Postnatal Chick Choroids Exhibit Increased Retinaldehyde Dehydrogenase Activity During Recovery From Form Deprivation Induced Myopia

Abstract

PurposeIncreases in retinaldehyde dehydrogenase 2 (RALDH2) transcript in the chick choroid suggest that RALDH2 may be responsible for increases observed in all-trans-retinoic acid (atRA) synthesis during recovery from myopic defocus. The purpose of the present study was to examine RALDH2 protein expression, RALDH activity, and distribution of RALDH2 cells in control and recovering chick ocular tissues.MethodsMyopia was induced in White Leghorn chicks for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of RALDH isoforms in chick ocular tissues was evaluated by Western blot. Catalytic activity of RALDH was measured in choroidal cytosol fractions using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Distribution of RALDH2 cells throughout the choroid was evaluated by immunohistochemistry.ResultsRALDH2 was expressed predominately in the chick choroid (P < 0.001) and increased after 24 hours and 4 days of recovery (76%, 74%, and 165%, respectively; P < 0.05). Activity of RALDH was detected solely in the choroid and was elevated at 3 and 7 days of recovery compared to controls (70% and 48%, respectively; P < 0.05). The number of RALDH2 immunopositive cells in recovering choroids was increased at 24 hours and 4 to 15 days of recovery (P < 0.05) and were concentrated toward the RPE side compared to controls.ConclusionsThe results of this study suggest that RALDH2 is the major RALDH isoform in the chick choroid and is responsible for the increased RALDH activity seen during recovery.

Design, synthesis, and ex vivo evaluation of a selective inhibitor for retinaldehyde dehydrogenase enzymes

“…We and others 37,38,86 have previously shown that RALDH2 protein expression and RALDH enzymatic activity are detectible in the choroids of chick eyes, with negligible amounts in the retina/RPE and sclera. Moreover, ATRA synthesis is increased in the chick choroid during recovery from form-deprivation induced myopia, which results from an increase in RALDH2 protein expression.…”

“…We have previously established that RALDH2 expression and activity are increased significantly in recovering choroids 37 and that RALDH2 is responsible for elevated ATRA concentrations in recovering choroids. 38, 86 Moreover, when placed in vitro , only recovering choroids are capable of de novo ATRA synthesis. 37 The EC 50 value for inhibition of ATRA formation in living recovering choroids by compound 2 was estimated to be 119.7 nM, but DAR only inhibited a maximum of 51% of ATRA formation in recovering choroids (with maximum inhibition at 1 μM).…”

“…Relative RALDH2 protein expression was determined by western blot, as previously described. 38 RALDH2 protein expression using the (Dox) RALDH2-eGFP 293 HEK cell line ranged from 25% – 37% following incubation with 0.05 – 2 μM DAR, as determined from three independent experiments, each performed in triplicate.…”

“…38 Briefly, an 8 mm punch was taken from the posterior pole of the chick eye using a dermal biopsy punch (Miltex, Inc.; York, PA). Punches were located nasal to the exit of the optic nerve, with care to exclude the optic nerve and pecten.…”

“…RALDH1 has been linked to obesity, inflammation, and Crohn’s disease, 32-36 while RALDH2 has been associated with abnormal ocular growth (myopia). 37, 38 Changes in the expression of all three RALDH proteins in tumors have been associated with poor cancer Prognoses. 39-44 …”