Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process

Patel, Jatin, Seppanen, Elke J., Rodero, Mathieu, Wong, Ho Yi, Donovan, Prudence, Neufeld, Zoltan, Fisk, Nicholas, Francois, Mathias and Khosrotehrani, Kiarash (2017) Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process. Circulation, 135 8: 786-805. doi:10.1161/CIRCULATIONAHA.116.024754


Author Patel, Jatin
Seppanen, Elke J.
Rodero, Mathieu
Wong, Ho Yi
Donovan, Prudence
Neufeld, Zoltan
Fisk, Nicholas
Francois, Mathias
Khosrotehrani, Kiarash
Title Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process
Journal name Circulation   Check publisher's open access policy
ISSN 0009-7322
1524-4539
Publication date 2017-02-21
Year available 2016
Sub-type Article (original research)
DOI 10.1161/CIRCULATIONAHA.116.024754
Open Access Status Not yet assessed
Volume 135
Issue 8
Start page 786
End page 805
Total pages 67
Place of publication Philadelphia, United States
Publisher Lippincott Williams & Wilkins
Language eng
Subject 2705 Cardiology and Cardiovascular Medicine
2737 Physiology (medical)
Abstract Background: During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. Methods: Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. Results: Lineage tracing by using Cdh5cre /Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr, and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf, and Notch. The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on Sox18Cre/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Conclusions: Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.
Formatted abstract
Background—During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neo-vessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, as well as wound healing, tumors and placenta.

Methods—Based on protein expression levels of common endothelial markers using flow cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells.

Results—Lineage tracing using Cdh5creERt2/Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony forming capacity in limiting dilution and by transplantation in MatrigelTM plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf and Notch. The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. SOX18's role in the differentiation process was validated using lineage-tracing experiments based on Sox18CreERt2/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced.

Conclusions—Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.
Keyword Cardiac & Cardiovascular Systems
Peripheral Vascular Disease
Cardiovascular System & Cardiology
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID APP1023368
APP1011242
Institutional Status UQ

 
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Created: Fri, 06 Jan 2017, 21:13:07 EST by Susan Allen on behalf of Institute for Molecular Bioscience