Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments

Abstract

BackgroundIt 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.MethodsPeripheral 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.ResultsPatients 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.ConclusionsLevels 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.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…Several studies have been published, but no consensus has thus far been reached on either the markers that should be used to identify these cells, or on the necessity of a pre‐analytical enrichment (by density gradient, buffy coat, and/or magnetic enrichment). The enrichment, however, can have negative effects if rare cells are lost, or these effects may be positive, if unwanted cells are removed . Unfortunately, quite often, the lack of well standardized methods influences the decision regarding the number of markers, which are necessary for the identification of the population of interest.…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies*

“…Several studies have been published, but no consensus has thus far been reached on either the markers that should be used to identify these cells, or on the necessity of a pre-analytical enrichment (by density gradient, buffy coat and/or magnetic enrichment). The enrichment, however, can have negative effects if rare cells are lost, or these effects may be positive, if unwanted cells are removed [230][231][232][233][234]. Unfortunately, quite often, the lack of well standardized methods influences the decision regarding the number of markers, which are necessary for the identification of the population of interest.…”

iNKT Cells in Secondary Progressive Multiple Sclerosis Patients Display Pro-inflammatory Profiles

“…In order to identify circulating iNKT whose percentage in peripheral blood is typically much less than 1%, it is mandatory to acquire a huge number of cells (28, 33). Thus, for the phenotypic and functional analysis, we made use of a novel acoustic flow cytometer able to align cells in the flow chamber by using ultrasounds and to acquire up to 35,000 events/second.…”