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Mineralization In Age-Related Macular Degeneration: Cause Or Consequence?

Age-related macular degeneration (AMD) is one of the leading causes of irreversible visual impairment in the elderly. Due to the demographic shift towards an aging global population, the frequency of AMD is expected to reach 196 million worldwide by 2020, increasing to a staggering 288 million by the year 2040 (1). This far exceeds the 30-50 million cases reported in 2006 (2).

The projected growth of this disease will lead to severe socioeconomic effects, especially considering that current treatment strategies are expensive and only slow progression to irreversible loss of sight, rather than prevent it. This suggests that intervention, and, if possible, prevention, should target the earliest stages of AMD.

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One of the earliest appearing hallmarks of AMD, and dangerous in terms of risk for severe vision loss, are drusen (3-5). Drusen are extracellular deposits that are visible during ophthalmic examinations. Drusen accumulate between the retinal pigment epithelium (RPE) – a polarised cell monolayer that supports the light-sensitive photoreceptor cells – and the inner wall of the choroid – a blood vessel complex that supplies the RPE and photoreceptor cells with nutrients and oxygen. Drusen have a heterogeneous composition that contains proteins (6-8), lipids (9-11), and trace metals (12, 13).

Seminal works by Dr. Bertha A. Klein and Dr. W Richard Green described the accumulation of mineral constituents within drusen (14, 15), whilst subsequent studies described these as spherical particles formed of calcium and phosphate (16, 17). More recently, an international team of researchers identified these spherical particles as hydroxyapatite (HAP), the mineral component of bones and teeth (18). Moreover, this was the first study to show that spherical particles contained a lipid core surrounded by a HAP “crust” and a proteinaceous coating (18). Consequently, it was proposed that HAP spherules play an essential role in drusen biogenesis and disease progression (18).

For many decades, drusen have been readily visible on low-resolution fundus photographs. However, with recent advancements to clinical imaging modalities, especially to spectral domain-optical coherence tomography (SD-OCT), drusen can now be imaged at higher resolution and tracked over long periods of time. Using this high-resolution and non-invasive method, heterogeneous internal reflectivity within drusen (HIRD) was identified as signifying a high risk for progression to late stages of disease (19, 20).

However, the morphology and composition of HIRD remained unknown. The previous identification of HAP within drusen led us to hypothesize that HIRD might also be formed of this inorganic calcium phosphate. Using a multi-disciplinary approach that combined clinical imaging, histopathology, and state of the art materials science technologies, our team of researchers led by Imre Lengyel Ph.D. (Queen’s University Belfast, UK), Anna C.S. Tan, MD (Singapore Eye Institute), K. Bailey Freund MD (New York University, USA), Srinvas R. Sadda MD (Doheny Eye Institute, USA), and Christine A. Curcio Ph.D. (University of Alabama at Birmingham, USA) investigated the morphology and composition of HIRD.

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Our initial investigations correlated OCT signatures of HIRD with histology. These correlations identified HIRD as multi-lobed nodules that were ~100 µm in diameter (roughly the size of a human hair). Remarkably, the shape of HIRD fit with the shape of previously described “calcified” drusen (14, 21, 22). However, conclusive evidence of calcium content was lacking. Using energy dispersive X-ray spectroscopy, we confirmed that calcium and phosphorus were major constituents of HIRD. Subsequently, the time of flight-secondary ion mass spectrometry provided the first evidence that these nodules might be formed by inorganic HAP, the mineral component of spherules.

To determine whether the mineral constituents of HIRD were identical to those previously identified as forming small spherules, we utilized selected area electron diffraction. This technique is able to differentiate between mineral phases, which might be an important factor for disease progression. Based on the given diffraction patterns it was concluded that nodules were formed of polycrystalline HAP, whilst some spherules contained a magnesium-substituted calcium phosphate called whitlockite. The identification of magnesium in spherules, but not in nodules, might be an important finding when considering AMD disease progression.

It is known that hydroxyapatite crystal growth can be attenuated in the presence of magnesium. Therefore, in an environment where magnesium concentration is low, crystal growth might occur unchecked leading to the formation of these large nodules. As HIRD have been associated with increased risk of progression to irreversible vision loss within a year, the need for appropriate magnesium availability might be critical for the prevention of AMD disease progression. The reason why HIRD signify a risk for progression is linked to the reduced signal observed on fundus autofluorescence photographs, a diagnostic clinical imaging technique, from the RPE cells overlying drusen with HIRD. RPE degeneration may thus result in changes in the extracellular environment promoting the formation of HIRD.

Our study, therefore, highlighted the importance of metal ions in AMD pathogenesis. Understanding how metal ions contribute to the initiation and progression of AMD may facilitate the development of novel therapeutic interventions. For example, in calcific cardiovascular disorders, magnesium supplementation is being trialed as a potential treatment strategy (23-25). If this is successful, a similar approach could be trialed for AMD. However, the function of metal ions at the choroid-RPE interface appears highly complex.

A recent investigation by Tisdale and colleagues, as part of the ARED study, suggested increased calcium intake provides protection against progression to AMD. While reinforcing the role of diet also found in our work (12, 26, 27), these new results are reminiscent of paradoxical findings previously reported for zinc: zinc supplementation has an apparent protective effect for AMD, whilst accumulation of zinc is also reported within drusen.

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While these discrepancies have yet to be resolved, it is possible that the accumulation of metal ions within drusen are a sign of an overall imbalance, which could potentially be remedied by nutrition or supplementation. Our study further demonstrated the remarkable capability of modern multimodal ophthalmic imaging for longitudinal follow-up of molecularly-defined processes, in living patients.

These findings are described in the article entitled Calcified nodules in retinal drusen are associated with disease progression in age-related macular degeneration, recently published in the journal Science Translational Medicine.

References:

  1. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. The Lancet Global health. 2014;2(2):e106-16.
  2. Gehrs KM, Anderson DH, Johnson LV, Hageman GS. Age‐related macular degeneration—emerging pathogenetic and therapeutic concepts. Annals of Medicine. 2006;38(7):450-71.
  3. Ferris FL, 3rd, Wilkinson CP, Bird A, Chakravarthy U, Chew E, Csaky K, et al. Clinical classification of age-related macular degeneration. Ophthalmology. 2013;120(4):844-51.
  4. Wang JJ, Rochtchina E, Lee AJ, Chia EM, Smith W, Cumming RG, et al. Ten-year incidence and progression of age-related maculopathy: the blue Mountains Eye Study. Ophthalmology. 2007;114(1):92-8.
  5. Joachim N, Mitchell P, Burlutsky G, Kifley A, Wang JJ. The Incidence and Progression of Age-Related Macular Degeneration over 15 Years: The Blue Mountains Eye Study. Ophthalmology. 2015;122(12):2482-9.
  6. Hageman GS, Mullins RF, Russell SR, Johnson LV, Anderson DH. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 1999;13(3):477-84.
  7. Anderson DH, Ozaki S, Nealon M, Neitz J, Mullins RF, Hageman GS, 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. 2001;131(6):767-81.
  8. Mullins RF, Russell SR, Anderson DH, Hageman GS. Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2000;14(7):835-46.
  9. Ruberti JW, Curcio CA, Millican CL, Menco BP, Huang JD, Johnson M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch’s membrane. Invest Ophthalmol Vis Sci. 2003;44(4):1753-9.
  10. Suzuki M, Kamei M, Itabe H, Yoneda K, Bando H, Kume N, et al. Oxidized phospholipids in the macula increase with age and in eyes with age-related macular degeneration. Molecular vision. 2007;13:772-8.
  11. Pauleikhoff D, Harper CA, Marshall J, Bird AC. Aging changes in Bruch’s membrane. A histochemical and morphologic study. Ophthalmology. 1990;97(2):171-8.
  12. Lengyel I, Flinn JM, Peto T, Linkous DH, Cano K, Bird AC, et al. High concentration of zinc in sub-retinal pigment epithelial deposits. Exp Eye Res. 2007;84(4):772-80.
  13. Flinn JM, Kakalec P, Tappero R, Jones B, Lengyel I. Correlations in distribution and concentration of calcium, copper and iron with zinc in isolated extracellular deposits associated with age-related macular degeneration. Metallomics. 2014;6(7):1223-8.
  14. Green WR, Key SN, 3rd. Senile macular degeneration: a histopathologic study. Transactions of the American Ophthalmological Society. 1977;75:180-254.
  15. Klien BA. The Heredodegeneration of the Macula Lutea*: Diagnostic and Differential Diagnostic Considerations and A Histopathologic Report. American Journal of Ophthalmology. 1950;33(3, Part 1):371-9.
  16. Ulshafer RJ, Allen CB, Nicolaissen JB, Rubin ML. Scanning electron microscopy of human drusen. Investigative Ophthalmology & Visual Science. 1987;28(4):683-9.
  17. van der Schaft TL, de Bruijn WC, Mooy CM, Ketelaars DM, de Jong PM. Element analysis of the early stages of age-related macular degeneration. Archives of Ophthalmology. 1992;110(3):389-94.
  18. Thompson RB, Reffatto V, Bundy JG, Kortvely E, Flinn JM, Lanzirotti A, et al. Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye. Proceedings of the National Academy of Sciences. 2015;112(5):1565-70.
  19. Veerappan M, El-Hage-Sleiman A-KM, Tai V, Chiu SJ, Winter KP, Stinnett SS, et al. Optical Coherence Tomography Reflective Drusen Substructures Predict Progression to Geographic Atrophy in Age-related Macular Degeneration. Ophthalmology. 2016;123(12):2554-70.
  20. Bonnet C, Querques G, Zerbib J, Oubraham H, Garavito RB, Puche N, et al. Hyperreflective pyramidal structures on optical coherence tomography in geographic atrophy areas. Retina (Philadelphia, Pa). 2014;34(8):1524-30.
  21. Sarks JP, Sarks SH, Killingsworth MC. Evolution of geographic atrophy of the retinal pigment epithelium. Eye (London, England). 1988;2 ( Pt 5):552-77.
  22. Suzuki M, Curcio CA, Mullins RF, Spaide RF. REFRACTILE DRUSEN: Clinical Imaging and Candidate Histology. Retina (Philadelphia, Pa). 2015;35(5):859-65.
  23. Turgut F, Kanbay M, Metin MR, Uz E, Akcay A, Covic A. Magnesium supplementation helps to improve carotid intima media thickness in patients on hemodialysis. International Urology and Nephrology. 2008;40(4):1075.
  24. Bots ML, Hoes AW, Koudstaal PJ, Hofman A, Grobbee DE. Common Carotid Intima-Media Thickness and Risk of Stroke and Myocardial Infarction. The Rotterdam Study. 1997;96(5):1432-7.
  25. Bressendorff I, Hansen D, Schou M, Kragelund C, Brandi L. The effect of magnesium supplementation on vascular calcification in chronic kidney disease-a randomised clinical trial (MAGiCAL-CKD): essential study design and rationale. BMJ open. 2017;7(6):e016795.
  26. A.R.E.D.S. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Archives of ophthalmology (Chicago, Ill : 1960). 2001;119(10):1417-36.
  27. Tisdale AK, Agrón E, Sunshine SB, Clemons TE, Ferris FL, III, Chew EY, et al. Association of Dietary and Supplementary Calcium Intake With Age-Related Macular Degeneration: Age-Related Eye Disease Study Report 39Association of Dietary and Supplementary Calcium Intake With Age-Related Macular DegenerationAssociation of Dietary and Supplementary Calcium Intake With Age-Related Macular Degeneration. 2019.

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