Quantification of αvβ6 Integrin Expression in Normal and Fibrotic Human Lung Tissue

The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β that is hypothesized to be pivotal in the development of idiopathic pulmonary fibrosis (IPF) (1). In this study a radiolabelled αvβ6 small molecule RGD-mimetic was used in a filtration binding assay to quantify the amount of αvβ6 integrin present in normal (non-fibrotic) and IPF human lung tissue.

Radioligand binding experiments were performed by filtration with [3H]compound 1 (patent WO2014154725 A1) and membrane fragments generated from normal and IPF human lung parenchyma (human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents). Normal (non-fibrotic) human lungs (designated by medical history) from organ donors (transplant reject) were obtained from NDRI (PA, USA). Post-transplant lung tissue samples from IPF patients (disease state defined by medical history and post-transplant pathology) were obtained from Newcastle University (Tyne and Wear, UK). Human lung parenchyma tissue (between 2 and 15 g) obtained from 6 normal and 6 IPF donors was frozen as soon as possible following surgical removal from donors and stored at -80°C until plasma membrane preparations were generated as described in (2). Radioligand binding studies completed with human lung parenchyma membranes were in a total volume of 1.5 ml consisting of 50 µl/well of either 10μM vehicle (1 % DMSO) or A20FMDV2 (selective αvβ6 peptide described in (3)), 50 µl of [3H]compound 1 and 1.4 ml/well of membranes (μg/well dependent on the number of binding sites (Bmax) of individual preparations). All radioligand binding experiments were performed at 37ºC in 25 mM HEPES, 100 mM NaCl, 1 mM CHAPS and either 2 mM MgCl2 or MnCl2 at pH 7.4 (NaOH).

Table 1. Saturation binding parameters for [3H]compound 1 with the αvβ6 integrin in normal and IPF human lung parenchyma membranes.

A significant increase in Bmax was observed in IPF membranes in the presence of Mn2+ compared with normal membranes in the presence of Mg2+ and Mn2+ and IPF membranes in the presence of Mg2+ (ANOVA, Bonferroni post-test p < 0.05) (Table 1). An average 2.9-fold increase was observed for αvβ6 between normal to IPF lungs (in the presence of Mn2+). A significant difference in pKD was observed in IPF membranes in the presence of Mn2+ compared with IPF membranes in the presence of Mg2+, normal membranes in the presence of 2 mM Mg2+ and 2 mM Mn2+ (ANOVA, Bonferroni post-test p < 0.05) (Table 1).

In conclusion, this data supports the literature findings that αvβ6 integrin expression is increased in IPF patients (3). In addition, there is evidence for a larger population of αvβ6 in IPF membranes that can be activated by Mn2+ suggesting the integrin has the potential to exist in a more activated state in IPF.

References

1. Goodwin and Jenkins (2009). Biochem Soc Trans 37: 849-854.

2. Slack (2014), Pharmacology and Pharmacy 5:30-36.

3. Hausner et al. (2009). Cancer Res 69: 5843-5850.

4. Horan et al. (2008). Am J Respir Crit Care Med 177:56–65.