The Actin Cytoskeleton Mediates Hyperoxia Response of Patient-Derived Primary Endothelial Progenitor Cells through Thymosin BETA-4 and Hypoxia-Inducible Factor Signaling

Abstract

Disruptions to pro-growth, proliferation, and angiogenesis signaling pathways within endothelial progenitor cells (EPC) may play key role in the pathogenesis of multiple pediatric pulmonary diseases, such as bronchopulmonary dysplasia. Using high-throughput, single-molecule, single-cell imaging of multiple RNA and protein species, we have created spatiotemporal “stop-motion” movies of F-actin structure, thymosin beta-4 (Tβ4), hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) expression in a unique resource of primary, patient-derived EPC isolated from the umbilical cord blood of preterm- and term-birth infants, grown in vitro using differing oxygen environments. In particular, a hyperoxic growth environment mimics preterm birth, raising the environmental oxygen level at an abnormal time point during fetal development. We find that EPC isolated from preterm- versus term-birth infants have different spatial expression of Tβ4 and HIF-1α protein at basal conditions and that upon exposure to hyperoxia, the two classes of EPC respond with markedly different spatiotemporal expression of Tβ4/HIF-1α protein and VEGF/eNOS RNA. Additionally, hyperoxia causes a remodeling of the actin cytoskeleton that releases excess Tβ4, an actin sequestering protein, into the cytosol. Because preterm EPC have an elevated cytosolic level of Tβ4 at basal conditions compared to term EPC, this excess of Tβ4 activates an inhibitory feedback network to reduce Tβ4 levels and subsequently limits the translocation of HIF-1α, a potent transcription factor for the VEGF-eNOS signaling pathway, to the nucleus. Overall these signaling changes due to hyperoxia have the effect of down-regulating growth, proliferation, and angiogenesis in EPC isolated from preterm-birth infants. Our results suggest a new potential pathway for molecular intervention that may restore normal angiogenesis after preterm birth.