Oxygen-induced pathological angiogenesis promotes intense lipid synthesis and remodeling in the retina

Description

The retina is a notable tissue with high metabolic needs which relies on specialized vascular networks to protect the neural retina while maintaining constant supplies of oxygen, nutrients and dietary essential fatty acids. Here we determined the lipid content of the developing mouse retina under healthy and pathological angiogenesis using the oxygen-induced retinopathy model (OIR). In the OIR model, pathological angiogenesis is induced in mice through their exposure to variable oxygen levels. Thus, mouse pups with their nursing mothers were kept at 75% O2 from postnatal day 7 (P7) until day 12 (P12). Mice were then returned to ambient air (20.8% O2) and retinas (N=6) were collected at different time points (P12, P12.5, P15 and P17) and used in a lipid extraction procedure. Retinas from mouse pups under physiological development (controls) were also collected. Total lipid extracts were then used for a comprehensive non-targeted lipidomic analysis, whose quantitative results for 300 lipid species are presented here (‘Inague and Alecrim et al - Lipidomics Data’ spreadsheet). Lipidomics data was integrated with previously obtained transcriptomics data (SRA: SRP155931; BioProject: PRJNA483866). By matching the lipid profile to changes in the mRNA transcriptome, we identified a lipid signature associated with oxygen-induced retinopathy. Our data show that pathological angiogenesis leads to intense lipid remodeling favoring pathways for neutral lipid synthesis, cholesterol import/export and lipid droplet formation. The lipid signature also indicates that, from its early stages, pathological angiogenesis induces profound changes in pathways for the production of long-chain fatty acids, vital for retinal homeostasis. The net result is the production of large quantities of mead acid, a marker of essential fatty acid deficiency, which might also be an important marker for retinopathy severity. In sum, our lipid signature might contribute to a better understanding of many diseases of the retina that lead to vision impairment or blindness. This lipid signature might also be important for the development of additional translational therapies for retinopathy and other angiogenesis-dependent diseases.

Steps to reproduce

Retinas in 600 μL of 50 mM phosphate buffer (pH 7.4), containing 100 μM deferoxamine mesylate (Cat# D9533, Sigma-Aldrich), were lysed with zirconia beads and a Mixer Mill MM 301 homogenizer (Retsch GmbH, Germany). Lysis was performed with a cycle of 1 min at a frequency of 30 s-1. 500 μL aliquots were then used in the lipid extraction procedure. Each aliquot was mixed with 400 μL of ice-cold methanol containing 100 μM butylated hydroxytoluene (Cat# W218405, Sigma-Aldrich) and 100 μL of lipid internal standards (10 ng/µL). 2.0 mL of chloroform: ethyl acetate (4:1) were added to each mixture, followed by vortexing during 30 s. After centrifugation at 1,500 x g for 2 min at 4 °C, the lower phase containing the total lipid extracts (TLE) was transferred to new tubes, dried under N2 gas and dissolved in 100 μL of isopropanol. Non-targeted lipidomic analysis of major lipids was performed by reverse-phase liquid chromatography coupled to mass spectrometry (RPLC-MS). In detail, TLE were analyzed by electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS, Triple TOF 6600, Sciex, USA) interfaced with ultra high-performance liquid chromatography (UHPLC Nexera, Shimadzu, Japan). In UHPLC, the flow rate was set to 0.2 mL/min and oven temperature maintained at 35°C. 1 µL of each TLE was loaded into a C18 column (1.6 µm, 2.1 mm i.d. x 100 mm, CORTECS®, Waters Corporation, USA). Mobile phase A consisted of water: acetonitrile (60:40) while mobile phase B was composed of isopropanol: acetonitrile: water (88:10:2). Mobile phases A and B were supplemented with ammonium acetate or ammonium formate at a final concentration of 10 mM for experiments performed in negative or positive ionization modes, respectively. The linear gradient used was 40% to 100% B over the first 10 min, held at 100% B for additional 2 min, decreased from 100 to 40% B during the next 1 min, and held at 40% B for the remaining 7 min. The MS was operated in both positive and negative ionization modes, and the scan range set at a mass-to-charge ratio of 200-2000 Da. Data for lipid species identification were obtained by Information Dependent Acquisition (IDA®). Analyst®TF 1.7.1 was used for data acquisition with a period cycle time of 1.05 s, 100 ms acquisition time for MS1 scan, 25 ms acquisition time to obtain the top 36 precursor ions, ion spray voltage of -4.5 kV and 5.5 kV for negative and positive modes, respectively, and the cone voltage at +/- 80 V. The curtain gas was set at 25 psi, nebulizer and heater gases at 45 psi and interface heater at 450 °C. The MS/MS data were analyzed with PeakView® 2.2 and lipid molecular species were manually identified with the help of an in-house manufactured Excel-based macro. Lipid quantification was performed with MultiQuant® 3.0.3, and peak areas of precursor ions were normalized to those of their respective lipid internal standards. Final data were expressed as mass of lipid species per total mass of proteins in each sample.

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