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Characterization and identification of measurable endpoints in a mouse model featuring age-related retinal pathologies: a platform to test therapies

Abstract

Apolipoprotein B100 (apoB100) is the structural protein of cholesterol carriers including low-density lipoproteins. It is a constituent of sub-retinal pigment epithelial (sub-RPE) deposits and pro-atherogenic plaques, hallmarks of early dry age-related macular degeneration (AMD), an ocular neurodegenerative blinding disease, and cardiovascular disease, respectively. Herein, we characterized the retinal pathology of transgenic mice expressing mouse apoB100 in order to catalog their functional and morphological ocular phenotypes as a function of age and establish measurable endpoints for their use as a mouse model to test potential therapies. ApoB100 mice were found to exhibit an age-related decline in retinal function, as measured by electroretinogram (ERG) recordings of their scotopic a-wave, scotopic b-wave; and c-wave amplitudes. ApoB100 mice also displayed a buildup of the cholesterol carrier, apolipoprotein E (apoE) within and below the supporting extracellular matrix, Bruch’s membrane (BrM), along with BrM thickening, and accumulation of thin diffuse electron-dense sub-RPE deposits, the severity of which increased with age. Moreover, the combination of apoB100 and advanced age were found to be associated with RPE morphological changes and the presence of sub-retinal immune cells as visualized in RPE-choroid flatmounts. Finally, aged apoB100 mice showed higher levels of circulating and ocular pro-inflammatory cytokines, supporting a link between age and increased local and systemic inflammation. Collectively, the data support the use of aged apoB100 mice as a platform to evaluate potential therapies for retinal degeneration, specifically drugs intended to target removal of lipids from Bruch’s membrane and/or alleviate ocular inflammation.

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Fig. 1: In vivo imaging of apoB100 mouse eyes.
Fig. 2: Retinal function of apoB100 mice with age.
Fig. 3: ApoE accumulates below the RPE and within BrM of aged apoB100 mice.
Fig. 4: Ultrastructural changes in apoB100 as a function of age.
Fig. 5: Distribution of sub-retinal immune cells and RPE degenerative changes in apoB100 mice increases in severity with age.
Fig. 6: Aged apoB100 mice exhibit a significant upregulation of pro-inflammatory cytokines in the systemic circulation.

References

  1. Friedman, D. S., O’Colmain, B. J., Munoz, B., Tomany, S. C., McCarty, C., de Jong, P. T. et al. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 122, 564-572 (2004).

  2. Ambati, J. & Fowler, B. J. Mechanisms of age-related macular degeneration. Neuron 75, 26-39 (2012).

  3. Li, J. Q., Welchowski, T., Schmid, M., Mauschitz, M. M., Holz, F. G. & Finger, R. P. Prevalence and incidence of age-related macular degeneration in Europe: a systematic review and meta-analysis. Br J Ophthalmol 104, 1077-1084 (2020).

  4. Klein, R., Peto, T., Bird, A. & Vannewkirk, M. R. The epidemiology of age-related macular degeneration. Am J Ophthalmol 137, 486-495 (2004).

  5. DeAngelis, M. M., Owen, L. A., Morrison, M. A., Morgan, D. J., Li, M., Shakoor, A. et al. Genetics of age-related macular degeneration (AMD). Hum Mol Genet 26, R246 (2017).

  6. Jonas, J. B., Cheung, C. M. G. & Panda-Jonas, S. Updates on the Epidemiology of Age-Related Macular Degeneration. Asia Pac J Ophthalmol (Phila) 6, 493-497 (2017).

  7. Grassmann, F., Fauser, S. & Weber, B. H. The genetics of age-related macular degeneration (AMD)-Novel targets for designing treatment options? Eur J Pharm Biopharm 95, 194-202 (2015).

  8. Malek, G., Yao, P.-L. & Choudhary, M. Models of Pathologies Associated with Age-Related Macular Degeneration and Their Utilities in Drug Discovery. In: Christopher L. Cioffi (ed). Drug Delivery Challenges and Novel Therapeutic Approaches for Retinal Diseases 10.1007/7355_2020_93 83-123 (Springer International Publishing: Cham, 2020).

  9. Elizabeth Rakoczy, P., Yu, M. J., Nusinowitz, S., Chang, B. & Heckenlively, J. R. Mouse models of age-related macular degeneration. Exp Eye Res 82, 741-752 (2006).

  10. Sorsby, A. Experimental Pigmentary Degeneration of the Retina by Sodium Iodate. Br J Ophthalmol 25, 58-62 (1941).

  11. Kannan, R. & Hinton, D. R. Sodium iodate induced retinal degeneration: new insights from an old model. Neural Regen Res 9, 2044-2045 (2014).

  12. Moriguchi, M., Nakamura, S., Inoue, Y., Nishinaka, A., Nakamura, M., Shimazawa, M. et al. Irreversible Photoreceptors and RPE Cells Damage by Intravenous Sodium Iodate in Mice Is Related to Macrophage Accumulation. Invest Ophthalmol Vis Sci 59, 3476-3487 (2018).

  13. Reisenhofer, M. H., Balmer, J. M. & Enzmann, V. What Can Pharmacological Models of Retinal Degeneration Tell Us? Curr Mol Med 17, 100-107 (2017).

  14. Malek, G., Busik, J., Grant, M. B. & Choudhary, M. Models of retinal diseases and their applicability in drug discovery. Expert Opin Drug Discov 13, 359-377 (2018).

  15. Hadziahmetovic, M. & Malek, G. Age-Related Macular Degeneration Revisited: From Pathology and Cellular Stress to Potential Therapies. Front Cell Dev Biol 8, 612812 (2020).

  16. Choudhary, M. & Malek, G. A Review of Pathogenic Drivers of Age-Related Macular Degeneration, Beyond Complement, with a Focus on Potential Endpoints for Testing Therapeutic Interventions in Preclinical Studies. Adv Exp Med Biol 1185, 9-13 (2019).

  17. Choudhary, M. & Malek, G. Rethinking Nuclear Receptors as Potential Therapeutic Targets for Retinal Diseases. J Biomol Screen 21, 1007-1018 (2016).

  18. Curcio, C. A., Johnson, M., Huang, J. D. & Rudolf, M. Apolipoprotein B-containing lipoproteins in retinal aging and age-related macular degeneration. J Lipid Res 51, 451-467 (2010).

  19. Wang, L., Clark, M. E., Crossman, D. K., Kojima, K., Messinger, J. D., Mobley, J. A. et al. Abundant lipid and protein components of drusen. PLoS One 5, e10329 (2010).

  20. Rudolf, M. & Curcio, C. A. Esterified cholesterol is highly localized to Bruch’s membrane, as revealed by lipid histochemistry in wholemounts of human choroid. J Histochem Cytochem 57, 731-739 (2009).

  21. Curcio, C. A., Millican, C. L., Bailey, T. & Kruth, H. S. Accumulation of cholesterol with age in human Bruch’s membrane. Invest Ophthalmol Vis Sci 42, 265-274 (2001).

  22. Curcio, C. A., Johnson, M., Rudolf, M. & Huang, J. D. The oil spill in ageing Bruch membrane. Br J Ophthalmol 95, 1638-1645 (2011).

  23. Klaver, C. C., Kliffen, M., van Duijn, C. M., Hofman, A., Cruts, M., Grobbee, D. E. et al. Genetic association of apolipoprotein E with age-related macular degeneration. Am J Hum Genet 63, 200-206 (1998).

  24. Ebrahimi, K. B. & Handa, J. T. Lipids, lipoproteins, and age-related macular degeneration. J Lipids 2011, 802059 (2011).

  25. Malek, G., Li, C. M., Guidry, C., Medeiros, N. E. & Curcio, C. A. Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy. Am J Pathol 162, 413-425 (2003).

  26. Li, C. M., Clark, M. E., Chimento, M. F. & Curcio, C. A. Apolipoprotein localization in isolated drusen and retinal apolipoprotein gene expression. Invest Ophthalmol Vis Sci 47, 3119-3128 (2006).

  27. Pikuleva, I. A. & Curcio, C. A. Cholesterol in the retina: the best is yet to come. Prog Retin Eye Res 41, 64-89 (2014).

  28. Holz, F. G., Sheraidah, G., Pauleikhoff, D. & Bird, A. C. Analysis of lipid deposits extracted from human macular and peripheral Bruch’s membrane. Arch Ophthalmol 112, 402-406 (1994).

  29. Ruberti, J. W., Curcio, C. A., Millican, C. L., Menco, B. P., Huang, J. D. & Johnson, M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch’s membrane. Invest Ophthalmol Vis Sci 44, 1753-1759 (2003).

  30. Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C. & Williams, D. S. A comparison of some organizational characteristics of the mouse central retina and the human macula. PLoS One 10, e0125631 (2015).

  31. Farese, R. V., Jr., Veniant, M. M., Cham, C. M., Flynn, L. M., Pierotti, V., Loring, J. F. et al. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100. Proc Natl Acad Sci U S A 93, 6393-6398 (1996).

  32. Fujihara, M., Cano, M. & Handa, J. T. Mice that produce ApoB100 lipoproteins in the RPE do not develop drusen yet are still a valuable experimental system. Invest Ophthalmol Vis Sci 55, 7285-7295 (2014).

  33. Hu, P., Herrmann, R., Bednar, A., Saloupis, P., Dwyer, M. A., Yang, P. et al. Aryl hydrocarbon receptor deficiency causes dysregulated cellular matrix metabolism and age-related macular degeneration-like pathology. Proc Natl Acad Sci U S A 110, E4069-4078 (2013).

  34. Mehalow, A. K., Kameya, S., Smith, R. S., Hawes, N. L., Denegre, J. M., Young, J. A. et al. CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum Mol Genet 12, 2179-2189 (2003).

  35. Choudhary, M., Ismail, E. N., Yao, P. L., Tayyari, F., Radu, R. A., Nusinowitz, S. et al. LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target. JCI Insight 5 (2020).

  36. Prusky, G. T., Alam, N. M., Beekman, S. & Douglas, R. M. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol Vis Sci 45, 4611-4616 (2004).

  37. Claybon, A. & Bishop, A. J. Dissection of a mouse eye for a whole mount of the retinal pigment epithelium. J Vis Exp https://doi.org/10.3791/2563 (2011).

  38. Choudhary, M., Safe, S. & Malek, G. Suppression of aberrant choroidal neovascularization through activation of the aryl hydrocarbon receptor. Biochim Biophys Acta Mol Basis Dis 1864, 1583-1595 (2018).

  39. Mattapallil, M. J., Wawrousek, E. F., Chan, C. C., Zhao, H., Roychoudhury, J., Ferguson, T. A. et al. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci 53, 2921-2927 (2012).

  40. Yonehara, K., Shintani, T., Suzuki, R., Sakuta, H., Takeuchi, Y., Nakamura-Yonehara, K. et al. Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse. PLoS One 3, e1533 (2008).

  41. Kretschmer, F., Tariq, M., Chatila, W., Wu, B. & Badea, T. C. Comparison of optomotor and optokinetic reflexes in mice. J Neurophysiol 118, 300-316 (2017).

  42. Oyster, C. W., Takahashi, E. & Collewijn, H. Direction-selective retinal ganglion cells and control of optokinetic nystagmus in the rabbit. Vision Res 12, 183-193 (1972).

  43. Curcio, C. A., Johnson, M., Huang, J. D. & Rudolf, M. Aging, age-related macular degeneration, and the response-to-retention of apolipoprotein B-containing lipoproteins. Prog Retin Eye Res 28, 393-422 (2009).

  44. Anderson, D. H., Ozaki, S., Nealon, M., Neitz, J., Mullins, R. F., Hageman, G. S. et al. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol 131, 767-781 (2001).

  45. Lu, C. K., Chen, S. J., Niu, D. M., Tsai, C. C., Lee, F. L. & Hsu, W. M. Electrophysiological changes in lipaemia retinalis. Am J Ophthalmol 139, 1142-1145 (2005).

  46. Ong, J. M., Zorapapel, N. C., Aoki, A. M., Brown, D. J., Nesburn, A. B., Rich, K. A. et al. Impaired electroretinogram (ERG) response in apolipoprotein E-deficient mice. Curr Eye Res 27, 15-24 (2003).

  47. Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J. G. et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 71, 343-353 (1992).

  48. Curcio, C. A. Soft Drusen in Age-Related Macular Degeneration: Biology and Targeting Via the Oil Spill Strategies. Invest Ophthalmol Vis Sci 59, AMD160–AMD181 (2018).

    Article  Google Scholar 

  49. Hayes, K. C., Lindsey, S., Stephan, Z. F. & Brecker, D. Retinal pigment epithelium possesses both LDL and scavenger receptor activity. Invest Ophthalmol Vis Sci 30, 225-232 (1989).

  50. Ryeom, S. W., Silverstein, R. L., Scotto, A. & Sparrow, J. R. Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36. J Biol Chem 271, 20536-20539 (1996).

  51. Ryeom, S. W., Sparrow, J. R. & Silverstein, R. L. CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium. J Cell Sci 109 (Pt 2), 387-395 (1996).

  52. Reis, A., Mateus, C., Melo, P., Figueira, J., Cunha-Vaz, J. & Castelo-Branco, M. Neuroretinal dysfunction with intact blood-retinal barrier and absent vasculopathy in type 1 diabetes. Diabetes 63, 3926-3937 (2014).

  53. Feng, L., Ju, M., Lee, K. Y. V., Mackey, A., Evangelista, M., Iwata, D. et al. A Proinflammatory Function of Toll-Like Receptor 2 in the Retinal Pigment Epithelium as a Novel Target for Reducing Choroidal Neovascularization in Age-Related Macular Degeneration. Am J Pathol 187, 2208-2221 (2017).

  54. Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S. et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 24, 353-389 (2006).

  55. Huh, H. Y., Pearce, S. F., Yesner, L. M., Schindler, J. L. & Silverstein, R. L. Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. Blood 87, 2020-2028 (1996).

  56. Miller, J. W., Bagheri, S. & Vavvas, D. G. Advances in Age-related Macular Degeneration Understanding and Therapy. US Ophthalmic Rev 10, 119-130 (2017).

  57. Hollyfield, J. G., Bonilha, V. L., Rayborn, M. E., Yang, X., Shadrach, K. G., Lu, L. et al. Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat Med 14, 194-198 (2008).

  58. Cruz-Guilloty, F., Saeed, A. M., Echegaray, J. J., Duffort, S., Ballmick, A., Tan, Y. et al. Infiltration of proinflammatory m1 macrophages into the outer retina precedes damage in a mouse model of age-related macular degeneration. Int J Inflam 2013, 503725 (2013).

  59. Ban, N., Lee, T. J., Sene, A., Choudhary, M., Lekwuwa, M., Dong, Z. et al. Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss. JCI Insight 3 (2018).

  60. Sennlaub, F., Auvynet, C., Calippe, B., Lavalette, S., Poupel, L., Hu, S. J. et al. CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice. EMBO Mol Med 5, 1775-1793 (2013).

  61. Combadiere, C., Feumi, C., Raoul, W., Keller, N., Rodero, M., Pezard, A. et al. CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration. J Clin Invest 117, 2920-2928 (2007).

  62. Kobayashi, Y. The role of chemokines in neutrophil biology. Front Biosci 13, 2400-2407 (2008).

  63. Kaplan, R. C., McGinn, A. P., Pollak, M. N., Kuller, L. H., Strickler, H. D., Rohan, T. E. et al. Association of total insulin-like growth factor-I, insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-3 levels with incident coronary events and ischemic stroke. J Clin Endocrinol Metab 92, 1319-1325 (2007).

  64. Gomez, J. M., Maravall, F. J., Gomez, N., Navarro, M. A., Casamitjana, R. & Soler, J. The IGF-I system component concentrations that decrease with ageing are lower in obesity in relationship to body mass index and body fat. Growth Horm IGF Res 14, 91-96 (2004).

  65. Kielczewski, J. L., Hu, P., Shaw, L. C., Li Calzi, S., Mames, R. N., Gardiner, T. A. et al. Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury. Am J Pathol 178, 1517-1528 (2011).

  66. Cai, S., Yang, Q., Cao, Y., Li, Y., Liu, J., Wang, J. et al. PF4 antagonizes retinal neovascularization via inhibiting PRAS40 phosphorylation in a mouse model of oxygen-induced retinopathy. Biochim Biophys Acta Mol Basis Dis 1866, 165604 (2020).

  67. Crane, I. J., Wallace, C. A., McKillop-Smith, S. & Forrester, J. V. CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha. J Immunol 165, 4372-4378 (2000).

  68. Wooff, Y., Man, S. M., Aggio-Bruce, R., Natoli, R. & Fernando, N. IL-1 Family Members Mediate Cell Death, Inflammation and Angiogenesis in Retinal Degenerative Diseases. Front Immunol 10, 1618 (2019).

  69. Sahu, B., Chavali, V. R., Alapati, A., Suk, J., Bartsch, D. U., Jablonski, M. M. et al. Presence of rd8 mutation does not alter the ocular phenotype of late-onset retinal degeneration mouse model. Mol Vis 21, 273-284 (2015).

  70. Chavali, V. R., Khan, N. W., Cukras, C. A., Bartsch, D. U., Jablonski, M. M. & Ayyagari, R. A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration. Hum Mol Genet 20, 2000-2014 (2011).

  71. Cao, X., Sanchez, J. C., Dinabandhu, A., Guo, C., Patel, T. P., Yang, Z. et al. Aqueous proteins help predict the response of patients with neovascular age-related macular degeneration to anti-VEGF therapy. J Clin Invest 132 (2022).

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Acknowledgements

J.T.H. is the Robert Bond Welch Professor. We thank Dr. Neal Peachy for assistance with the c-wave recording protocols and Mr. Michael Lekwuwa for technical support.

Funding

This research was supported by funding from the National Eye Institute: EY027802 (G.M.), EY028160 (G.M.), EY032751 (G.M.), EY027691 (J.T.H.), P30 EY005722 (Duke Eye Center), a Research to Prevent Blindness, Inc (RPB) Core grant (Duke Eye Center), and an unrestricted RPB grant (Wilmer Eye Institute).

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M.C., J.T.H., and G.M. contributed to the conception and design of the study, data interpretation, and paper editing. M.C., F.T., and J.T.H. conducted experiments, collected, and analyzed data. M.C. and G.M. contributed to paper writing. All authors approved the final version of the submitted paper.

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Correspondence to Goldis Malek.

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J.T.H. received grant funding and royalties from Bayer Pharmaceuticals, Inc, and grant funding and stock options as a member of the Scientific Advisory Board for Clover Pharmaceuticals, Inc, both for unrelated projects.

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Study protocols were approved by the Duke University and Johns Hopkins Institutional Animal Care and Use Committees. All animal experiments were performed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.

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Choudhary, M., Tayyari, F., Handa, J.T. et al. Characterization and identification of measurable endpoints in a mouse model featuring age-related retinal pathologies: a platform to test therapies. Lab Invest (2022). https://doi.org/10.1038/s41374-022-00795-7

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