Abstract 1458: Transcriptional regulation of Bim by FOXO3a and Akt Mediates Scleroderma Serum-Induced Apoptosis in Endothelial Progenitor Cells
Objectives: Scleroderma (SSc) is a connective tissue disease with a complex pathobiology involving vasculopathy, fibrosis, and autoimmunity. Endothelial progenitor cells (EPC) contribute to vascular regeneration and repair, thereby protecting against vasculopathy. We aimed to determine (i) whether circulating EPCs were reduced in SSc patients, (ii) whether EPCs were susceptible to apoptosis in the presence of SSc sera, and (iii) whether Akt/FOXO3a/bim signaling pathway mediated SSc sera-induced EPC apoptosis.
METHODS and RESULTS: Circulating EPC levels were quantified in 44 patients with SSc, 48 patients with primary pulmonary hypertension (PPH) and 18 matching healthy controls by colony forming unit (CFU) assay and flow cytometry using CD133/CD34 and CD34/VEGFR2 as EPC markers. Both methods revealed markedly decreased EPC levels in SSc patients relative to patients with PPH and healthy subjects. To determine whether excessive destruction of EPCs upon their mobilization from the bone marrow by factors present in SSc sera contributed to the EPC reduction, EPCs derived from lineage negative umbilical cord blood (UCB) were cultured in the presence of SSc, PPH and normal sera. SSc sera resulted in substantial EPC apoptosis as compared with PPH (7.3±1.6% vs. 2.8±0.9%, p<0.04) and normal sera (7.3±1.6% vs. 1.1±0.7%, p=0.001), as detected by terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) assay. These results were confirmed by DNA fragmentation and caspase3/7 activation assays. Intriguingly, depletion of IgG fraction from SSc sera completely abolished the apoptotic effects. Furthermore, SSc sera inhibited the activation/phosphorylation of the survival kinase Akt, which in turn suppressed the phosphorylation and degradation of forkhead transcription factor FKHRL1 (FOXO3a), resulting in the upregulation of apoptotic proteins bim and, to a much lesser degree, PUMA expression. Importantly, siRNA mediated FOXO3a knockdown inhibited SSc sera-induced bim expression and EPC apoptosis.
SSc patients have lower levels of circulating EPCs.
SSc sera-induced EPC apoptosis is mediated by the Akt-FOX3a-bim pathway, which may account, at least in part, for the decreased circulating EPC levels in SSc patients.