Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung.
In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7-11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, beta-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, beta-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-alpha from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.
Hermanns
Maria Iris
MI
Institute of Pathology, Mainz University Clinic, Johannes Gutenberg University, Langenbeckstrasse 1, 55101, Mainz, Germany. hermanni@uni-mainz.de
Fuchs
Sabine
S
Bock
Michaela
M
Wenzel
Katja
K
Mayer
Eckhard
E
Kehe
Kai
K
Bittinger
Fernando
F
Kirkpatrick
C James
CJ
D055371:Acute Lung Injury / Q000209:etiology* / Q000473:pathology; D051402:Aquaporin 5 / Q000235:genetics / Q000378:metabolism; D015824:Blood-Air Barrier / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products / Q000235:genetics / Q000378:metabolism
C496036:AQP5 protein, human; D051402:Aquaporin 5; D037721:Pulmonary Surfactant-Associated Protein C; D000067759:Receptor for Advanced Glycation End Products; C489679:SFTPC protein, human