Circulating fibrocytes are increased in children and young adults with pulmonary hypertension

Abstract

Chronic inflammation is an important component of the fibroproliferative changes that characterise pulmonary hypertensive vasculopathy. Fibrocytes contribute to tissue remodelling in settings of chronic inflammation, including animal models of pulmonary hypertension (PH). We sought to determine whether circulating fibrocytes were increased in children and young adults with PH. 26 individuals with PH and 10 with normal cardiac anatomy were studied. Fresh blood was analysed by flow cytometry for fibrocytes expressing CD45 and procollagen. Fibrocyte numbers were correlated to clinical and haemodynamic parameters, and circulating CC chemokine ligand (CCL)2 and CXC chemokine ligand (CXCL)12 levels. We found an enrichment of circulating fibrocytes among those with PH. No differences in fibrocytes were observed among those with idiopathic versus secondary PH. Higher fibrocytes correlated to increasing mean pulmonary artery pressure and age, but not to length or type of treatment. Immunofluorescence analysis confirmed flow sorting specificity. Differences in plasma levels of CCL2 or CXCL12, which could mobilise fibrocytes from the bone marrow, were not found. We conclude that circulating fibrocytes are significantly increased in individuals with PH compared with controls. We speculate that these cells might play important roles in vascular remodelling in children and young adults with pulmonary hypertension.

FIGURE 1

Representative flow cytometric analysis for circulating CD45+/procollagen I C-terminal peptide (PICP)+ cells in controls and pulmonary hypertension (PH) patients. a) CD45/Pacific Blue (PB) versus intracellular isotype control/fluorescein isothiocyanate (FITC) analysis on blood from a representative normal subject. This control was used to set the negative gates. b) CD45+/PICP+ cells in normal blood. x axis: procollagen stained with goat anti-rabbit-FITC secondary antibody. c) CD45/PB versus intracellular isotype control/FITC analysis on blood from a patient with PH. This control was used to set the negative gates for the PH sample, same axes as in panel a. d) CD45+/PICP+ cells in PH blood, same axes as in panel b. e) Side scatter (SS) versus forward scatter (FS) analysis of buffy coat cells from the sample in panel d).

FIGURE 2

Circulating fibrocytes are enriched in pulmonary hypertension (PH) compared with controls and correlate to mean pulmonary artery pressure (P̄_pa) and age. Fibrocyte counts for 10 controls and 26 PH patients were mean±sd a) 0.84±0.23% versus 4.24±3.06%, and b) 0.21 × 10⁶±0.10 × 10⁶ versus 0.42 × 10⁶±0.21 × 10⁶ CD45+ procollagen I+ cells per millilitre, respectively. Analysis of correlation of fibrocyte c and e) differential and d and f) absolute cell counts in patients with PH to c and d) P̄_pa and e and f) age. e) Note that the correlation between age and fibrocytes expressed as a percentage did not reach significance at α=0.05. c) r=0.575, p<0.05; d) r=0.695, p<0.05; e) r=0.354, p>0.05; f) r=0.401, p<0.05. *: p<0.05 versus control.

FIGURE 3

Immunofluorescent analysis of circulating fibrocytes. Equivalent numbers of cytospun buffy coat cells were examined from a) controls and b) individuals with pulmonary hypertension for expression of procollagen I N-terminal peptide (PINP) stained with Alexa488 (Invitrogen, Carlsbad, CA, USA) (arrows). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).