title: Alveolocapillary model system to study alveolar re-epithelialization.
abstract: In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process.
authors.affiliation: Department of Paediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: Coen H M P
authors.initials: CH
authors.lastname: Willems
authors.forename: Luc J I
authors.initials: LJ
authors.lastname: Zimmermann
authors.forename: Patricia J L T
authors.initials: PJ
authors.lastname: Sanders
authors.forename: Margot
authors.initials: M
authors.lastname: Wagendorp
authors.forename: Nico
authors.initials: N
authors.lastname: Kloosterboer
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Cohen Tervaert
authors.forename: Hans J Q
authors.initials: HJ
authors.lastname: Duimel
authors.forename: Fons K C P
authors.initials: FK
authors.lastname: Verheyen
authors.forename: J Freek
authors.initials: JF
authors.lastname: van Iwaarden
mesh_terms: D055371:Acute Lung Injury / Q000473:pathology* / Q000503:physiopathology; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology* / Q000473:pathology; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology / Q000503:physiopathology; D011650:Pulmonary Alveoli / Q000098:blood supply* / Q000166:cytology* / Q000473:pathology; D020545:Respiratory Mucosa / Q000098:blood supply* / Q000166:cytology / Q000473:pathology