TY - JOUR
T1 - Alveolocapillary model system to study alveolar re-epithelialization
AU - Willems, Coen H. M. P.
AU - Zimmermann, Luc J. I.
AU - Sanders, Patricia J. L. T.
AU - Wagendorp, Margot
AU - Kloosterboer, Nico
AU - Tervaert, Jan Willem Cohen
AU - Duimel, Hans J. Q.
AU - Verheyen, Fons K. C. P.
AU - van Iwaarden, J. Freek
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
KW - Pulmonary alveolus
KW - Alveolocapillary model system
KW - Co-culture
KW - Re-epithelialization
KW - Protrusions
KW - Junctions
KW - Communication
KW - ATII
KW - A549
KW - Pulmonary microvascular endothelium
KW - HPMEC
U2 - 10.1016/j.yexcr.2012.09.010
DO - 10.1016/j.yexcr.2012.09.010
M3 - Article
C2 - 23022369
SN - 0014-4827
VL - 319
SP - 64
EP - 74
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 1
ER -