Retinoic acid synthesis by a population of choroidal stromal cells

Choroidal all- trans -retinoic acid (atRA) may play a key role in the control of postnatal eye growth in a variety of vertebrates through modulation of scleral extracellular matrix synthesis and may therefore play a crucial role in the development of myopia. In the chick eye, choroidal atRA synthesis is exclusively regulated by its synthesizing enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In chicks and humans, RALDH2 has been detected in a population of hitherto uncharacterized choroidal cells.Therefore, the aim of this study was to identify the RALDH2+ cell type(s) in the choroid and determine how these cells modulate atRA concentrations during periods of visually guided eye growth. Chicks wore translucent goggles on one eye for 10 days and choroids were analyzed for RALDH activity and RALDH2 protein expression at days 0, 1, 4, 7, 15 following removal of the goggle ("recovery"); choroids from contralateral eyes served as controls. The presence of RALDH2+ cells was assessed in chick choroid wholemounts using multiphoton microscopy. RALDH2 protein expression was measured by western blot and RALDH2 activity was assessed via HPLC quantification of atRA. Cell proliferation was assessed by BrdU-labelling in combination with RALDH2-immunohistochemistry. For characterization of RALDH2+ cells, immunohistochemistry for various tissue specific markers was applied in chicken (Ia antigen, CD5, Col1-propeptide, desmin, IgY, L-Cam, Cadherin1, MHC-II; Tcr-γδ, vimentin) and human donor tissue (α-smooth-muscle-actin, CD's 31/34/68/146, desmin, IBA1, LYVE-1, PGP9.5, vimentin) followed by confocal microscopy. In the chick and human choroid, RALDH2+ cells with variable morphology were present in the stroma and adjacent to choroidal blood vessels. In chick wholemounts, RALDH2+ cells were concentrated toward the choriocapillaris, and their number increased nearly linearly between 1 and 7 days of recovery and plateaued between 7 and 15 days compared to corresponding controls. A significant increase in choroidal RALDH2 protein concentration and atRA synthetic activity was observed by four days of recovery (↑107% and ↑120%) by western blot and HPLC, respectively. A 3-fold increase in RALDH2+/BrDU+ cells was observed following 4 days of recovery compared to controls (12.43 ± 0.73% of all RALDH2+ cells in recovering eyes as compared with 4.46 ± 0.63% in control eyes, p < 0.001). In chick choroids, the vast majority of RALDH2+ cells co-expressed Col1-propetide, but did not co-label with any other antibodies tested. In human choroid, some, but not all RALDH2+ cells colocalized with vimentin, but were negative for all other antibodies tested. RALDH2+ cells represent a novel cell type in the chick and human choroid. Our findings that some human RALDH2+ cells were positive for vimentin and all chick RALDH2+ cells were positive for Col1, suggest that RALDH2+ cells most closely resemble perivascular and stromal fibroblasts. The increased number of RALDH2+/BRDU+ cells following 4 days of recovery suggests that choroidal atRA concentrations are partially controlled by proliferation of RALDH2+ cells. The identification of this choroidal cell type will provide a broader understanding of the cellular events responsible for the regulation of postnatal ocular growth, and may provide new avenues for specifically targeted strategies for the treatment of myopia.