Jatin Patel1, Elke J Seppanen1, Mathieu P Rodero1, Ho Yi Wong1, Prudence Donovan1, Zoltan Neufeld1, Nicholas M Fisk1, Mathias Francois1, Kiarash Khosrotehrani2. 1. From The University of Queensland, UQ Centre for Clinical Research, Experimental Dermatology Group, Brisbane, QLD, Australia (J.P., E.J.S., M.P.R., H.Y.W., N.M.F., K.K.); The University of Queensland, UQ Diamantina Institute, Translational Research Institute, Woolloongabba, QLD, Australia (P.D., K.K.); The University of Queensland, School of Mathematics and Physics, Brisbane, QLD, Australia (Z.N.); and The University of Queensland, Institute of Molecular Biosciences, Brisbane, QLD, Australia (M.F.). 2. From The University of Queensland, UQ Centre for Clinical Research, Experimental Dermatology Group, Brisbane, QLD, Australia (J.P., E.J.S., M.P.R., H.Y.W., N.M.F., K.K.); The University of Queensland, UQ Diamantina Institute, Translational Research Institute, Woolloongabba, QLD, Australia (P.D., K.K.); The University of Queensland, School of Mathematics and Physics, Brisbane, QLD, Australia (Z.N.); and The University of Queensland, Institute of Molecular Biosciences, Brisbane, QLD, Australia (M.F.). k.khosrotehrani@uq.edu.au.
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 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 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 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.
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 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 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 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.
Authors: Patrycja Nowak-Sliwinska; Kari Alitalo; Elizabeth Allen; Andrey Anisimov; Alfred C Aplin; Robert Auerbach; Hellmut G Augustin; David O Bates; Judy R van Beijnum; R Hugh F Bender; Gabriele Bergers; Andreas Bikfalvi; Joyce Bischoff; Barbara C Böck; Peter C Brooks; Federico Bussolino; Bertan Cakir; Peter Carmeliet; Daniel Castranova; Anca M Cimpean; Ondine Cleaver; George Coukos; George E Davis; Michele De Palma; Anna Dimberg; Ruud P M Dings; Valentin Djonov; Andrew C Dudley; Neil P Dufton; Sarah-Maria Fendt; Napoleone Ferrara; Marcus Fruttiger; Dai Fukumura; Bart Ghesquière; Yan Gong; Robert J Griffin; Adrian L Harris; Christopher C W Hughes; Nan W Hultgren; M Luisa Iruela-Arispe; Melita Irving; Rakesh K Jain; Raghu Kalluri; Joanna Kalucka; Robert S Kerbel; Jan Kitajewski; Ingeborg Klaassen; Hynda K Kleinmann; Pieter Koolwijk; Elisabeth Kuczynski; Brenda R Kwak; Koen Marien; Juan M Melero-Martin; Lance L Munn; Roberto F Nicosia; Agnes Noel; Jussi Nurro; Anna-Karin Olsson; Tatiana V Petrova; Kristian Pietras; Roberto Pili; Jeffrey W Pollard; Mark J Post; Paul H A Quax; Gabriel A Rabinovich; Marius Raica; Anna M Randi; Domenico Ribatti; Curzio Ruegg; Reinier O Schlingemann; Stefan Schulte-Merker; Lois E H Smith; Jonathan W Song; Steven A Stacker; Jimmy Stalin; Amber N Stratman; Maureen Van de Velde; Victor W M van Hinsbergh; Peter B Vermeulen; Johannes Waltenberger; Brant M Weinstein; Hong Xin; Bahar Yetkin-Arik; Seppo Yla-Herttuala; Mervin C Yoder; Arjan W Griffioen Journal: Angiogenesis Date: 2018-08 Impact factor: 9.596
Authors: Aleix Ribas-Latre; Rafael Bravo Santos; Baharan Fekry; Yomna M Tamim; Samay Shivshankar; Alaa M T Mohamed; Corrine Baumgartner; Christopher Kwok; Claudia Gebhardt; Angielyn Rivera; Zhanguo Gao; Kai Sun; John T Heiker; Brad E Snyder; Mikhail G Kolonin; Kristin L Eckel-Mahan Journal: Nat Commun Date: 2021-06-09 Impact factor: 14.919
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