title: Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments.
abstract: BACKGROUND
  It has been suggested that circulating fibrocytes and endothelial cells actively participate in the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis (IPF). Indeed, fibrotic areas exist that have fewer blood vessels, whereas adjacent non-fibrotic tissue is highly vascularized. The number of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair. Thus, fibrocytes as well as endothelial cells could potentially be used as biomarkers of disease progression and treatment outcome.
  
  
  METHODS
  Peripheral blood samples were collected from 67 patients with a multidisciplinary diagnosis of IPF and from 45 age-matched and sex-matched healthy volunteers. Buffy coat was isolated according to standard procedures and at least 20 million cells were stained with different monoclonal antibodies for the detection of CEC, EPC and circulating fibrocytes. For the detection of CEC and EPC, cells were stained with anti-CD45, anti-CD34, anti-CD133, anti-CD14, anti-CD309 and with the viability probe Far-Red LIVE/DEAD. For the detection of circulating fibrocytes, cells were first stained with LIVE/DEAD and the following monoclonal antibodies: anti-CD3, anti-CD19, anti-CD45, anti-CD34 and anti-CD14, then cells were fixed, permeabilized and stained with fluorochrome-conjugated anti-collagen I monoclonal antibodies.
  
  
  RESULTS
  Patients with IPF displayed almost undetectable levels of circulating fibrocytes, low levels of CEC, and normal levels of EPC. Patients treated with nintedanib displayed higher levels of CEC, but lower levels of endothelial cells expressing CD309 (the type II receptor for vascular endothelial growth factor). Treatment with both nintedanib and pirfenidone reduced the percentage of CEC and circulating fibrocytes.
  
  
  CONCLUSIONS
  Levels of CEC were reduced in patients with IPF as compared to healthy individuals. The anti-fibrotic treatments nintedanib and pirfenidone further reduced CEC levels. These findings might help explain the mechanism of action of these drugs and should be explored as predictive biomarkers in IPF.
authors.affiliation: Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi, 287-41125, Modena, Italy. debiasisara@yahoo.it.
authors.forename: Sara
authors.initials: S
authors.lastname: De Biasi
authors.affiliation: Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy. stefania.cerri@unimore.it.
authors.forename: Stefania
authors.initials: S
authors.lastname: Cerri
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy. elena.bianchini88@gmail.com.
authors.forename: Elena
authors.initials: E
authors.lastname: Bianchini
authors.affiliation: Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi, 287-41125, Modena, Italy. lara.gibellini@unimore.it.
authors.forename: Lara
authors.initials: L
authors.lastname: Gibellini
authors.affiliation: Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy. elisa.persioni@unimore.it.
authors.forename: Elisa
authors.initials: E
authors.lastname: Persiani
authors.affiliation: Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy. gloriamontanari@tiscali.it.
authors.forename: Gloria
authors.initials: G
authors.lastname: Montanari
authors.affiliation: Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy. fabrizio.luppi@unimore.it.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Luppi
authors.affiliation: Pulmonology Unit, Department of Cardiology, Thoracic and Vascular Surgery and Critical Care Medicine, IRCCS - Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. cristianoMatteo.carbonelli@asmn.re.it.
authors.forename: Cristiano Matteo
authors.initials: CM
authors.lastname: Carbonelli
authors.affiliation: Pulmonology Unit, Department of Cardiology, Thoracic and Vascular Surgery and Critical Care Medicine, IRCCS - Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. zucchi.luigi@asmn.re.it.
authors.forename: Luigi
authors.initials: L
authors.lastname: Zucchi
authors.affiliation: Respiratory Medicine Section, Department of Clinical Medicine and Surgery, "Federico II" University of Naples, Naples, Italy. marialuisa.bocchino@unina.it.
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Bocchino
authors.affiliation: Respiratory Medicine Section, Department of Clinical Medicine and Surgery, "Federico II" University of Naples, Naples, Italy. sanduzzi@unina.it.
authors.forename: Alessandro Sanduzzi
authors.initials: AS
authors.lastname: Zamparelli
authors.affiliation: Regional Centre for Rare Lung Diseases, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy. vancheri@unict.it.
authors.forename: Carlo
authors.initials: C
authors.lastname: Vancheri
authors.affiliation: Department of Respiratory Medicine, University of Southampton, Southampton, UK. giacomo.sgalla@uhs.nhs.uk.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Sgalla
authors.affiliation: Department of Respiratory Medicine, University of Southampton, Southampton, UK. l.richeldi@soton.ac.uk.
authors.forename: Luca
authors.initials: L
authors.lastname: Richeldi
authors.affiliation: Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi, 287-41125, Modena, Italy. andrea.cossarizza@unimore.it.|Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, 42122, Reggio Emilia, Italy. andrea.cossarizza@unimore.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cossarizza
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood*; D018450:Disease Progression; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000235:genetics*; D008297:Male; D011728:Pyridones; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D015415:Biomarkers; D011728:Pyridones; D042461:Vascular Endothelial Growth Factor A; C093844:pirfenidone