Study of Gene Activity in Endothelial Cells Aims to Better Treat PAH

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by Steve Bryson PhD |

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PAH endothelial cells | Pulmonary Hypertension News | mouse model experiment

Potential targets to treat pulmonary arterial hypertension (PAH) were identified by analyzing the gene activity of individual lung endothelial cells isolated from a mouse model of the disease.

Study results were validated by cross-referencing them with rat and human gene activity datasets.

“The identification of distinct molecular mechanisms and potential therapeutic targets is crucial for the future development of pharmacological interventions targeting endothelial dysfunction,” the researchers wrote.

The study, “Single-cell RNA-seq profiling of mouse endothelial cells in response to pulmonary arterial hypertension,” was published in the journal Cardiovascular Research.

PAH is thought to be triggered by a combination of genetic and environmental factors that damage endothelial cells, the thin layer of cells that line blood vessels and are “involved in the primary vascular changes leading to PAH.” This damage causes structural alterations in pulmonary vessels and inflammation, increasing blood pressure.

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Study Pinpoints Altered Biological Pathways in Endothelial Cells

Changes in gene activity (or gene expression) in PAH have been investigated at the whole-organ and tissue level. Still, many different subtypes of endothelial cells contribute to the disease throughout its development. Single-cell RNA-sequencing allows for gene expression to be measured in individual cell types, helping scientists to identify specific cell populations directly involved in PAH.

Using a mouse model of pulmonary hypertension, researchers at the University of Edinburgh, along with colleagues, carried out single-cell RNA sequencing of isolated lung endothelial cells to examine disease-related changes in gene expression that may help to identify potential therapeutic targets.

“We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution,” the team wrote.

Using mice with induced PAH and a control group without it, they isolated distinct subpopulations of endothelial cells, including two types from tiny blood vessels known as capillaries (called capillaryA and capillaryB), and artery and vein ECs. A subgroup with high levels of lymphatic markers was also found, plus another defined as proliferating (growing) due to high cell cycle-related gene expression.

Gene expression analysis, conducted on each type of endothelial cell, was compared between PAH and control mice to identify genes with different levels of activity, called differentially expressed genes (DEGs).

Overall, 222 DEGs were detected with more differentially expressed genes found in the artery ECs, and both capillary EC types compared to the vein and lymphatic ECs. Proliferating endothelial cells did not show PAH-specific expression changes.

The expression of 17 genes involved in immune responses was higher (upregulated) in artery ECs in PAH. The greatest increase was seen in genes that encode for proteins that are part of the major histocompatibility complex class II (MHC-II) and the related protein Cd74 in the artery and capillaryA ECs, which supported “a role for ECs in the inflammatory response in PAH,” the researchers wrote.

In capillaryB cells, 37 genes showed a stronger response to PAH, which involved cell localization, cell death, and the growth of new blood vessels (angiogenesis). Among these 37 genes, the presence of three genes, Cd34, Plaur, Apln, are known to be active in cells at the tip of growing blood vessels, suggesting “angiogenic regulatory pathways are activated in CapillaryB ECs and enhanced in PAH,” the scientists wrote.

To confirm the relevance of these data, the team examined whether the expression of human genes with PAH-associated mutations were also altered in the mouse data.

From 12 genes known to drive PAH, four common genes were identified: Aquaporin (Aqp1), Caveolin1 (Cav1), Bone Morphogenetic Protein Receptor Type (Bmpr2), and Endoglin (Eng). Aqp1 had the greatest change in expression and was upregulated in artery, vein, capillaryA, and lymphatic ECs. Cav1 was also upregulated, whereas Bmpr2 and Eng were down-regulated.

Further analysis compared these data to previously determined EC responses to PAH in rats and humans conducted by whole-lung single-cell RNA-sequencing. Overall, the team found 51% of the genes seen to be upregulated in the mice were also differentially expressed in rats or humans, with 20 genes commonly regulated in all three species.

As artery and capillaryA cells had a high number of upregulated genes in the PAH mouse, DEGs that were in common in these cell types were assessed. Three genes (Cd74, Sparc, Slc6a6) were upregulated in artery ECs in all three species, and five (Sparc, Cd81, Anxa2, Id3, Slc9a3r2) were upregulated in capillaryA ECs in mice, rats, and people as well.

Along with CD74, the MHC-II complex genes were also upregulated in human PAH, “suggesting the importance of this pathway,” the scientists added.

The role of CD74 was then characterized by suppressing its expression in human cells. CD74 deficiency led to a decrease in endothelial cell growth and a loss of barrier function, which separates blood from tissue, that supported an “important contribution of CD74 to EC function,” the researchers wrote.

“Overall, our study provides high resolution insights into the diversity of EC subpopulation responses to pulmonary hypertension and highlights novel candidates for future therapeutic development,” the team concluded.