Export file:


  • RIS(for EndNote,Reference Manager,ProCite)
  • BibTex
  • Text


  • Citation Only
  • Citation and Abstract

Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells

1 Department of Physics, University of Colorado Denver, Denver, CO 80212, USA
2 Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Denver Anschutz Medical Campus, Aurora CO 80045, USA
† Current address: Department of Physiology, School of Medicine, University of Arizona, Tucson, USA

Special Issues: Single Cell analysis

Disruptions in the response of endothelial progenitor cells to changes in oxygen environment may present a possible mechanism behind multiple pediatric pulmonary disease models, such as bronchopulmonary dysplasia. Using high-throughput fixed single-cell protein and RNA imaging, we have created “stop-motion” movies of Thymosin β4 (Tβ4) and Hypoxia Inducible Factor 1α (HIF-1α) protein expression and vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) mRNA in human umbilical cord-derived endothelial colony-forming cells (ECFC). ECFC were grown in vitro under both room air and hyperoxia (50% O2). We find elevated basal Tβ4 protein expression in ECFC derived from prematurely born infants versus full term infants. Tβ4 is a potent growth hormone that additionally acts as an actin sequestration protein and regulates the stability of HIF-1α. This basal level increase of Tβ4 is associated with lower HIF-1α nuclear localization in preterm versus term ECFC upon exposure to hyperoxia. We find altered expression in the pro-angiogenic genes vegf and eNOS, two genes that HIF-1α acts as a transcription factor for. This provides a potential link between a developmentally regulated protein and previously observed impaired function of preterm ECFC in response to hyperoxia.
  Article Metrics

Keywords single-molecule; single-cell; fluorescence microscopy; endothelial colony-forming cells; endothelial cell biology

Citation: Megan A. Ahern, Claudine P. Black, Gregory J. Seedorf, Christopher D. Baker, Douglas P. Shepherd. Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells. AIMS Biophysics, 2017, 4(2): 284-297. doi: 10.3934/biophy.2017.2.284


  • 1. Munsky B, Fox Z, Neuert G (2015) Integrating single-molecule experiments and discrete stochastic models to understand heterogeneous gene transcription dynamics. Methods 85: 12–21.    
  • 2. Munsky B, Neuert G, Oudenaarden AV (2012) Using gene expression noise to understand gene regulation. Science 336: 183–187.    
  • 3. Neuert G, Munsky B, Tan RZ, et al. (2013) Systematic identification of signal-activated stochastic gene regulation. Science 339: 584–587.    
  • 4. Shepherd DP, Li N, Micheva-Viteva SN, et al. (2013) Counting small RNA in pathogenic bacteria. Anal Chem 85: 4938–4943.    
  • 5. Hill JM, Zalos G, Halcox JP, et al. (2003) Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 348: 593–600.    
  • 6. Ingram DA, Mead LE, Tanaka H, et al. (2004) Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord. Blood 104: 2752–2760.    
  • 7. Prater DN, Case J, Ingram DA, et al. (2007) Working hypothesis to redefine endothelial progenitor cells. Leukemia 21: 1141–1149.    
  • 8. Critser PJ, Yoder MC (2010) Endothelial colony forming cell role in neoangiogenesis and tissue repair. Curr Opin Organ Transplant 15: 68–72.    
  • 9. Fujinaga H, Baker CD, Ryan SL, et al. (2009) Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants. Am J Physiol-Lung C 297: L1160–L1169.    
  • 10. Baker CD, Balasubramaniam V, Mourani PM, et al. (2012) Cord blood angiogenic progenitor cells are decreased in bronchopulmonary dysplasia. Eur Respir J 40: 1516–1522.    
  • 11. Gumina DL, Black CP, Balasubramaniam V, et al. (2016) Umbilical cord blood circulating progenitor cells and endothelial colony-forming cells are decreased in preeclampsia. Reprod Sci: 1933719116678692.
  • 12. Jo JO, Kim SR, Bae MK, et al. (2010) Thymosin β4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1α-dependent manner. BBA-Mol Cell Res 1803: 1244–1251.
  • 13. Kim NS, Kang YJ, Jo JO, et al. (2011) Elevated expression of thymosin β4, vascular endothelial growth factor (VEGF), and hypoxia inducible factor (HIF)-1α in early-stage cervical cancers. Pathol Oncol Res 17: 493–502.    
  • 14. Moon EY, Im YS, Ryu YK, et al. (2010) Actin-sequestering protein, thymosin beta-4, is a novel hypoxia responsive regulator. Clin Exp Metastasis 27: 601–609.    
  • 15. Oh JM, Moon EY (2010) Actin-sequestering protein, thymosin beta-4, induces paclitaxel resistance through ROS/HIF-1α stabilization in HeLa human cervical tumor cells. Life Sci 87: 286–293.    
  • 16. Milosevic J, Adler I, Manaenko A, et al. (2009) Non-hypoxic stabilization of hypoxia-inducible factor alpha (HIF-α): relevance in neural progenitor/stem cells. Neurotox Res 15: 367–380.    
  • 17. Abman SH (2010) Impaired vascular endothelial growth factor signaling in the pathogenesis of neonatal pulmonary vascular disease. Adv Exp Med Biol 661: 323–335.    
  • 18. Ferrara N (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25: 581–611.    
  • 19. Drummond GR, Cai H, Davis ME, et al. (2000) Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res 86: 347–354.    
  • 20. Dudzinski DM, Michel T (2007) Life history of eNOS: partners and pathways. Cardiovasc Res 75: 247–260.    
  • 21. Femino AM, Fay FS, Fogarty K, et al. (1998) Visualization of single RNA transcripts in situ. Science 280: 585–590.    
  • 22. Raj A, Bogaard P van den, Rifkin SA, et al. (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5: 877–879.    
  • 23. Baker CD, Ryan SL, Ingram DA, et al. (2009) Endothelial colony-forming cells from preterm infants are increased and more susceptible to hyperoxia. Am J Respir Crit Care Med 180: 454–461.    
  • 24. Edelstein AD, Tsuchida MA, Amodaj N, et al. (2014) Advanced methods of microscope control using μManager software. J Biol Methods 1: e10.    
  • 25. Bruce MA, Butte MJ (2013) Real-time GPU-based 3D deconvolution. Opt Express 21: 4766–4773.    
  • 26. Mueller F, Senecal A, Tantale K, et al. (2013) FISH-Quant: automatic counting of transcripts in 3d fish images. Nat Methods 10: 277–278.    
  • 27. Perillo EP, De Haro L, Phipps L, et al. (2014) Enhanced 3D localization of individual RNA transcripts via astigmatic imaging. Proc SPIE 8950: 895003.    
  • 28. Székely GJ, Rizzo ML (2013) The distance correlation-test of independence in high dimension. J Multivar Anal 117: 193–213.    
  • 29. Prasain N, Lee MR, Vemula S, et al. (2014) Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat Biotechnol 32: 1151–1157.    


Reader Comments

your name: *   your email: *  

Copyright Info: 2017, Douglas P. Shepherd, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

Download full text in PDF

Export Citation

Copyright © AIMS Press All Rights Reserved