased lipid levels, cause tissue damage and are recognized by the immune system as foreign bodies because of their shape, firmness and inability to be dissolved. In this study, we will test whether retinal CC can cause retina damage leading to the development of diabetic retinopathy.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Shen, Grace L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Alabama Birmingham
Schools of Medicine
United States
Zip Code
Shaw, Lynn Calvin; Li Calzi, Sergio; Li, Nan et al. (2018) Enteral Arg-Gln Dipeptide Administration Increases Retinal Docosahexaenoic Acid and Neuroprotectin D1 in a Murine Model of Retinopathy of Prematurity. Invest Ophthalmol Vis Sci 59:858-869
Malek, Goldis; Busik, Julia; Grant, Maria B et al. (2018) Models of retinal diseases and their applicability in drug discovery. Expert Opin Drug Discov 13:359-377
Beli, Eleni; Yan, Yuanqing; Moldovan, Leni et al. (2018) Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice. Diabetes 67:1867-1879
Kady, Nermin M; Liu, Xuwen; Lydic, Todd A et al. (2018) ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability. Diabetes 67:769-781
Huang, Chao; Fisher, Kiera P; Hammer, Sandra S et al. (2018) Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway. Diabetes 67:1639-1649
Baumler, Stephen M; McHale, Andrew M; Blanchard, G J (2018) Surface Charge and Overlayer pH Influence the Dynamics of Supported Phospholipid Films. J Electroanal Chem (Lausanne) 812:159-165
Yan, Yuanqing; Gao, Ruli; Trinh, Thao L P et al. (2017) Immunodeficiency in Pancreatic Adenocarcinoma with Diabetes Revealed by Comparative Genomics. Clin Cancer Res 23:6363-6373
Baumler, Stephen M; Blanchard, Gary J (2017) The Influence of Metal Ions on the Dynamics of Supported Phospholipid Langmuir Films. Langmuir 33:2986-2992
Kady, Nermin; Yan, Yuanqing; Salazar, Tatiana et al. (2017) Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes. J Clin Lipidol 11:694-703
Basavarajappa, Halesha D; Sulaiman, Rania S; Qi, Xiaoping et al. (2017) Ferrochelatase is a therapeutic target for ocular neovascularization. EMBO Mol Med 9:786-801

Showing the most recent 10 out of 25 publications