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Pulmonary Microvascular Endothelial Cells Form a Tighter Monolayer when Grown in Chronic Hypoxia

¹ Center for Lung Biology; Departments of ² Pharmacology and ³ Physiology; and ⁴ Division of Pulmonary Medicine, University of South Alabama School of Medicine, Mobile, Alabama

Correspondence and requests for reprints should be addressed to Brian Fouty, Center for Lung Biology, MSB 3340, University of South Alabama, Mobile, AL 36688. E-mail: bfouty{at}jaguar1.usouthal.edu.

Unique among the vascular beds, loss of endothelial integrity in the pulmonary microcirculation due to injury can lead to rapidly fatal hypoxemia. The ability to regain confluence and re-establish barrier function is central to restoring proper gas exchange. The adult respiratory distress syndrome (ARDS) is a heterogeneous disease, however, meaning that endothelial cells within different regions of the lung do not likely see the same oxygen tension as they attempt to proliferate and re-establish an intact endothelial monolayer; the effect of hypoxia on the integrity of this newly formed endothelial monolayer is not clear. Immortalized human pulmonary microvascular endothelial cells (PMVEC) (ST1.6R cells) were sparsely plated and grown to confluence over 4 days in either normoxia (21% oxygen) or hypoxia (5% oxygen). Confluence attained in a hypoxic environment resulted in a tighter, less permeable endothelial monolayer (as determined by an increase in transendothelial electrical resistance, decreased permeability to fluorescently labeled macromolecules, and decreased hydraulic conductance). PMVEC grown to confluence under hypoxia had decreased RhoA activity; consistent with this finding, inhibition of Rho kinase, a well-described downstream target of RhoA, markedly increased electrical resistance in normoxic, but not hypoxic, PMVEC. These results were confirmed in primary human and rat PMVEC. These data suggest that PMVEC grown to confluence under hypoxia form a tighter monolayer than similar cells grown under normoxia. This tighter barrier appears to be due, in part, to the inhibition of RhoA activity in hypoxic cells.

Key Words: human pulmonary microvascular endothelial cells • hypoxia • permeability • RhoA/Rho kinase

CLINICAL RELEVANCE Re-establishing endothelial confluence in the pulmonary circulation is critical to restoring proper gas exchange after acute lung injury/acute respiratory distress syndrome. These studies demonstrate that the oxygen tension at which this occurs impacts the integrity of the monolayer formed.