Abstract Rationale The alveolar epithelial glycocalyx is a complex layer of glycosaminoglycans (GAGs) and glycoproteins that is crucial to lung homeostasis. Degradation of the epithelial glycocalyx contributes to the pathogenesis of many forms of acute lung injury (ALI); however, the duration of glycocalyx damage and its contribution to chronic lung diseases is unknown. Chronic lung allograft dysfunction (CLAD) is a common cause of morbidity and mortality for lung transplant recipients (LTRs). Given that episodes of ALI are a dominant risk factor for CLAD, we hypothesized that epithelial glycocalyx degradation may contribute to CLAD pathogenesis. Methods LTRs with CLAD and stable controls without CLAD underwent bronchoscopy with bronchial brushings and bronchioalveolar lavage (BAL) as part of their routine post-transplant care at UPMC (n = 37 patients/group). Flow cytometry was performed to characterize the cellular composition of the bronchial brushings. RNA was isolated from the bronchial brushings and bulk RNA sequencing was performed. BAL fluid samples were analyzed using commercially available ELISAs. In complementary studies, 8-12-week-old male mice underwent orthotopic lung transplant (F1 (DBA x B6) into B6). Whole lung tissue was harvested at day 28 and single cell RNA sequencing was performed on both the allograft and the native lung from each mouse (n = 2). Results Flow cytometry demonstrated that the bronchial brush samples predominantly contained epithelial cells (54.5%) and monocytes/macrophages (39.2%). Bulk RNA sequencing of bronchial brushes revealed broad up-regulation of several known glycocalyx sheddases including heparanase and matrix metalloproteinases (MMPs) (Figure 1A). ELISAs on the BAL fluid demonstrated that glycocalyx sheddases (MMP7 and MMP9) and shed glycocalyx fragments (hyaluronic acid) were elevated in the airspaces in CLAD patients relative to controls. In mice, single cell RNA sequencing of whole lung tissue revealed upregulation of heparanase and MMPs (MMP12, and MMP14) in macrophages in the allografts relative to the native lungs (Figure 1B). Conclusions Glycocalyx sheddases were upregulated in bronchial brush samples, which contained predominantly epithelial cells and macrophages, from CLAD patients. Complementary studies using a murine orthotopic lung transplant model demonstrated that these sheddases were likely produced by macrophages. Additionally, both MMPs and glycocalyx fragments were present in the BAL fluid of CLAD patients, which demonstrates that alveolar epithelial glycocalyx shedding occurs in CLAD. Further work is needed in order to further delineate the underlying mechanisms and determine whether therapeutic agents aimed at blocking epithelial glycocalyx degradation could either prevent or treat CLAD. This abstract is funded by: National Institute of Health and the Parker B Francis Foundation
Rizzo et al. (Fri,) studied this question.