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.

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.

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.

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.

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