Integrative Metabolomics and Transcriptomics Identify Lipid Metabolism as a Key Mediator in Retinopathy of Prematurity

Abstract

Purpose : Hypoxic insults during early developmental stage of preterm infants affect neurovascular growth in the retina. Earlier, we showed a significant association of genes involved in cholesterol metabolism, and immune regulation in retinopathy of prematurity (ROP) pathogenesis, although their mechanisms are elusive. Herein, we present an integrative analysis of vitreous humour (VH) metabolome and ROP retina and fibrovascular membranes (FVMs) transcriptome in order to decipher the roles of lipid metabolizing enzymes in ROP.

Methods : VH (n=30), FVM (n-10) and retina (n=3) were collected from severe ROP cases during vitrectomy, membranectomy and evisceration surgeries, respectively. The age-matched non-ROP retina (n=3) were collected from eyes with anterior staphyloma. VH samples were analysed for global metabolomics by using the methanol chloroform method followed by LC-MS. Global transcriptome analysis was performed on FVM, ROP and non-ROP retina using microarray. Metabolomics data was analysed using XCMS software and MS/MS fragmentation pattern was confirmed on the human metabolome database (HMDB). Gene expression data was analysed using GeneSpring software and were subsequently validated by qPCR and immunohistochemistry (IHC).

Results : We observed 1031 differentially expressed metabolites (fold change: +2, p-value <0.05) of which, 43.2% (445) belonged to the lipid class, 27.2% (285) belonged to amino acids, peptides and analogues, while the rest were organic/benzenoid derivatives. Transcriptomics revealed differentially regulated (with a fold change of > +2) genes for ROP compared to non-ROP retina (4891), FVM to ROP retina (4136) and FVM compared to the non-ROP retina (4662), respectively. However, only 10-12% of differentially regulated genes were significant (p < 0.05). Retinal cell specific genes were downregulated in ROP retina. Genes involved in lipid metabolism (EPHX2, ALOX5, CERS2, CYP1B1) etc. were significantly dysregulated in ROP retina. Overall, the major pathways identified in ROP pathogenesis included the TCA cycle, arachidonic acid metabolism, cytokine and VEGF-MAPK2 signalling that were further confirmed by targeted gene expression and IHC of retinal tissues.

Conclusions : An interplay of hypoxia and altered lipid metabolism contributed to inflammation in ROP pathogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.