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LC-MS/MS targeted analysis of ferroptosis-associated phospholipid peroxidation in KIF11-depleted HRECs

ABSTRACT:

Human retinal vascular endothelial cells (HRECs) were transfected with lentivirus encoding either KIF11-targeting shRNA or a non-targeting control. KIF11-depleted and control cells were then subjected to lipid extraction followed by LC–MS/MS–based targeted analysis of ferroptosis-associated phospholipid peroxidation.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase

PROVIDER: MTBLS12912 | MetaboLights | 2026-02-13

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS12912_LC-MS_negative_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS12912_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular
	s_MTBLS12912.txt	Txt

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Project description:Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, we hypothesized that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from 6 human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasite:cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8747 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false discovery rate set at 5 %. For selected genes, differences in level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphical descriptive analysis demonstrated strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 of 8,747 transcripts (8.9 %) were differentially represented. Notably, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. Our findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte. Experiment Overall Design: Retinal and choroidal vascular endothelial cells were isolated from both eyes of 6 human cadaver donors. Endothelial cells of each subtype from each donor were separately pooled and cultured. In a first series of experiments (Experiment 1), donor-matched cultures of retinal and choroidal endothelial cells derived from 3 donors were incubated with either T. gondii tachyzoites or medium alone. In a second set of experiments (Experiment 2), cultured retinal and choroidal endothelial cells derived from the other 3 donors were stimulated with LPS or medium alone. Whenever possible, 2 replicate dishes of endothelial cells were subjected to each set of conditions. Subsequent to incubations, mRNA was extracted from a cell lysate prepared from each dish of cells, and cRNA derived from each mRNA preparation was separately hybridized to one oligonucleotide expression array.

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LC-MS/MS targeted analysis of ferroptosis-associated phospholipid peroxidation in KIF11-depleted HRECs

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