Molecular Imaging of Very Late Antigen-4 (VLA-4) in Acute Lung Injury

Abstract

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and the progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for non-invasive approaches to monitor lung inflammation through targeting immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intra-tracheal administration of lipopolysaccharide (10, 20 or 40 µg per mouse) in C57BL/6J mice. Control mice were intra-tracheally instilled with sterile phosphate-buffered saline. VLA-4 targeted PET/CT was performed 24 hours after intravenous injection of a copper-64 (⁶⁴Cu) labeled high-affinity peptidomimetic ligand, ⁶⁴Cu-CB-TE1A1P-PEG4-LLP2A (⁶⁴Cu-LLP2A), at day 2 after the induction of ALI. Ex vivo biodistribution of ⁶⁴Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intra-tracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in ⁶⁴Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and ⁶⁴Cu-LLP2A uptake, as quantified by in vivo PET (R=0.69, P<0.01). Expression levels of both subunits of VLA-4, i.e., integrins α4 and β1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1β and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo ⁶⁴Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically-relevant target in ALI, and the accuracy of VLA-4 targeted PET in quantification of ongoing lung inflammation in a murine model.