Short-Term Omega-3 Supplementation Modulates Novel Neurovascular and Fatty Acid Metabolic Proteome Changes in the Retina and Ophthalmic Artery of Mice with Targeted Cyp2c44 Gene Deletion

Abstract

Cytochrome P450 (CYP) gene mutations are a common predisposition associated with glaucoma. Although the molecular mechanisms are largely unknown, omega-3 polyunsaturated fatty acids (ω-3 PUFA) and their CYP-derived bioactive mediators play crucial roles in the ocular system. Here, we elucidated the proteome and cell-signalling alterations attributed to the main human CYP2C gene deficiency using a homologous murine model (Cyp2c44−/−), and unravelled the effects of acute ω-3 PUFA supplementation in two ocular vascular beds comprising the retrobulbar ophthalmic artery (OA) and retina (R). Male Cyp2c44−/− mice (KO) and their floxed littermates (WT) were gavaged daily for 7 days with 0.01 mL/g of ω-3 PUFA composed of menhaden fish oil. Another group in respective strains served as vehicle-treated controls. OA and R were isolated at day 8 post-treatment (n = 9/group) and subjected to mass spectrometry (MS)-based proteomics and in silico bioinformatics analyses. Cyp2c44−/− resulted in significant detrimental proteome changes associated with compromised vascular integrity and degeneration in the OA and R, respectively. However, notable changes in the OA after ω-3 PUFA intake were associated with the maintenance of intercellular junctional and endothelial cell functions, as well as activation of the fatty acid metabolic pathway in the KO mice. Conversely, ω-3 PUFA supplementation profoundly influenced the regulation of a large majority of retinal proteins involved in the preservation of neuronal and phototransduction activities in WT mice, namely synaptophysin, phosducin and guanylate cyclase-1, while significantly abrogating degenerative processes in the KO mice via the regulation of, namely, synaptotagmin-1 and beta-crystallin B2. In gist, this study demonstrated that dietary supplementation with ω-3 PUFA for a short period of seven days regulated specific neuro-vasculoprotective mechanisms to preserve the functionality of the OA and R in the absence of Cyp2c44. The potential adjunct use of ω-3 PUFA for glaucoma therapy needs further investigation.

Adaptive responses of neuronal cells to chronic endoplasmic reticulum (ER) stress

“…Proteome analysis was performed using the nanoLC-ESI-MS/MS system as previously described [ 35 , 36 ]. Label-free quantification analysis of designated samples identified 1578 proteins with a false discovery rate (FDR) of 1 % (Supplementary data 05).…”

“…Label-free quantitative discovery proteomics analysis and the corresponding steps such as sample preparation, protein extraction, nano-liquid chromatography–electrospray ionization–MS/MS (nLC-ESI-MS/MS) analysis, and bioinformatics analyses to elucidate the distinct functional annotation and pathways employing the Ingenuity Pathway Analysis (IPA) tool were carried out according to the procedures described elsewhere [ 35 , 36 ]. Detailed parameters for this analysis are described in Supplementary Data S6.…”

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate

“…The nano-liquid chromatography (nLC)-MS system utilized consisted of an EASY-nLC 1,200 system (Thermo Scientific, Rockford, United States) with an Acclaim PepMap RSLC, 75 µm × 50 cm, nanoViper analytical column (Thermo Scientific, Rockford, United States) directly coupled to ESI-LTQ-Orbitrap-XL MS (Thermo Scientific, Bremen, Germany), as described elsewhere ( Perumal et al, 2022 ). Two microliters of each sample (500 ng) were used to fractionate peptides at a flow of 300 μL/min.…”

“…In brief, the LTQ-Orbitrap operated in a data-dependent mode of acquisition, and survey full-scan MS spectra from m/z 300 to 2000 were acquired in the Orbitrap with a resolution of 30,000 at m/z 400 and a target automatic gain control (AGC) setting of 1.0 × 10 6 ions. The ten most intense precursor ions were sequentially isolated for fragmentation and recorded in the LTQ ( Perumal et al, 2022 ).…”

Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma

“…After 24 h, the cells were morphologically examined by light microscopy (Leica Microsystems GmbH, Wetzlar, Germany), harvested by scraping and stored at −20 • C in 1.5 mL tubes. Prior to homogenization, the tubes were filled with 300 µL of Tissue Protein Extraction Reagent, T-PER (Thermo Fisher Scientific, Rockford, IL, USA), as well as a 1% protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA), and subsequently subjected to homogenization with a Bullet Blender ® Storm 24 homogenizer (Next Advance, Inc., New York, NY, USA) as described in our previous studies [29,30]. The samples were then centrifuged, and the supernatant containing the proteins was collected in new 1.5 mL tubes.…”