Based on animal models that implicate fibrocytes in the pathobiology of PH and on human studies demonstrating increased fibrocyte numbers in patients with interstitial lung disease, we tested the hypothesis that fibrocytes would be increased in the circulation of patients with PH. We found a significant enrichment of circulating fibrocytes (defined as CD45+ procollagen+ cells) in patients with PH compared with control individuals undergoing catheterisation for arrhythmia. In addition, we found a correlation between fibrocyte counts and pa
as well as age. Taken together with previous findings from our group and others of exuberant decoration of fibrocytes in and around pulmonary vessels in a neonatal calf model of hypoxic PH [18
], our data here support the notion that the presence of these progenitor cells may contribute to pathological pulmonary vascular remodelling in the setting of inflammation. This is the first demonstration that individuals with PH harbour increased numbers of circulating fibrocytes, a cell type that has been identified in adults with chronic inflammatory disorders and is associated with poor prognosis, inflammation and tissue remodelling in the lung. Our study might, therefore, represent the first step in understanding the putative pathological roles of fibrocytes in the course of paediatric PH.
The biology of fibrocytes is rapidly coming into sharper focus. Originally described as a leukocyte subpopulation that effected tissue repair [21
], fibrocytes are critically involved in normal wound healing and fibrosis [22
]. Their differentiation from precursor cells is complex but may be inhibited by bacterial infection through Toll-like receptor 2 activation [23
]. In other contexts, fibrocytes can be differentiated to myofibroblast and adipocyte phenotypes, by transforming growth factor-β [24
] and peroxisome proliferator-activated receptor-γ [25
], respectively. Apart from tissue remodelling, fibrocytes have been negatively implicated in chronic inflammatory conditions. Fibrocytes are mobilised from the bone marrow and accumulate in regions of chronic inflammation, perhaps through the SDF-1/CXC chemokine receptor 4 signalling axis [26
]. In vivo
, fibrocytes have been studied in animal models of PH, and are associated with vascular remodelling, elevated pulmonary artery pressures and accumulation in bronchovascular regions [4
]. Although the extent of monocytic cell influx into the lung has been well documented in adults with PH [27
], ours is the first study to quantitatively assess circulating fibrocytes in children and young adults with PH. The present study has confirmed that fibrocytes are present in the blood of patients with PH. How (or whether) fibrocytes contribute to the remodelling of pulmonary vessels in the human condition remains to be determined. A more precise functional characterisation of fibrocytes, at least in the clinical setting, is currently hampered by the seeming requirement of (intracellular) collagen as the “gold standard” for fibrocyte identification. Magnetic bead or flow sorting using extracellular markers as surrogates for the expression of collagen would obviate the need for fixation and permeabilisation of putative fibrocytes, a process incompatible with many downstream functional assays. Recently, fibrocytes isolated from patients with chronic inflammation have been shown to display an activated phenotype [28
]. Additionally, the expression profile for cultured fibrocytes is distinct from monocytes, macrophages and fibroblasts [29
]. These findings highlight the complexity of fibrocyte differentiation, the details of which (stem cell, process, temporal mechanics, tissue influence, etc.
) remain shrouded in obscurity. Identification of cell surface markers with a high specificity for fibrocytes will facilitate studies designed to understand the functional roles of fibrocytes in vivo
, as has already been established for serumamyloid P [30
]. Our group has previously shown that vascular remodelling and inflammation of the rat pulmonary vasculature during hypoxia requires circulating cells of a monocytic/macrophage lineage [13
]. Use of fibrocyte-specific agonists and antagonists, based on in vitro
and in vivo
immunophenotyping of surface markers, will more sharply define the cell type most responsible for the remodelling.
Correlation analysis between fibrocyte numbers and pa
and increasing age showed a weak but positive relationship. Fibrocyte counts did not correlate with other patient demographic data. The lack of a stronger correlative relationship between fibrocyte count and pa
is not altogether surprising for several reasons. First, the age range of our PH patient cohort spanned 22 yrs and there was a large standard deviation (6.18 yrs). There are no studies available that define a link between fibrocyte counts and age. We assumed a priori
that (young) individuals in the control group would have low fibrocyte numbers (≤1% of total white blood cells), as is observed in control adults. However, this is an assumption that needs additional testing. In any case, we found low numbers of fibrocytes in peripheral blood of controls, numbers similar to those documented in older controls [16
]. Secondly, many of the PH patients in our study are on significantly long-standing and varied treatment regimens, including calcium channel blockers, endothelin receptor antagonists, phosphodiesterase inhibitors and prostanoids. In addition, some of the children responded to acute vasodilator challenge and others did not. As such, the variable clinical presentation and underlying pathobiology (e.g.
APAH-CHD) of our study group probably reduced the power of fibrocyte number to more closely correlate with severity of PH as measured by pa
. In fact, it remains possible that, since treatments with prostacyclin agonists appear to increase the number of late EPCs, such treatment could also lead tomobilisation of fibrocytes into the circulation. In future studies, restricting the analysis of circulating fibrocytes to exclude patients with PH not associated with extensive lung inflammation and remodelling will probably prove fruitful, as will studying patients at diagnosis (untreated patients). Fibrocytes are involved in both normal wound healing and inflammation but at present we are unable to say which of these mechanisms is involved in these patients. We speculate that the mechanism is pro-inflammatory and, perhaps, the data that the patients with the most severe disease (on triple therapy) also have the highest fibrocyte numbers offers a clue in support of this assumption. This is in contrast to EPCs, which are thought to be decreased with increased severity of PH [4
]. It is thus plausible that the collective contributions of distinct progenitor cell populations govern lung homeostasis, and by extension, deviation from homeostasis during pathogenesis.
In conclusion, we have shown quantitative differences in circulating fibrocytes between individuals with PH and those without PH. Fibrocyte count correlated with increased pa
and age. This study suggests a potential role for these cells in the pathogenesis of PH, but more studies are needed to confirm our findings. A great deal of renewed research emphasis over the past decade has been placed on mechanisms of lung inflammation and vascular remodelling in PH. Our study points to the potential contributions of circulating mesenchymal progenitors to those inflammatory processes.