500 Translationally Controlled Tumor Protein (TCTP) Revealed by Proteomic Analysis of Patient-Specific Blood outgrowth Endothelial Cells in Heritable Pulmonary Arterial Hypertension

Abstract

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal disease, characterized by excessive proliferation of pulmonary vascular cells. Hereditary PAH is mainly caused by “lossof-function” mutations in the bone morphogenetic protein type II receptor (Bmpr2). However, the mechanisms by which these mutations cause PAH remain unclear. AIM: To identify dysregulated proteins in blood-derived endothelial cells of PAH patients with Bmpr2 mutations compared with healthy controls that may contribute to the pathogenesis of this disease. METHODS: Blood-outgrowth endothelial cells (ECs) were expanded ex vivo from peripheral blood mononuclear cell samples of four patients with heritable PAH and four healthy subjects. Protein isolates were subjected to 2D gel electrophoresis and stained for total proteins with Sypro Ruby. Gels were scanned and subjected to quantitative computer-assisted analysis (PDQuest software) and differentially regulated proteins determined by statistical tests (p-value 0.05) were identified by mass spectrometry (LC-MS/MS). RESULTS: Of the 416 proteins detected, 11 were significantly downregulated in PAH cells, including proteins in pathways previously implicated in PAH (i.e. PKA and guanine nucleotide-binding protein). 11 proteins were significantly upregulated, including a number of cell growth regulatory proteins, notably translationally controlled tumor protein (TCTP). TCTP has previously been shown to be enriched in EC apoptotic nanovesicles and to mediate pro-survival signaling in adjacent cells. Increased expression of TCTP in PAH compared to healthy control cells was demonstrated in patient-derived blood outgrowth ECs in vitro. In addition, the potential role of TCTP in PAH was studied in vivo in the SU5416 (SU) rat model of severe, angioproliferative PAH. SU is a VEGFR2inhibitor which induces EC apoptosis followed by reactive pulmonary vascular cell proliferation causing obliterative pulmonary vascular lesions closely mimicking the human condition. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. CONCLUSIONS: Bmpr2 mutant ECs exhibited dysregulation of key proteins controlling vasodilation and EC growth. TCTP could play an important role in PAH, possibly mediating prosurvival signaling in smooth muscle cells in response to EC apoptosis and thereby contributing to the process of pulmonary vascular remodeling. CIHR, FRQS, Cambridge NIHR Biomedical Research Centre, UK