Nogo-B Receptor Modulates Pulmonary Artery Smooth Muscle Cell Function in Developing Lungs

Abstract

Nogo-B and its receptor (NgBR) are involved in blood vessel growth in developing lungs, but their role in pulmonary artery smooth muscle cell (PASMC) growth is unknown. We hypothesized that NgBR regulates growth of PASMCs by modulating the function of endoplasmic reticulum (ER) and formation of reactive oxygen species (ROS). In utero constriction of the ductus arteriosus created pulmonary hypertension in fetal lambs (hypertensive fetal lamb [HTFL]). PASMCs isolated 8 days after surgery were assessed for the alteration of protein levels by immunoblots and ROS formation by dihydroethidium and Cell ROX deep red fluorescence. NgBR small interfering RNA and plasmid DNA were used to manipulate NgBR levels. Proliferation and wound healing were assessed by cell counts and scratch recovery assay, respectively. Acute ER stress was induced by tunicamycin. Differences of mitogen-activated protein kinase and Akt pathway activation in HTFL versus control PASMCs were evaluated. Results showed that HTFL PASMCs had decreased NgBR levels and increased proliferation, wound healing, ER stress, and ROS formation compared with controls. Knockdown of NgBR in control PASMCs generated a phenotype similar to HTFL, and overexpression in HTFL restored the defective phenotype to control. Decreased NgBR levels were associated with increased ROS formation in HTFL PASMCs. Subsequently, scavenging ROS decreased proliferation and wound healing. Mechanistically, ROS formation decreases NgBR expression, which induces ER stress. This leads to extracellular signal-regulated kinase pathway activation and PASMC phenotype alteration. Our data suggest that decreased NgBR expression in pulmonary hypertension of the newborn contributes to increased PASMC proliferation and oxidative stress, which lead to the pathogenesis of lung injury.

Persistent Pulmonary Hypertension of the Newborn: Pathophysiological Mechanisms and Novel Therapeutic Approaches

“…Decreased NgBR expression is observed in PAECs and PASMCs of PPHN fetal lambs, contributing to abnormal angiogenesis, mediated through increased levels of ROS ( Figure 6). Its knockdown reproduces PPHN phenotype, and its overexpression reverts the abnormalities in both cell types of PPHN lamb models (145,146).…”

“…Recently, it has been shown that Nogo-B/NgBR pathway, involved in pulmonary vascular development, is altered in fetal lambs with PPHN ( 145 , 146 ) ( Figure 1 ). Decreased NgBR expression is observed in PAECs and PASMCs of PPHN fetal lambs, contributing to abnormal angiogenesis, mediated through increased levels of ROS ( Figure 6 ).…”

“…In PAECs, NgBR overexpression increases both AKT, eNOS activities and SOD2 expression and activity, diminishing oxidative stress in PPHN lamb models ( 145 ). In PASMCs, NgBR regulates proliferation by modulating endoplasmic reticulum (ER) stress and ROS formation ( 146 ). Increased ROS decrease NgBR expression, further contributing to abnormal PASMC phenotype, while lower NgBR expression further increases ROS formation, leading to PASMCs hyperproliferation ( 146 ).…”

“…In PASMCs, NgBR regulates proliferation by modulating endoplasmic reticulum (ER) stress and ROS formation ( 146 ). Increased ROS decrease NgBR expression, further contributing to abnormal PASMC phenotype, while lower NgBR expression further increases ROS formation, leading to PASMCs hyperproliferation ( 146 ).…”

Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation

“…Follow-up studies suggest that the cytoplasmic domain of NgBR resembles hydrophobic pockets to bind prenylated Ras and promotes its accumulation and activation at the membrane. Recently, it was reported that the decrease of NgBR expression promoted PASMC proliferation through regulation of reactive oxygen species (ROS) production in an intrauterine pulmonary hypertension (IPH) model [16]. However, the underlying mechanism remains unclear.…”