L‐Citrulline Attenuates Effects of Serotonin Signalling During Proliferation of Pulmonary Artery Smooth Muscle Cells in Pulmonary Hypertension

Abstract

IntroductionPremature infants are at risk for bronchopulmonary dysplasia (BPD), a disorder characterized by abnormal pulmonary development. Inflammation is one major component in causing BPD, which can further lead to abnormal vascular remodelling, causing pulmonary hypertension (PH). During this process, pulmonary artery smooth muscle cell (PASMC) proliferation and hypertrophy contribute to the thickening of the pulmonary artery wall, leading to heightened blood pressures. 5‐hydroxytryptamine (5‐HT) or serotonin has been implicated in PH and is involved in the pathogenesis of PH in adults. Meanwhile, L‐citrulline, a nitric oxide donor, has been effective in reducing PH in acute models of BPD. The role of 5‐HT signalling on PASMC proliferation during early pulmonary vascular development is currently unknown. Thus, the objective of this study is to establish the effect of 5‐HT on PASMC proliferation in a newborn model of early lung development, and to elucidate the interaction of 5‐HT on proliferation of PASMCs in the presence of LPS and L‐citrulline.MethodsSprague Dawley rat pups were grown and euthanized on postnatal day 4, after which the pulmonary arteries (PA) were isolated. Primary cultures of PASMCs were grown from PA explants and treated with varying concentrations of 5‐HT. Proliferation and cell viability assays were performed to measure growth and cell viability after exposure to 5‐HT and L‐citrulline (4mM). Lysate samples were also prepared with the cells to measure protein expression via Western blotting for Cyclin D1, an important protein involved in regulation of the cell cycle, and phosphorylated AKT (p‐AKT), which is involved in cell survival. To investigate the effects of inflammation on 5‐HT signalling, PASMCs were exposed to lipopolysaccharide (LPS, 3 µg/mL) and lysate samples were prepared to measure changes in the expression of the 5‐HT2A receptor.ResultsProliferation assays illustrated a dose‐dependent increase in growth of PASMCs exposed to 5‐HT, reaching a peak 150% growth rate (p < 0.05). Western blots revealed a three‐fold increase in Cyclin D1 expression (p < 0.01) and two‐fold increase in p‐AKT expression (p < 0.05) in PASMCs exposed to 5‐HT compared to protein expression of control PASMCs. Both changes were abrogated in PASMCs exposed to both L‐citrulline and 5‐HT, bringing expression of both proteins to control levels. When exposed to LPS, PASMCs have heightened expression of 5‐HT2A, reaching nearly two times higher than in control PASMCs. This effect is also reversed in PASMCs exposed to LPS and L‐citrulline (p < 0.05).ConclusionOur preliminary data support a role for 5‐HT in pathogenesis on BPD‐PH in the presence and absence of inflammation in a newborn model of lung development, and L‐citrulline may be an attractive safe therapeutic option to reverse or mitigate inflammation induced PH in a newborn model, which could later be translated to human infants.