title: Expression and function of toll-like receptors in human circulating endothelial colony forming cells.
abstract: Mature endothelial cells are known to sense microbial products through toll-like receptors (TLRs), a family of membrane proteins which serve as pathogen recognition and signaling elements; however, there are limited data in the literature about the expression and function of TLRs in human circulating endothelial colony forming cells (ECFCs), which are considered the most likely endothelial precursors. We expanded and differentiated in vitro umbilical cord blood (UCB) and adult peripheral blood (PB) ECFCs and studied the expression of TLR1 to TLR10 mRNA by qPCR analysis, and we further characterized TLR function in ECFCs through functional assays including in vitro ECFC growth and differentiation, assessment of cytokine production, and measurement of intracellular Ca(2+) signals. Both UCB- and PB-ECFCs had detectable mRNA levels of all the TLRs from 1 to 10; TLR4, a sensor of Gram-negative bacterial lipopolysaccharide (LPS), had a higher level compared to other TLRs. Exposure to LPS induced cytokine production, although with less efficiency compared to PB-mononuclear cells. However, no effect of LPS was seen on ECFC growth and differentiation, and no increase in intracellular Ca(2+) concentrations, which is essential for ECFC proliferation, was observed after exposure to increasing amounts of LPS. Our data show that all TLRs from 1 to 10 are constitutively expressed in ECFCs, and suggest that TLR4 is functional in ECFCs, but its activation through its ligand LPS does not affect ECFC growth and differentiation.
authors.affiliation: Laboratory of Neonatal Immunology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy; Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy.
authors.forename: Iolanda
authors.initials: I
authors.lastname: Mazzucchelli
authors.affiliation: Laboratory of Transplant Immunology/Cell Factory, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy; Cell Therapy Production Unit, UO Cerebrovascolar Disease, IRCCS Neurological Institute C. Besta Foundation, Milano, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Lisini
authors.affiliation: Laboratory of Neonatal Immunology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Garofoli
authors.affiliation: Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Dragoni
authors.affiliation: Laboratory of Neonatal Immunology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Micol
authors.initials: M
authors.lastname: Angelini
authors.affiliation: Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Pozzi
authors.affiliation: Center for the Study of Myelofibrosis, Biotechnology Laboratory Research Area, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bonetti
authors.affiliation: Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Chryssoula
authors.initials: C
authors.lastname: Tzialla
authors.affiliation: Department of Pediatrics, School of Mental Health and Neuroscience, School of Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Boris W
authors.initials: BW
authors.lastname: Kramer
authors.affiliation: Department of Obstetrics and Gynecology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Arsenio
authors.initials: A
authors.lastname: Spinillo
authors.affiliation: Laboratory of Transplant Immunology/Cell Factory, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Maccario
authors.affiliation: Center for the Study of Myelofibrosis, Biotechnology Laboratory Research Area, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Rosti
authors.affiliation: Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Moccia
authors.affiliation: Laboratory of Neonatal Immunology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy; Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy. Electronic address: alessandroborghesi@yahoo.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Borghesi
authors.affiliation: Laboratory of Neonatal Immunology, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy; Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico "San Matteo", Pavia, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Stronati
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D049109:Cell Proliferation / Q000235:genetics; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000097:blood / Q000235:genetics / Q000276:immunology; D051193:Toll-Like Receptors / Q000097:blood / Q000235:genetics / Q000276:immunology*
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D002118:Calcium
keywords: Ca(2+); ECFC; Endothelial colony forming cells; LPS; TLR; Toll-like receptors