Pulmonary arteriole gene expression signature in idiopathic pulmonary fibrosis

RESULTS

We included eight patients with PH-IPF, eight with NPH-IPF and seven controls (table 1). IPF patients were older than the controls and predominantly male. Patients with PH-IPF and NPH-IPF were similar with regards to age, sex, race, smoking history and lung function. Pulmonary function data revealed the presence of a restrictive impairment and the absence of obstructive physiology in both groups. One patient had mild upper lobe emphysema by computed tomography (CT); no patients had significant emphysema histologically or by chest CT. Notably, the degree of PH present in the PH-IPF group was mild, with mPAP of 29 mmHg.

The raw gene expression values for four commonly expressed endothelial and epithelial genes were examined to confirm the isolation of pulmonary arterioles while minimising the inclusion of lung parenchyma or airways. Endothelial gene expression was elevated across all samples, while epithelial gene expression was minimal (online supplementary table E1), confirming the successful isolation of pulmonary arterioles.

We first compared PH-IPF arterioles to NPH-IPF arterioles. Unsupervised hierarchical clustering is a method of grouping gene microarray data according to similarity of content between groups. The dendogram resulting from unsupervised hierarchical clustering analysis of PH-IPF and NPH-IPF arterioles revealed no separation between the two groups (fig. 1), and there were no significant differences in gene expression by univariate analysis (online supplementary table E2).

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Figure 1–

Vessels from patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) versus vessels from IPF patients without PH (NPH-IPF). Unsupervised hierarchical clustering analysis using uncentered correlation and average linkage. The dendogram shows no separation of gene expression between PH-IPF and NPH-IPF samples.

We then analysed the gene expression of the combined IPF samples (PH-IPF + NPH-IPF) versus controls. Unsupervised hierarchical clustering demonstrated a significant separation between the IPF samples and the controls (fig. 2). 255 genes were differentially expressed in IPF arterioles as compared to control vessels using univariate analysis (p<0.001, q<10%) (fig. 3), with selected results shown in table 2 (full list in online supplementary table E4). Two mediators of Wnt signalling, SFRP2 and CCND1, were upregulated in IPF arterioles, along with members of the complement family (C4A, C4B and CFH). Expression of STAT1 and SMOC2, a transcription factor and extracellular protein associated with vascular proliferation, was increased in IPF arterioles.

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Figure 2–

Idiopathic pulmonary fibrosis (IPF) vessels versus control vessels. Unsupervised hierarchical clustering analysis, using uncentered correlation and average linkage. The dendogram demonstrates clustering of gene expression of IPF samples separately from controls.

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Figure 3–

Heat map of differentially expressed genes in idiopathic pulmonary fibrosis (IPF) vessels versus controls. 259 genes differentially expressed at p<0.001 with false discovery rate <10%. Red: upregulated genes; green: downregulated genes.

Conversely, members of the Kruppel-like family of transcription factors (KLF6 and 10), which modulate endothelial function, were downregulated in IPF arterioles compared with controls. S1PR1, whose ligand sphingosine 1 phosphate is an effector of endothelial cell barrier integrity, was also downregulated. Expression of arginase II, a mediator of nitric oxide signalling [11], was decreased in IPF arterioles. The effectors of apoptosis, IGFBP3 and TIMP3, were downregulated in IPF arterioles.

Lastly, we analysed the gene expression of the PH-IPF group versus controls and the NPH-IPF group versus controls. The analysis of PH-IPF versus controls revealed no statistically significant genes. Results of analysis of the NPH-IPF group versus controls are displayed in online supplementary table E3.

Validation of gene array data with immunohistochemical staining

Immunohistochemical staining of selected gene products of biological interest was performed to confirm the gene expression data (fig. 4). STAT1 and SMOC2 protein expression was significantly upregulated in IPF arterioles compared to controls (p<0.05). SFRP2 protein expression also tended to be more highly expressed in IPF arterioles (p=0.10). These results corroborate the gene expression data.

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Figure 4–

a) Blood vessel from usual interstitial pneumonia (UIP) tissue showing marked nuclear and cytoplasmic staining for signal transducer and activator of transcription 1 in endothelium and medial smooth muscle cells, while b) normal vessel shows only endothelial staining. c) blood vessel from UIP tissue shows intense cytoplasmic staining for secreted protein, acidic, cysteine-rich-related modular calcium binding (SMOC)-2 in medial smooth muscle cells and endothelial cells, while d) normal vessel is negative. e) blood vessel wall from UIP tissue shows cytoplasmic staining for secreted frizzled-related protein 2 in medial smooth muscle cells, but no staining in f) normal vessel. a, b) Magnification×100, c–f) magnification×150.

DISCUSSION

Our findings provide direct evidence that genes involved in vascular proliferation and aberrant apoptosis are differentially expressed in pulmonary arterioles of IPF patients with or without PH compared with controls. Furthermore, patients with IPF who were free of PH and those with coexistent PH had similar gene expression in their pulmonary arterioles, suggesting that pre-clinical pulmonary vascular disease may be present in IPF patients with normal haemodynamics.

The mechanisms that mediate pulmonary vascular remodelling in IPF are unknown. To our knowledge, this is the first study to utilise gene expression profiling of LCM pulmonary arterioles, a technically demanding process in fibrotic lung disease, to examine vascular gene expression in IPF. The patients in our study also underwent extensive and precise clinical phenotyping.

Two previous studies of gene expression profiling in PH-IPF have utilised whole-lung homogenates. Gagermeier et al. [5] reported gene expression data from 13 patients with PH-IPF. They found decreased expression of VEGF, endothelin (ET)-1 and platelet endothelial cell adhesion molecule, while phospholipase A2 and other inflammatory genes were upregulated. We also found that genes associated with VEGF signalling were downregulated, although inflammatory genes were not highly represented in our IPF samples. Rajkumar et al. [4] compared whole-lung gene expression from 18 subjects with PAH to that of eight subjects with PH-IPF. Rho-kinases and mediators of cAMP signalling were upregulated in PAH compared with PH-IPF. It is difficult to make a direct comparison between this study and ours, as we did not examine patients with PAH. More recently, Mura et al. [12] examined lung gene expression in patients with varying aetiologies of pulmonary fibrosis (UIP and non-UIP) with and without severe PH (mPAP >40 mmHg). Cellular proliferation and fibroblast migration pathways were highly expressed in the PH group. While the former pathway was also highly represented in the vasculature of IPF patients in our study, there was no overlap in the most highly upregulated genes. There were significant phenotypic differences between the patients in this study and ours. Differences in results may further be explained by our direct focus on isolated pulmonary arterioles rather than the mix of parenchyma, airways and vessels in prior studies. Investigation of isolated pulmonary arterioles enabled us to determine gene expression directly in the pulmonary vasculature, without potential artefacts of lung parenchyma affected by IPF.

Genes that promote cellular proliferation were upregulated in IPF pulmonary arterioles regardless of the presence of coexistent PH. SMOC2 promotes angiogenesis and endothelial proliferation [13], and SMOC2 ablation inhibits DNA synthesis in mouse fibroblasts exposed to PDGF-β and other growth factors implicated in the pathogenesis of IPF [14]. Additionally, SMOC2 promotes cell cycle progression via cyclinD1, which was also upregulated in our samples [15]. Given these findings, it is plausible that SMOC2 contributes to vascular remodelling in IPF. In addition, gene and protein expression of STAT1 was increased in IPF pulmonary arterioles regardless of the presence of coexistent PH. STAT1 increases vascular smooth muscle cell proliferation in response to angiotensin II and serotonin, proposed mediators of PAH [16, 17]. Woods et al. [18] demonstrated that STAT1 mediates increased ET-1 production in human vascular smooth muscle cells (HVSMCs) [18]. Additionally, PLAU, which was overexpressed in our IPF samples, has been shown to activate STAT1 in both human vascular endothelial cells and HVSMCs [19, 20]. These data suggest that PLAU and other mediators may activate STAT1 in IPF, leading to vascular proliferation. Our finding of increased SFRP2 expression in IPF arterioles may indicate a role for Wnt signalling in promoting the development of PH-IPF. SFRP2, a mediator of both canonical and noncanonical Wnt signalling, promotes angiogenesis and inhibits hypoxia-induced endothelial cell apoptosis in experimental models [21]. Moreover, noncanonical Wnt signalling has recently been established to be active in the development of idiopathic PAH [22]. Lastly, genes that inhibit proliferation were downregulated in pulmonary arterioles of IPF patients with and without coexistent PH. KLF10 mediates antiproliferative and antiapoptotic effects via TGF-β–Smad signalling [23]. KLF6 functions as a tumour suppressor which is expressed in endothelial cells, and is induced by hypoxia and other forms of cellular stress [24]. Downregulation of KLF10 and KLF6 may contribute to cellular proliferation and deregulation of apoptosis in the vasculature in IPF. YY1, a zinc-finger transcription factor that inhibits vascular smooth muscle cell growth and functions downstream from haem oxygenase (HO)-1, was downregulated in our samples [25, 26]. Reduced HO-1 expression characterises severe PAH, suggesting a potential association between the pathobiology of PAH and vascular change in IPF [26, 27]. Similar alterations in expression of genes that promote and inhibit cellular proliferation in pulmonary arterioles of IPF patients with and without coexistent PH strongly suggest that vascular function is altered early in the course of IPF. Such findings concur with previous reports indicating the development of clinical PH in the vast majority of IPF patients over time [2]. Alternatively, similar gene expression in pulmonary arterioles of IPF patients with or without PH may indicate that the biologic processes that account for pulmonary vascular changes in IPF may be a manifestation of IPF as a disease rather than PH.

Interestingly, the genes that were differentially expressed in IPF vessels compared to controls largely fell into categories of cellular proliferation and cancer. Shared pathogenic ties between cancer biology and IPF, including aberrant cellular proliferation and impaired apoptosis, have been increasingly noted [28]. In PAH, STAT3 activation contributes to the propagation of hyperproliferative, apoptosis-resistant endothelial cells [29]. Several modulators of apoptosis were underexpressed in our samples. Conversely, mediators of proliferation, such as STAT1 and SMOC2, were upregulated [19, 20]. These findings suggest that uncontrolled vascular proliferation and impaired apoptosis may contribute to vascular remodelling in IPF as well. Lastly, increased SFRP2 expression in our samples suggests an active role for Wnt signalling in the development of vascular changes in IPF [22].

There were several limitations in our study. We studied a small number of patients, albeit a typical sample size for gene microarray studies. We used LCM to isolate vessels for analysis, a technologically sophisticated approach that is novel in studies of the vasculature in IPF. Reverse transcriptase (RT)-PCR was not utilised for validation, as accurate dissection of pulmonary arteriolar tissue from architecturally distorted, fibrotic parenchyma precluded extraction of a sufficient quantity of tissue for both expression microarray analysis and RT-PCR. Patients with PH-IPF had only mild elevations in pulmonary artery pressure, but fulfilled the WHO criteria and are typical of PH-IPF [3, 30]. Patients with more severe PH-IPF might have shown significant differences in gene expression compared to the NPH-IPF group. Combined pulmonary fibrosis with emphysema is associated with PH in IPF [31]; however, only one patient in our study had evidence of mild upper-lobe emphysema [31]. Moreover, spirometry and lung volumes were similar between the groups. We used specimens obtained from normal lung in patients undergoing carcinoid resection for a portion of control samples, and acknowledge that while the tissue appeared normal architecturally, it may have been abnormal at the molecular level. Lastly, while high-throughput gene expression analysis may be subject to Type I error, we constrained our false-positive rate and confirmed selected gene expression findings using immunohistochemistry.

In conclusion, the pulmonary vascular gene expression signature is similar in patients with coexistent IPF and PH and those with IPF alone and is characterised by aberrant cellular growth and deregulated apoptosis. Characterisation of pre-clinical pulmonary vascular alterations in IPF may lead to the development of novel therapeutic strategies for coexistent PH in these patients.