Laser-capture microdissection, mass spectrometry and immunohistochemistry reveal pathologic alterations in the extracellular matrix of transplanted lungs

About 50% of lung transplanted patients develop chronic rejection in the form of bronchiolitis obliterans syndrome (BOS) within 5 years. The patho-anatomical correlate is progressive, fibrotic occlusion of the small airways (OB-lesion) ultimately leading to organ failure. The cause and development of this drastic remodeling of epithelium and extracellular matrix (ECM) is currently unknown. We hypothesize that there are ECM alterations long before the diagnosis of BOS and that the so far unknown protein content of the OB-lesion is likely to give insight into both early and late pathologic events. By combining laser-capture microdissection (LCM), mass spectrometry (MS) and immunohistochemistry (IHC) we analyzed the distribution of ECM proteins in transbronchial biopsies as well as in explants. We established a sample preparation protocol to analyze the protein content in the minute amount of tissue obtained by LCM from single OB-lesions from tissue sections (0.00024mm3) by MS. We identified distinct ECM-affiliated proteins, ECM regulators, glycoproteins, collagens and secreted factors. Quantitative analysis of IHC for collagen, biglycan and periostin on biopsies (3 vs. 12 months after transplantation) revealed early ECM alterations in alveoli and small airways, distinguishing patients who did or did not develop BOS, and predicted which patients would have a severe disease course. The combined analysis of early and end-stage ECM alterations in the transplanted lung by LCM-MS and IHC provides molecular and morphological insights essential for better understanding of the development of chronic rejection after lung transplantation.