Cholesterol crystal formation is a unifying pathogenic mechanism in the development of diabetic retinopathy.

Publisher: Springer Verlag Country of Publication: Germany NLM ID: 0006777 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-0428 (Electronic) Linking ISSN: 0012186X NLM ISO Abbreviation: Diabetologia Subsets: MEDLINE

Original Publication: Berlin Springer Verlag

Diabetic Retinopathy*/metabolism ; alpha-Cyclodextrins*/adverse effects ; alpha-Cyclodextrins*/metabolism ; Diabetes Mellitus, Experimental*/metabolism; Animals ; Cattle ; Mice ; Humans ; Swine ; Endothelial Cells/metabolism ; Retina/metabolism ; Disease Models, Animal ; Cholesterol/metabolism

Aims/hypothesis: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown.
Methods: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using ² H 2 O and ² H 7 -cholesterol.
Results: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy.
Conclusions/interpretation: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.
(© 2023. The Author(s).)

Ramachandra Rao S, Fliesler SJ (2021) Cholesterol homeostasis in the vertebrate retina: biology and pathobiology. J Lipid Res 62:100057. https://doi.org/10.1194/jlr.TR120000979. (PMID: 10.1194/jlr.TR120000979336623848010701)
Diaz-Coranguez M, Ramos C, Antonetti DA (2017) The inner blood-retinal barrier: cellular basis and development. Vision Res 139:123–137. https://doi.org/10.1016/j.visres.2017.05.009. (PMID: 10.1016/j.visres.2017.05.009286195165723228)
Fields MA, Del Priore LV, Adelman RA, Rizzolo LJ (2020) Interactions of the choroid, Bruch’s membrane, retinal pigment epithelium, and neurosensory retina collaborate to form the outer blood-retinal-barrier. Prog Retin Eye Res 76:100803. https://doi.org/10.1016/j.preteyeres.2019.100803. (PMID: 10.1016/j.preteyeres.2019.10080331704339)
Zheng W, Reem RE, Omarova S et al (2012) Spatial distribution of the pathways of cholesterol homeostasis in human retina. PLoS One 7(5):e37926. https://doi.org/10.1371/journal.pone.0037926. (PMID: 10.1371/journal.pone.0037926226294703358296)
Zheng W, Mast N, Saadane A, Pikuleva IA (2015) Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments. J Lipid Res 56(1):81–97. https://doi.org/10.1194/jlr.M053439. (PMID: 10.1194/jlr.M053439252935904274074)
Petrov AM, Astafev AA, Mast N, Saadane A, El-Darzi N, Pikuleva IA (2019) The interplay between retinal pathways of cholesterol output and its effects on mouse retina. Biomolecules 9(12):867. https://doi.org/10.3390/biom9120867. (PMID: 10.3390/biom9120867318423666995521)
Hammer SS, Beli E, Kady N et al (2017) The mechanism of diabetic retinopathy pathogenesis unifying key lipid regulators, sirtuin 1 and liver X receptor. EBioMedicine 22:181–190. https://doi.org/10.1016/j.ebiom.2017.07.008. (PMID: 10.1016/j.ebiom.2017.07.008287747375552206)
Abela GS (2010) Cholesterol crystals piercing the arterial plaque and intima trigger local and systemic inflammation. J Clin Lipidol 4(3):156–164. https://doi.org/10.1016/j.jacl.2010.03.003. (PMID: 10.1016/j.jacl.2010.03.00321122648)
Vedre A, Pathak DR, Crimp M, Lum C, Koochesfahani M, Abela GS (2009) Physical factors that trigger cholesterol crystallization leading to plaque rupture. Atherosclerosis 203(1):89–96. https://doi.org/10.1016/j.atherosclerosis.2008.06.027. (PMID: 10.1016/j.atherosclerosis.2008.06.02718703195)
Fragiotta S, Fernandez-Avellaneda P, Breazzano MP et al (2019) The fate and prognostic implications of hyperreflective crystalline deposits in nonneovascular age-related macular degeneration. Invest Ophthalmol Vis Sci 60(8):3100–3109. https://doi.org/10.1167/iovs.19-26589. (PMID: 10.1167/iovs.19-2658931323680)
Li M, Dolz-Marco R, Huisingh C et al (2019) Clinicopathologic correlation of geographic atrophy secondary to age-related macular degeneration. Retina 39(4):802–816. https://doi.org/10.1097/IAE.0000000000002461. (PMID: 10.1097/IAE.0000000000002461308394956445604)
Li M, Dolz-Marco R, Messinger JD et al (2019) Clinicopathologic correlation of aneurysmal type 1 neovascularization in age-related macular degeneration. Ophthalmol Retina 3(2):99–111. https://doi.org/10.1016/j.oret.2018.08.008. (PMID: 10.1016/j.oret.2018.08.00831014774)
Pang CE, Messinger JD, Zanzottera EC, Freund KB, Curcio CA (2015) The onion sign in neovascular age-related macular degeneration represents cholesterol crystals. Ophthalmology 122(11):2316–2326. https://doi.org/10.1016/j.ophtha.2015.07.008. (PMID: 10.1016/j.ophtha.2015.07.00826298717)
Curcio CA, Zanzottera EC, Ach T, Balaratnasingam C, Freund KB (2017) Activated retinal pigment epithelium, an optical coherence tomography biomarker for progression in age-related macular degeneration. Invest Ophthalmol Vis Sci 58(6):BIO211–BIO226. https://doi.org/10.1167/iovs.17-21872. (PMID: 10.1167/iovs.17-21872287857695557213)
Ong SS, Cummings TJ, Vajzovic L, Mruthyunjaya P, Toth CA (2019) Comparison of optical coherence tomography with fundus photographs, fluorescein angiography, and histopathologic analysis in assessing coats disease. JAMA Ophthalmol 137(2):176–183. https://doi.org/10.1001/jamaophthalmol.2018.5654. (PMID: 10.1001/jamaophthalmol.2018.565430476946)
Venturelli C, Jeannin G, Sottini L, Dallera N, Scolari F (2006) Cholesterol crystal embolism (atheroembolism). Heart Int 2(3–4):155. https://doi.org/10.4081/hi.2006.155. (PMID: 10.4081/hi.2006.155219772653184670)
Kovach JL, Isildak H, Sarraf D (2019) Crystalline retinopathy: unifying pathogenic pathways of disease. Surv Ophthalmol 64(1):1–29. https://doi.org/10.1016/j.survophthal.2018.08.001. (PMID: 10.1016/j.survophthal.2018.08.00130144456)
Das R, Spence G, Hogg RE, Stevenson M, Chakravarthy U (2018) Disorganization of inner retina and outer retinal morphology in diabetic macular edema. JAMA Ophthalmol 136(2):202–208. https://doi.org/10.1001/jamaophthalmol.2017.6256. (PMID: 10.1001/jamaophthalmol.2017.6256293270335838716)
Yamaguchi M, Nakao S, Wada I et al (2022) Identifying hyperreflective foci in diabetic retinopathy via VEGF-induced local self-renewal of CX3CR1 ⁺ vitreous resident macrophages. Diabetes 71(12):2685–2701. https://doi.org/10.2337/db21-0247. (PMID: 10.2337/db21-024736203331)
Jaffe GJ, Chakravarthy U, Freund KB et al (2021) Imaging features associated with progression to geographic atrophy in age-related macular degeneration: classification of Atrophy Meeting Report 5. Ophthalmol Retina 5(9):855–867. https://doi.org/10.1016/j.oret.2020.12.009. (PMID: 10.1016/j.oret.2020.12.00933348085)
Miura M, Makita S, Sugiyama S et al (2017) Evaluation of intraretinal migration of retinal pigment epithelial cells in age-related macular degeneration using polarimetric imaging. Sci Rep 7(1):3150. https://doi.org/10.1038/s41598-017-03529-8. (PMID: 10.1038/s41598-017-03529-8286005155466639)
Inoue M, Arakawa A, Yamane S, Kadonosono K (2014) Imaging of sub-retinal pigment epithelial linear structures in patients with age-related macular degeneration. Eur J Ophthalmol 24(5):744–750. https://doi.org/10.5301/ejo.5000433. (PMID: 10.5301/ejo.500043324474382)
Yoshitake T, Murakami T, Suzuma K, Dodo Y, Fujimoto M, Tsujikawa A (2020) Hyperreflective foci in the outer retinal layers as a predictor of the functional efficacy of ranibizumab for diabetic macular edema. Sci Rep 10(1):873. https://doi.org/10.1038/s41598-020-57646-y. (PMID: 10.1038/s41598-020-57646-y319649706972781)
Waldstein SM, Vogl WD, Bogunovic H, Sadeghipour A, Riedl S, Schmidt-Erfurth U (2020) Characterization of drusen and hyperreflective foci as biomarkers for disease progression in age-related macular degeneration using artificial intelligence in optical coherence tomography. JAMA Ophthalmol 138(7):740–747. https://doi.org/10.1001/jamaophthalmol.2020.1376. (PMID: 10.1001/jamaophthalmol.2020.1376323792877206537)
Abela GS, Shamoun F, Vedre A et al (2012) The effect of ethanol on cholesterol crystals during tissue preparation for scanning electron microscopy. J Am Coll Cardiol 59(1):93. https://doi.org/10.1016/j.jacc.2011.08.065 . (author reply 93–94). (PMID: 10.1016/j.jacc.2011.08.06522192678)
Baumer Y, Mehta NN, Dey AK, Powell-Wiley TM, Boisvert WA (2020) Cholesterol crystals and atherosclerosis. Eur Heart J 41(24):2236–2239. https://doi.org/10.1093/eurheartj/ehaa505. (PMID: 10.1093/eurheartj/ehaa505325724757850045)
Bakke SS, Aune MH, Niyonzima N et al (2017) Cyclodextrin reduces cholesterol crystal-induced inflammation by modulating complement activation. J Immunol 199(8):2910–2920. https://doi.org/10.4049/jimmunol.1700302. (PMID: 10.4049/jimmunol.170030228855312)
Hasselbacher P, Hahn JL (1980) Activation of the alternative pathway of complement by microcrystalline cholesterol. Atherosclerosis 37(2):239–245. https://doi.org/10.1016/0021-9150(80)90009-x. (PMID: 10.1016/0021-9150(80)90009-x6903449)
Niyonzima N, Halvorsen B, Sporsheim B et al (2017) Complement activation by cholesterol crystals triggers a subsequent cytokine response. Mol Immunol 84:43–50. https://doi.org/10.1016/j.molimm.2016.09.019. (PMID: 10.1016/j.molimm.2016.09.01927692470)
Rajamaki K, Lappalainen J, Oorni K et al (2010) Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation. PLoS One 5(7):e11765. https://doi.org/10.1371/journal.pone.0011765. (PMID: 10.1371/journal.pone.0011765206687052909263)
Cochran BJ, Ong KL, Manandhar B, Rye KA (2021) High density lipoproteins and diabetes. Cells 10(4):850. https://doi.org/10.3390/cells10040850. (PMID: 10.3390/cells10040850339185718069617)
Lin JB, Mast N, Bederman IR et al (2016) Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis. J Lipid Res 57(2):258–264. https://doi.org/10.1194/jlr.M064469. (PMID: 10.1194/jlr.M064469266309124727421)
Gupta GK, Dhar K, Del Core MG, Hunter WJ 3rd, Hatzoudis GI, Agrawal DK (2011) Suppressor of cytokine signaling-3 and intimal hyperplasia in porcine coronary arteries following coronary intervention. Exp Mol Pathol 91(1):346–352. https://doi.org/10.1016/j.yexmp.2011.04.004. (PMID: 10.1016/j.yexmp.2011.04.004215400273139760)
Hammer SS, Vieira CP, McFarland D et al (2021) Fasting and fasting-mimicking treatment activate SIRT1/LXRalpha and alleviate diabetes-induced systemic and microvascular dysfunction. Diabetologia 64(7):1674–1689. https://doi.org/10.1007/s00125-021-05431-5. (PMID: 10.1007/s00125-021-05431-5337701948236268)
Wang Q, Bozack SN, Yan Y, Boulton ME, Grant MB, Busik JV (2014) Regulation of retinal inflammation by rhythmic expression of MiR-146a in diabetic retina. Invest Ophthalmol Vis Sci 55(6):3986–3994. https://doi.org/10.1167/iovs.13-13076. (PMID: 10.1167/iovs.13-13076248675824073658)
Antonetti DA, Wolpert EB (2003) Isolation and characterization of retinal endothelial cells. Methods Mol Med 89:365–374. https://doi.org/10.1385/1-59259-419-0:365. (PMID: 10.1385/1-59259-419-0:36512958433)
R Core Team (2013) R: a language and environment for statistical computing, v. 3.6.3. In: R Foundation for Statistical Computing, Vienna, Austria. Available from https://www.R-project.org/ . Accessed 14 Nov 2022.
Wickham H (2011) ggplot2. Wires Comput Stat 3(2):180–185. https://doi.org/10.1002/wics.147. (PMID: 10.1002/wics.147)
Dobin A, Davis CA, Schlesinger F et al (2013) STAR: ultrafast universal RNA-seq aligner, v. 2.5.2. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635. (PMID: 10.1093/bioinformatics/bts63523104886)
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, v. 1.26.0. Genome Biol 15:550. https://doi.org/10.1186/s13059-014-0550-8. (PMID: 10.1186/s13059-014-0550-8255162814302049)
Kady NM, Liu X, Lydic TA et al (2018) ELOVL4-mediated production of very long-chain ceramides stabilizes tight junctions and prevents diabetes-induced retinal vascular permeability. Diabetes 67(4):769–781. https://doi.org/10.2337/db17-1034. (PMID: 10.2337/db17-1034293622265860862)
ACCORD Study Group, ACCORD Eye Study Group, Chew EY et al (2010) Effects of medical therapies on retinopathy progression in type 2 diabetes. N Engl J Med 363(3):233–244. https://doi.org/10.1056/NEJMoa1001288. (PMID: 10.1056/NEJMoa1001288)
Keech AC, Mitchell P, Summanen PA et al (2007) Effect of fenofibrate on the need for laser treatment for diabetic retinopathy (FIELD study): a randomised controlled trial. Lancet 370(9600):1687–1697. https://doi.org/10.1016/S0140-6736(07)61607-9. (PMID: 10.1016/S0140-6736(07)61607-917988728)
North BE, Katz SS, Small DM (1978) The dissolution of cholesterol monohydrate crystals in atherosclerotic plaque lipids. Atherosclerosis 30(3):211–217. https://doi.org/10.1016/0021-9150(78)90047-3. (PMID: 10.1016/0021-9150(78)90047-3678318)
Abela GS, Kalavakunta JK, Janoudi A et al (2017) Frequency of cholesterol crystals in culprit coronary artery aspirate during acute myocardial infarction and their relation to inflammation and myocardial injury. Am J Cardiol 120(10):1699–1707. https://doi.org/10.1016/j.amjcard.2017.07.075. (PMID: 10.1016/j.amjcard.2017.07.07528867129)
Lechner J, O’Leary OE, Stitt AW (2017) The pathology associated with diabetic retinopathy. Vision Res 139:7–14. https://doi.org/10.1016/j.visres.2017.04.003. (PMID: 10.1016/j.visres.2017.04.00328412095)
Wright AD, Dodson PM (2011) Medical management of diabetic retinopathy: fenofibrate and ACCORD Eye studies. Eye (Lond) 25(7):843–849. https://doi.org/10.1038/eye.2011.62. (PMID: 10.1038/eye.2011.6221436845)
Chen Y, Hu Y, Lin M et al (2013) Therapeutic effects of PPARalpha agonists on diabetic retinopathy in type 1 diabetes models. Diabetes 62(1):261–272. https://doi.org/10.2337/db11-0413. (PMID: 10.2337/db11-041323043158)
Roy S, Kim D, Hernandez C, Simo R, Roy S (2015) Beneficial effects of fenofibric acid on overexpression of extracellular matrix components, COX-2, and impairment of endothelial permeability associated with diabetic retinopathy. Exp Eye Res 140:124–129. https://doi.org/10.1016/j.exer.2015.08.010. (PMID: 10.1016/j.exer.2015.08.010262976154646846)
Abela GS, Aziz K (2006) Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events—a novel insight into plaque rupture by scanning electron microscopy. Scanning 28(1):1–10. https://doi.org/10.1002/sca.4950280101. (PMID: 10.1002/sca.495028010116502619)
Abela GS, Aziz K, Vedre A, Pathak DR, Talbott JD, Dejong J (2009) Effect of cholesterol crystals on plaques and intima in arteries of patients with acute coronary and cerebrovascular syndromes. Am J Cardiol 103(7):959–968. https://doi.org/10.1016/j.amjcard.2008.12.019. (PMID: 10.1016/j.amjcard.2008.12.01919327423)

R01 EY028858 United States EY NEI NIH HHS; R01EY016077 United States EY NEI NIH HHS; R01EY030766 United States EY NEI NIH HHS; R01 EY012601 United States EY NEI NIH HHS; R01 EY025383 United States EY NEI NIH HHS; R01EY028049 United States EY NEI NIH HHS; P30 EY011373 United States EY NEI NIH HHS; R01EY025383 United States EY NEI NIH HHS; R01 EY032753 United States EY NEI NIH HHS

Keywords: Blood retinal barrier; Cholesterol crystals; Complement activation; Cyclodextrin; Diabetic retinopathy; Endothelial cells; Fibrate; Hyper-reflective crystalline deposits; Inflammation; Statin

0 (alpha-Cyclodextrins)
97C5T2UQ7J (Cholesterol)

Date Created: 20230613 Date Completed: 20230801 Latest Revision: 20240709

20240709

PMC10390399

10.1007/s00125-023-05949-w

37311879