Abstract 13520: Paradoxical Increase in Background Phosphorylation of Bmp-Responsive Proteins in Blood-Derived Endothelial Cells From Bmpr2 Mutation Carriers With Hereditary Pulmonary Arterial Hypertension
Background: Severe pulmonary arterial hypertension (PAH) is a rare but lethal disease, which is characterized by profound dysregulation in growth and survival of pulmonary endothelial cells (ECs). Hereditary PAH (HPAH) is usually caused by heterozygous mutations in bone morphogenetic protein type II receptor (Bmpr2), with “loss of function” in downstream Smad signaling. However, the precise mechanism by which mutation of this gene causes PAH is unclear. Aim: To elucidate the effects of Bmpr2 mutations on the activation of proteins in the BMP signaling pathway in ECs using an unbiased proteomic approach.
Methods: Blood-derived, late outgrowth ECs were expanded ex vivo from peripheral blood samples of four PAH patients with Bmpr2 mutations and four healthy control subjects. Proteins were isolated from quiescent cells with and without exposure to BMP-9 (1 ng/ml, 8 hours) and subjected to high-resolution 2D electrophoresis following by multiplex-staining for proteins (Sypro Ruby) and phosphoproteins (Pro-Q Diamond), and differential protein expression and phosphorylation was determined using PDQuest software.
Results: Boolean analysis of the expression profiles identified 18 differentially activated phosphoproteins. In healthy cells, phosphorylation was increased for 17 proteins and reduced for a single protein in response to BMP-9. In contrast, in cells from PAH patients carrying Bmpr2 mutations, the responses to BMP-9 were lost entirely. However, in all but one instance, there was a profound increase in basal phosphorylation of the same proteins to levels that were at, or above, the maximal activation seen in response to BMP in the healthy control cells. These proteins have been harvested and are being subjected to mass spectrometry analysis (LC-MS/MS) to allow protein identification.
Conclusions: These data suggest that while Bmpr2 mutations resulted in the loss of responsiveness to the BMP ligand, they caused a profound constitutive increase in phosphorylation of BMP signaling proteins which was independent of ligand availability. This could explain how a single copy of a mutant Bmpr2 gene can have such an important effect on EC biology.
- © 2011 by American Heart Association, Inc.