June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Biochemical Dissection of Pseudoexfoliative Glaucoma Deposit Formation
Author Affiliations & Notes
  • Anna Mueller
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States
  • Vanessa Collao
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States
  • Richard K Lee
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States
  • Anna K Junk
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States
    Veterans Health Administration, Miami, Florida, United States
  • Sanjoy K Bhattacharya
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Anna Mueller None; Vanessa Collao None; Richard Lee None; Anna Junk None; Sanjoy Bhattacharya None
  • Footnotes
    Support  This work was partially supported by an RPB unrestricted grant to the University of Miami and NIH R01 031292
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 56. doi:
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    • Get Citation

      Anna Mueller, Vanessa Collao, Richard K Lee, Anna K Junk, Sanjoy K Bhattacharya; Biochemical Dissection of Pseudoexfoliative Glaucoma Deposit Formation. Invest. Ophthalmol. Vis. Sci. 2023;64(8):56.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To determine whether vitamin D-binding protein (GC) serves as a nucleation center for the formation of pseudoexfoliative material (PEXM). Previous proteomic studies demonstrated the presence of GC in PEXM. We aim to capture the mechanistic steps that lead to the formation of PEXM in vitro.

Methods : 50-100 µl aqueous humor (AH) were obtained via paracentesis from patients with pseudoexfoliative glaucoma (PEXG), primary open angle glaucoma (POAG), and cataract only (n=10 per group) under institutional review board approved protocols adhering to the Tenets of the Declaration of Helsinki. All patients were Caucasian, >30 years old, evenly distributed by sex, and did not have systemic comorbidities or co-existing ocular disease. In each group, 10 µl AH were seeded with or without 1 µl GC (10 mg/ml, AB90920, Abcam Inc, Waltham, MA), covered with coverslip, sealed with an insert, and incubated for 3-7 days without disturbance. Slides were monitored daily and imaged with confocal microscopy (Stellaris 5, Leica Microsystems Inc., Deerfield, IL). Deposits were drained off, washed with 5 µl distilled water, digested with trypsin, and subjected to mass spectrometry (MS) on a Q-Exactive instrument (Thermo Fischer Scientific, Waltham, MA). C57BL/6J mice (n=6, 4 month old, even sex distribution), handled according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, were injected intracamerally with 1 µl GC or human serum albumin (10mg/ml; Abcam).

Results : We observed deposit formation only in the 10 PEXG samples (all 10x10) with GC—all formed by day 5. In 3/10 samples, >7 day incubation led to crystal formation. None of POAG and cataract only samples formed deposits. MS identified proteins previously reported in patient PEXM. Intracameral injection of GC (but not serum albumin) in mice led to deposit formation within 16-24 hours.

Conclusions : Our in vitro studies demonstrate deposit formation only in PEXG AH and only when GC is added. Increased GC levels are linked to solar radiation, which has been previously shown to accelerate PEXM formation. Our in vitro studies are consistent with our hypothesis that GC, with its large positive and negative patches, serves as a nucleation center for PEXM proteins in AH, i.e., GC optimizes the environment for proteins to form PEXM. Our in vivo experiments demonstrating deposit formation with GC injections in mice further support our hypothesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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