In an article published in the journal iScience, scientists from the Instituto de Química at Universidade de São Paulo (USP) described changes in retinal lipid metabolism in a model of oxygen-induced retinopathy (OIR), which mimics the retinopathy of prematurity (ROP). They analyzed the retinal lipid composition of mice with normal and pathological angiogenesis and combined this data with changes in the messenger RNA transcriptome. In this way, they identified the lipid signature of pathological angiogenesis. According to the researchers, a better understanding of retinal lipid metabolism may translate into better therapeutic and diagnostic alternatives for the treatment and prevention of human eye diseases.

The retina, considered an extension of the brain, is a tissue of the central nervous system. Located at the back of the eyeball, it receives light and transforms it into visual messages that are sent to the brain. To function well, it depends on the supply of oxygen, nutrients, and essential fatty acids from the diet, carried by blood vessels. Some diseases that affect vision, such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity have in common pathological angiogenesis, characterized by the abnormal growth of new blood vessels. Cancer is also considered an angiogenesis-dependent disease.

The main alterations found in pathological retinas lipidome were decreased levels of polyunsaturated fatty acids omega 3 and 6 and increased polyunsaturated fatty acids of the omega 9 class; alterations in neutral lipid metabolism, in particular, accumulation of lipid droplets rich in cholesterol esters and triglycerides; and alterations in lipoprotein-mediated intraretinal cholesterol transport.

“This is the first time that data from two “omics” approaches - lipidomics and transcriptomics - are integrated in the study of this pathology”, says researcher Lilian Costa Alecrim, who shares first authorship of the article with researcher Alex Inague. According to Inague, “the transcriptomics analysis was the key to integrate and make sense of the lipidomic data. In terms of the complexity of the lipid analysis data, I have not seen similar studies”.

The study was conducted during the Ph.D. research of Inague, under the supervision of Professor Sayuri Miyamoto, and of Alecrim, under the supervision of Professor Ricardo José Giordano, both from the Departamento de Bioquímica, Instituto de Química, USP. Miyamoto is also a researcher at RIDC Redoxoma.

According to Giordano, “This work represents an important contribution to the research field, as it brings a large amount of data, showing the dynamics of lipids in the retina”.

Graphical Abstract – Inague A., et al. iScience 26, 106777. 2023 Jun 16, under license CC BY 4.0

Lipid droplets

To induce pathological angiogenesis in oxygen-induced retinopathy model, newborn mice are submitted to 75% oxygen for five days. The high concentration of oxygen inhibits the production of vascular endothelial growth factor (VEGF) and the formation of blood vessels in the retina. When the animals return to the normal atmospheric oxygen concentration of 21%, poorly vascularized retinas experience a state of relative hypoxia, which increases VEGF expression beyond physiological levels, leading to exacerbated blood vessel growth.

“In the case of us humans, retinal vascularization is completed in the last week of gestation, hence the retinopathy problem in premature infants. In the animal model, the advantage is that the retina vascularization of mice begins after birth. That’s why we manage to introduce the disease into these animals”, explains Alecrim.

From samples collected at different times, the researchers identified and quantified 300 lipid species, both in the physiological and pathological retinas. Of these, 227, or 92% of the total, underwent remodeling, with part of the modifications occurring with the normal development of the animals. The lipidomic investigation had the collaboration of researcher Marcos Y. Yoshinaga, a former postdoc in Miyamoto’s laboratory.

Analyzing the data, what initially caught the researchers’ attention was that virtually all detected species of triacylglycerols and cholesterol esters were increased in retinopathy. “In general, not only in this disease but in a series of other models, including amyotrophic lateral sclerosis (ALS), we see that situations of oxidative stress cause an increase in lipid droplets. We saw a large increase of neutral lipids - cholesterol esters and triacylglycerols - present inside these droplets”, said Inague.

From there, to better understand the data, the researchers integrated the analysis of lipids with messenger RNAs. “With transcriptomics, we compare gene expression in disease and health to see how the DNA is being read. But here the change is much greater: 3,800 genes have differential expression. So, it was necessary to navigate all these genes, filter out those related to lipid metabolism, and, within that, map everything”, Giordano explained. This stage of the work had the collaboration of professor João Carlos Setubal, also from IQ-USP, and his doctoral student Jhonatas Monteiro, who carried out the statistics of the differentially expressed genes.

For Miyamoto, lipid droplet accumulation is a common phenotype in degenerative processes. “This is seen in several neurodegenerative diseases, it is a rather common phenotype in processes involving oxidative stress and in degenerative processes, that still needs to be better understood.“

Omega 9

In another important result, the researchers detected the accumulation of large amounts of Mead acid in the pathological retina. Mead acid is one of the omega-9 fatty acids and may be a marker of the severity of retinopathy of prematurity, a disease caused by abnormal growth of blood vessels in the retina of premature babies, which can lead to blindness.

“In our analysis, we found parameters that differentiate species of polyunsaturated fatty acids. So we found that omega 3 and 6 were reduced and the whole omega-9 pathway was increased in the pathological retina compared to controls. Apparently, in disease, these omega 3 and 6 are replaced by omega 9”, explains Inague. Omega 3 and 6 polyunsaturated fats provide high fluidity for the retina, being important for its function.

These data reinforce the importance of omega 3 and 6 supplementation for the prevention of the retinopathy of prematurity, as has been suggested in the literature. However, there are still issues to be explored, such as controlling the phosphorylation of enzymes known as elongases, which catalyze the extension of fatty acids. Inhibition of ELOVL5 elongase phosphorylation, for example, can cause the retina to use omega 9 instead of omega 3, decreasing the effect of supplementation. Modulation of this enzyme might be related to the vascular endothelial growth factor (VEGF) pathways.

Now, the idea, according to Giordano, is to try to measure Mead acid in biological samples and see if it has a relationship with the severity of retinopathy of prematurity. “It is possible to measure Mead acid in the baby’s blood or perhaps in saliva or even in tears”, he said.

The article Oxygen-induced pathological angiogenesis promotes intense lipid synthesis and remodeling in the retina, by Alex Inague, Lilian Costa Alecrim, Jhonatas Sirino Monteiro, Marcos Yukio Yoshinaga, João Carlos Setubal, Sayuri Miyamoto and Ricardo José Giordano, can be accessed here.