Novel Gene Implicated in PH Development in Preclinical Study

A gene called DNMT3B was found for the first time to be a mediator of blood vessel wall growth, and has been implicated in the development of pulmonary hypertension (PH) in a rat study.

This gene, which regulates inflammatory pathways by epigenetic mechanisms, may be a potential therapeutic target for the treatment of PH, scientists said. 

The study, “DNA methyltransferase 3B deficiency unveils a new pathological mechanism of pulmonary hypertension,” was published in the journal Science Advances.

Epigenetic mechanisms in biology are modifications to DNA that control the activation or silencing of genes. DNA methylation is one type of epigenetic mechanism in which enzymes called DNA methyltransferases (DNMTs) add so-called methyl groups to DNA to reduce or block gene activity. 

Studies have suggested that altered DNA methylation plays a critical role in the development of PH, which includes pathological alterations to the PH-related BMPR2 gene. However, the part played by DNMT enzymes in PH remains poorly understood.

Now, researchers based at the Tongji University School of Medicine in China examined the epigenetic changes in rat models of PH and the activity and influence of DNMT enzymes on disease processes. 

PH was induced in rats by injecting a compound called monocrotaline (MCT), which leads to higher blood pressure in the heart and pulmonary arteries (hypertension) as well as thickening of heart tissue after three weeks. PH was also induced in rats by depriving them of oxygen (hypoxia) in a hypobaric chamber for three weeks. 

DNA methylation analysis showed a 1.8 times increase of overall DNA methylation after 21 days in whole lungs of MCT-induced PH rats compared to controls. 

Likewise, hypoxia-induced PH rats had a 1.4-fold elevation in DNA methylation in lungs compared to non-hypoxic control animals, suggesting that “global DNA methylation levels are up-regulated in PH rodent models,” the researchers wrote. 

Next, the team tested the activity of four DNMT genes to determine which was responsible for increased methylation and found one — DNMT3B — that was highly active in the blood vessel walls of lung tissue of both PH rat models at 21 days. 

Consistently, significantly elevated human DNMT3B gene activity was seen in lung tissues of patients with pulmonary arterial hypertension (PAH) who have heart defects (congenital heart disease) compared to those without PAH. Moreover, the levels of DNMT3B in pulmonary artery smooth muscle cells (PASMCs) isolated from patients with PAH were found to be 24-times higher than in controls.

In contrast, no increase in DNMT3B activity was found in pulmonary arterial endothelial cells that line blood vessels suggesting that “DNMT3B is up-regulated in PASMCs of patients with PAH and experimental PH models,” the team wrote. 

To investigate the role of DNMT3B in PH, the gene was removed to create a DNMT3B-deficient rat model. Four weeks after their MCT injection, DNMT3B-deficient rats had significantly higher blood pressure in the heart and pulmonary arteries and heart tissue thickening than normal rats. 

Conversely, no differences in a heart function test were found, suggesting that “Dnmt3b is mainly involved in pulmonary vascular remodeling, while its effect on cardiac function is still uncertain,” the researchers wrote. “These data collectively suggest that Dnmt3b deficiency exacerbates the development of MCT-induced PH.”

Normal and DNMT3B-deficient rats were then treated in the low-oxygen chamber for three weeks. Compared to controls, DNMT3B–deficient rats showed a significant increase in heart and lung hypertension as well as in heart tissue thickening. 

Moreover, DNMT3B deficiency worsened vascular remodeling in hypoxic rats by increased pulmonary vascular wall thickness and muscularization. Altogether, these findings provided “the first evidence that Dnmt3b confers protection against PH development and progression,” the team wrote.

To test if increased enzyme activity could influence disease development, the human DNMT3B gene was delivered to the lungs of DNMT3B–deficient and control rats. Compared to control rats, overexpression of human DNMT3B significantly reduced hypertension in response to two weeks of hypoxia.

Furthermore, DNMT3B expression protected against blood vessel thickness and muscularization, “supporting a notion that DNMT3B may have a therapeutic potential to decelerate PH progression,” the researchers wrote. 

Excess growth of smooth muscle cells in lung blood vessels is considered an underlying mechanism of vascular remodeling in PH; thus, the team assessed the role of DNMT3B in muscle cell growth under PDGF-BB stimuli — a protein that triggers the growth of PASMCs. 

When PDGF-BB-treated cells were exposed to various concentrations of a molecule that blocks DNMT3B, cell growth increased in a dose-dependent manner. DNMT3B blockage also affected human PASMC migration. Overexpressing human DNMT3B together with PDGF-BB abolished the impact of PDGF-BB on cell growth and reduction of cell migration.

“Collectively, these results suggest that DNMT3B acts against PDGF-BB-induced proliferation and migration of PASMCs in a protective manner,” the researchers wrote. 

Experiments to find the underlying mechanism by which DNMT3B confers protection against PH identified genes related to inflammation as being the most affected by increased DNMT3B activity in human PASMCs. In particular, the activity of a pro-inflammatory gene called CCL5 was reduced upon DNMT3B expression. 

Consistently, increased CCL5 expression was observed in DNMT3B-deficient rats compared to controls after MCT or hypoxia exposure, demonstrating that “inflammatory regulation might contribute to the protective role of Dnmt3b in vascular remodeling,” the scientists wrote. 

Taken together, the “results reveal that DNMT3B is a previously undefined mediator in the pathogenesis of PH, which couples epigenetic regulations with vascular remodeling and represents a therapeutic target to tackle PH,” the team concluded.