A Molecular Mechanism Underlying Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension in Neonates

Nitric oxide can be life saving for many newborns with pulmonary hypertension (PH); however, there are subsets of patients who do not respond to current PH treatment. Therefore, scientists at Nationwide Children’s Hospital are investigating the enzymes associated with nitric oxide metabolism in search of novel treatments to cure bronchopulmonary dysplasia-associated PH (BPD-PH).

“Preterm babies with BPD-PH have thickened pulmonary blood vessels from vascular remodelling, and these patients can be particularly refractory to current treatment protocols. We decided to go looking for an answer in cases where nitric oxide didn’t help,” says first author Jennifer Trittmann, MD, MPH, principal investigator in the Center for Perinatal Research at the Abigail Wexner Research Institute at Nationwide Children’s Hospital.

According to the new study published in Physiological Reports, nitric oxide-mediated caspase-3 activation, which is important for apoptosis and angiogenesis in fetal lungs, is regulated by the enzyme DDAH1 (dimethylarginine dimethy-aminohydrolase-1). This finding suggests a potential novel therapeutic target for pulmonary hypertensive disorders in preterm infants.

The primary role of DDAH1 is to degrade asymmetric dimethylarginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. By regulating DDAH1, the team could modify endogenous endothelial nitric oxide production. For a patient with a chronic lung disease like BPD, this might be a long term treatment strategy. The investigators hypothesized that reducing the expression of the DDAH1 gene would lead to decreased nitric oxide levels and, in turn, reduced caspase-3 activation and less angiogenesis as measured by endothelial tube formation.

Indeed, when the team decreased DDAH1 expression using RNA interference in cultured human fetal pulmonary microvascular endothelial cells, the cells had lower nitric oxide levels, greater numbers of viable cells, and lower levels of cleaved caspase-3 and cleaved caspase-8 compared with the control cells. Furthermore, when these cells were cultured in a gel matrix that allows for three-dimensional growth to assess angiogenesis, the cells with decreased DDAH1 expression formed fewer tubes compared with the control cells.

“What we’re seeing is a problem with cells abnormally proliferating in the vascular wall,” says Dr. Trittmann, who is also an Assistant Professor in the Division of Neonatology, Department of Pediatrics at The Ohio State University College of Medicine. “These endothelial cells as well as surrounding cells proliferate inwardly to create the lesion of pulmonary vascular remodelling and that inward proliferation of the cells into the vessel lumen is what we think ultimately leads to pulmonary hypertension.”

The investigators were also able to confirm their results by treating the cell cultures with exogenous ADMA, which had a similar effect to knocking down DDAH1 with RNA interference. Additionally, rescue experiments demonstrated that treatment of cells with reduced DDAH1 expression with a nitric oxide donor molecule could restore the levels of activated caspase-3 and caspase-8, viable cell numbers, and tube formation to the levels seen in the controls.

Lastly, treating the cells with reduced DDAH1 expression that were subsequently treated with the nitric oxide donor with a caspase-3 inhibitor resulted in increased numbers of viable cells and decreased tube formation compared with the controls.

These results support the idea of using targeted therapies to promote apoptosis via caspase-3 activation in the vascular wall to improve angiogenesis and reverse or prevent the vascular wall proliferation in pulmonary hypertension that’s occurring in patients with BPD.

“We hope, that studies such as these will eventually lead to better outcomes for patients with BPD-PH,” says Dr. Trittmann. “We imagine that DDAH1 delivery to the pulmonary vascular endothelium by methods such as gene therapy could be used in BPD patients at high risk for PH as a preventative therapy, and/or as a rescue therapy in patients with known BPD-PH who might have an acute exacerbation.”

The team is currently conducting additional experiments over-expressing DDAH1 in the pulmonary endothelial cells and co-culturing these cells alongside pulmonary smooth muscle cells, which are thought to be the primary cell that contributes to the lesion of vascular remodelling underlying pulmonary hypertension, in order to determine the effects of gene expression alterations in these endothelial cell on smooth muscle cells. In vivo experiments using a DDAH1 endothelial conditional knockout mouse will determine if there are differences in pulmonary function, as well as physiologic measurements of pulmonary hypertension.

Reference

Trittmann JK, Almazroue H, Jin Y, Nelin LD. DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells. Physiological Reports. 2019;7(12):e14150. doi:10.14814/phy2.14150