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Quantification of VEGFRs, NRP1, and PDGFRs on Endothelial Cells and Fibroblasts Reveals Serum, Intra-Family Ligand, and Cross-Family Ligand Regulation

Abstract

Computational modeling of angiogenesis is limited by a lack of experimental data on angiogenic receptor levels. Recent receptor profiling quantified vascular endothelial growth factor receptors (VEGFRs); however data on other angiogenic receptors, such as platelet derived growth factor receptors (PDGFRs), are also necessary for the development of an accurate angiogenesis model. Here, we establish conditions for membrane PDGFR quantification. Additionally, we determine how several environmental conditions control membrane PDGFR levels on human dermal fibroblasts. We demonstrate that membrane PDGFRβ concentrations are negatively correlated with both media serum concentration and cell growth rate, in vitro. We also show VEGF-A165-mediated downregulation of membrane PDGFRα (~25%) and PDGFRβ (~30%), following a 24-h treatment. This supports the idea that VEGF-A165 acts independently of VEGFRs to signal through PDGFRα and PDGFRβ. We observe that PDGF-AA and PDGF-AB downregulate membrane PDGFRα by up to 55 and 75%, respectively, while having little to no effect on PDGFRβ or NRP1. We observe that PDGF-BB effects both PDGFRs and NRP1: membrane PDGFRα and PDGFRβ were downregulated by up to 70 and 90%, respectively, whereas membrane NRP1 was upregulated by up to 40%. These data provide the necessary insight to accurately represent PDGFRs in angiogenesis models, while offering new insight into the regulation of membrane PDGFRs.

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Abbreviations

VEGFR:

Vascular endothelial growth factor receptor

PDGFR:

Platelet derived growth factor receptor

NRP:

Neuropilin-1

FGF:

Fibroblast growth factor

TGF:

Transforming growth factor

PAD:

Peripheral artery disease

qFlow:

Quantitative flow

HDF:

Human dermal fibroblasts

HUVEC:

Human umbilical vein endothelial cells

FBS:

Fetal bovine serum

DMSO:

Dimethyl sulfoxide

FGM-2:

Fibroblasts growth medium-2

EGM-2:

Endothelial cell growth medium-2

rhFGF-B:

Human recombinant basic fibroblast growth factor

GA-1000:

Gentamicin and amphotericin diluted at a 11000 ratio

DMEM:

Dulbecco’s modified eagle medium

PES:

Polyethersulfone

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Acknowledgments

We would like to thank the American Cancer Society, Illinois Division Basic Research Grant, as well as the National Cancer Institute Grant for funding support. We would like to thank Audra Storm, Brendan Mathias, and Dipen Kumar for assistance with experiments, and Jared Weddell, Spencer Mamer, and Ali Ansari for insightful discussion. We also thank Marinos Kalafatis for careful and critical reading of the manuscript.

Conflict of interest

Si Chen, Xinyi Guo, Osazomon Imarenezor and PI Imoukhuede declare that they have no conflicts of interest.

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No human studies or animal studies were carried out by the authors for this article.

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Correspondence to P. I. Imoukhuede.

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This article is part of the 2015 Young Innovators Issue.

P.I. Imoukhuede Assistant Professor Imoukhuede is a native of Illinois, having attended Rich South High School and the Illinois Mathematics and Science Academy (IMSA). Assistant Professor Imoukhuede earned her SB in Chemical Engineering from the Massachusetts Institute of Technology (MIT) where her research earned her the coveted Class of 1972 award, presented annually to the project that most improves the quality of life through its impact on people and/or the environment. Assistant Professor Imoukhuede’s research was funded by the National Science Foundation’s Biotechnology Process Engineering Center at MIT and through a Bioengineering Undergraduate Research Award by the MIT Division of Bioengineering and Environmental Health. Assistant Professor Imoukhuede was also an NCAA All-American athlete, garnering these honors three times for placing at the NCAA Track and Field Championships. Assistant Professor Imoukhuede was honored with the 2002 Betsy Schumaker Award (also known as the MIT female athlete of the year), was selected to a COSIDA/VERIZON Academic All-America team, and was awarded an NCAA postgraduate scholarship. Assistant Professor Imoukhuede championed the importance of social responsibility in the midst of academic excellence by serving as the President of the MIT Committee on Multiculturalism, President of the MIT chapter of the American Institute of Chemical Engineers (AIChE), and held both chapter and zone offices in the National Society of Black Engineers (NSBE). After earning her undergraduate degree, Assistant Professor Imoukhuede pursued graduate study in Bioengineering at the California Institute of Technology (Caltech) in Pasadena, CA. Here, she combined sensitive techniques in biomedical optics with nanoparticle imaging towards understanding the structure, function, and trafficking of a key protein in epilepsy, the GABA transporter, GAT1. She also performed research in nicotine addiction through molecular imaging of nicotinic acetylcholine receptors. Assistant Professor Imoukhuede’s research in nanotechnology earned her the Kavli Nanoscience Institute Award and her graduate research was supported by the National Institutes of Health (NIDA). Assistant Professor Imoukhuede was the first African-American woman to be awarded a Bioengineering PhD by Caltech and was only the second African-American woman to earn a PhD from Caltech’s Division of Engineering and Applied Science. Assistant Professor Imoukhuede completed a Postdoctoral Fellowship in the Biomedical Engineering Department at the Johns Hopkins University School of Medicine. During her fellowship at Johns Hopkins, she was 1 of 10 postdoctoral fellows nationwide to earn the prestigious United Negro College Fund/Merck Postdoctoral Research Fellowship, 1 of 6 young investigators to earn the FASEB Postdoctoral Professional Development Award, and her work was awarded a Poster Award at the biennial Gordon Conference in Angiogenesis. Her postdoctoral work was also supported by the National Institutes of Health (NHLBI). Currently, Assistant Professor Princess Imoukhuede aims to advance our cellular and molecular understanding of cancer regulation through systems biology. Assistant Professor Imoukhuede has extensive training in bioengineering and biophysics; as such, her laboratory leads efforts to sense, model, predict, and ultimately tune tumor angiogenesis by developing novel tumor cell isolation platforms, mapping tumor heterogeneity, and integrating these tumor defining parameters through computational modeling. Towards these goals, Prof. Imoukhuede is currently developing novel approaches that can quickly, and efficiently isolate specific cell types without disrupting cell-surface receptor-levels. Prof. Imoukhuede has recently pioneered a novel quantitative fluorescence approach for sensitive cell isolation and mapping of angiogenic receptor surface-distributions. She has applied this technology to animal models of both breast cancer and ischemic disease. Prof. Imoukhuede incorporates these molecular and cellular data into multi-scale computational models. Her models have recently predicted the efficacy of anti-angiogenic therapeutics, predicted optimal anti-angiogenic therapeutic parameters, and predicted the effect of tumor heterogeneity on anti-angiogenic therapeutics. Her advancement of this experimental (cell isolation and profiling) and computational, paradigm accelerates discovery into the signaling cues mediating tumor growth and development. Assistant Professor Imoukhuede’s biography is featured in the book, A Hand Up: Women Mentoring Women in Science.

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Chen, S., Guo, X., Imarenezor, O. et al. Quantification of VEGFRs, NRP1, and PDGFRs on Endothelial Cells and Fibroblasts Reveals Serum, Intra-Family Ligand, and Cross-Family Ligand Regulation. Cel. Mol. Bioeng. 8, 383–403 (2015). https://doi.org/10.1007/s12195-015-0411-x

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Keywords

  • Flow cytometry
  • Cell-by-cell
  • Angiogenesis
  • Fibroblasts
  • Endothelial cells
  • Tyrosine kinase receptors