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Menaka Thounaojam, Shubhra Rajpurohit, Hossameldin Abouhish, Juan Zou, Ravirajsinh Jadeja, Pamela M Martin, Manuela Bartoli; Differential bile acids biosynthetic pathways in the developing retina and in the oxygen-induced model of retinopathy of prematurity. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3029.
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We recently reported the differential ability of various secondary conjugated bile acids (BAs) in ameliorating pathological neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Several studies have also shown the existence of primary BAs synthetic pathways in extrahepatic tissues, such as brain and retina, however no information is available on this metabolic pathway in the postnatal developing retina. Here, we have investigated retinal BAs synthesis in mice during normal postnatal development and in response to the OIR model of retinopathy of prematurity (ROP).
OIR was induced in mouse pups by subjecting them to different oxygen tensions, according to the protocol of Smith et al. Changes in sterol and BAs metabolic pathways in control and OIR mice were evaluated by RNA sequencing(RNAseq) using an Illumina HiSeq 2500 sequencer. Retinal expression of key enzymes of the classical (CYP7A1) and alternate (CYP46A1 and CYP27A1) BAs synthetic pathways were analyzed at different postnatal days (7, 12, 14, 17, and 23) in control mice and age-matched mice subjected to OIR by immunostaining (cell-specific localization), qPCR and immunoblotting. Retinal BAs content was evaluated using LC-MS/MS assay.
RNAseq data, followed by validation with QPCR and immunoblotting analyses, showed a significant increase in mRNA and protein levels of CYP46A1 (at P12, 14, 17 & 23) and CYP27A1 (at P14, 17 and 23) in normal postnatal retina. On the contrary, CYP7A1 was progressively down-regulated (at P14, 17 and 23). Dual immunofluorescence staining of these enzymes with neuronal (NeuN) and endothelial (CD31) cell markers revealed that while CYP27A1 is immunolocalized in endothelial cells, CYP46A1 is co-localizing with neuronal and endothelial cell markers. A significant dysregulation in retinal sterol and BAs synthetic pathways was found in OIR mice retinas in comparison to control. This was characterized by a significant loss of CYP46A1 and CYP27A1. LC-MS/MS assay further confirmed a significant dysregulation of BAs profiles content in OIR mice compared to control.
In summary, we have found that the developing retina relies primarily in the alternate BAs synthetic pathway. However, in the OIR condition BAs synthesis is significantly down-regulated, thus potentially implicating these signaling molecules also in ROP pathogenesis.
This is a 2021 ARVO Annual Meeting abstract.
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