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Comparative Study
. 2009 Feb 27;10(1):14.
doi: 10.1186/1465-9921-10-14.

Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis

Argyris Tzouvelekis  1 , Vassilis Aidinis, Vagelis Harokopos, Andreas Karameris, George Zacharis, Dimitrios Mikroulis, Fotios Konstantinou, Paschalis Steiropoulos, Ioannis Sotiriou, Marios Froudarakis, Ioannis Pneumatikos, Rodoula Tringidou, Demosthenes Bouros
Affiliations
Comparative Study

Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis

Argyris Tzouvelekis et al. Respir Res. .

Abstract

Background: Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).

Methods: Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.

Results: A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.

Conclusion: Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.

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Figures

Figure 1
Figure 1
Ing4 mRNA expression levels in BLM-induced pulmonary fibrosis. Ing4 gene expression levels quantified by qRT-PCR showed a trend to increase, compared to control untreated mice, at early disease stages (day 7 post-administration) whereas a gradual decline, compared to control and day 7 mice, following disease progression (days 7 and 15) was easily noted. All values were normalized with the reference gene B2m and presented as relative expression to the control sample as described in materials and methods. *p < 0.05, **p < 0.005, ***p < 0.001. (One way ANOVA).
Figure 2
Figure 2
Decreased ING4 expression in bleomycin (BLM)- induced pulmonary fibrosis (PF) following disease progression. (A) Representative immunohistochemistry with an anti-ING4 antibody on lung paraffin sections from BLM-treated mice (7, 15, and 23, days post-administration). ING4 was mainly expressed in alveolar epithelium (days 7 and 15) whereas showed weak staining within areas of dense fibrosis and collagen deposition at late disease stages (day 21). (B) Computerized image analysis of immunostained sections. *p < 0.05, **p < 0.005, ***p < 0.001. (One way ANOVA and unpaired t-test with Bonferroni correction, F = 71,126).
Figure 3
Figure 3
ING4 mRNA expression levels in patients with idiopathic pulmonary fibrosis (IPF), cryptogenic organizing pneumonia (COP) and control (ctrl) subjects. Significant reduction of ING4 gene expression levels in IPF patients compared to COP and control subjects, as quantified by qRT-PCR. Cycle threshold (Ct) values for each sample were converted to concentration values (through a standard curve of serial dilutions of a reference sample), normalized to the corresponding values of the reference gene B2M and presented as expression index. *p < 0.05, **p < 0.005, ***p < 0.001 (One way ANOVA).
Figure 4
Figure 4
Decreased ING4 expression within IPF lung compared to COP and normal lung. (A) Representative immunohistochemistry with an anti-ING4 antibody on lung paraffin sections from IPF and COP patients as well as control (CTRL) subjects. ING4 was extensively expressed in normal alveolar epithelial and endothelial cells in control lung samples and was also visualized in alveolar epithelial cells surrounding areas of active fibrosis, called Masson bodies, within COP lung. On the contrary, ING4 was almost absent in alveolar epithelium and fibrotic interstitium (fibroblastic foci) within IPF lung. (B) Computerized image analysis of immunostained sections. *p < 0.05, **p < 0.005, ***p < 0.001 (One way ANOVA and unpaired t-test with Bonferroni correction, F = 171,126).
Figure 5
Figure 5
Negative correlation between ING4 semi-quantitative expression levels and pulmonary function parameters in IPF patients. Spearman's correlation was performed and clearly demonstrated an almost linear negative association between ING4 down-regulation and parameters of disease progression including including forced vital capacity (FVC) (A), total lung capacity (TLC) (B) and diffuse lung capacity as expressed by KCO (carbon monoxide transfer coefficient) (C), in IPF patients.
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