Studies of All-trans Retinoic Acid Transport in the Eye.

PURPOSE

Myopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRA's weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOx 14, CAS:198696-03-6, referred as "floRA"), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues.

METHODS

Binding: We assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I), and retinol binding protein 4 (RBP4). Direct visualization: Wild-type C57BL/6J mice were euthanized, their eyes were enucleated, and wedges containing sclera and choroid were incubated for specific durations in 50 µM floRA + BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ software.

RESULTS

Association constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2 to 13 × 105 M-1, indicating nonspecific binding. floRA could be visualized in the sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei).

CONCLUSIONS

floRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I, and RBP4. Considering these proteins' relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement.