Elsevier

Microvascular Research

Volume 75, Issue 3, April 2008, Pages 391-402
Microvascular Research

Regular Article
Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions

https://doi.org/10.1016/j.mvr.2007.10.006Get rights and content

Abstract

Endothelial cells assemble unique barriers that confer specific permeability requirements at different vascular segments. We examined lung microvascular and artery endothelial cells to gain insight into mechanisms for segment-specific barrier functions. Transendothelial electrical resistance was significantly higher in microvascular barriers, and a 50% reduction in barrier function required 5-fold higher concentration of cytochalasin D in the microvascular compared to the arterial barrier. Transcriptional profiling studies identified N-cadherin and activated leukocyte cell adhesion molecule (ALCAM) to be most highly expressed in microvascular than in pulmonary artery endothelial cells. ALCAM was detected in microvascular endothelial cells in the alveolar septum but not in endothelial cells in larger pulmonary vessels in situ. This pattern was retained in culture as ALCAM was recruited to cell junctions in pulmonary microvascular endothelial cells but remained predominantly cytosolic in pulmonary artery endothelial cells. Confocal analysis revealed ALCAM in the lateral plasma membrane domain where it co-localized with N- and VE-cadherin. This finding was supported by co-immunoprecipitation studies demonstrating the presence of ALCAM in multiple adherens junction protein complexes. These functional, biophysical and molecular findings suggest specialization of the adherens junction as a basis for a highly restrictive endothelial barrier to control fluid flux into the alveolar airspace.

Introduction

The vascular system is a complex network of conduit and microvascular vessels exposed to different organ segments with unique local requirements for plasma protein, fluid, and leukocytes (Butcher et al., 1980, Cavender, 1990, Leach, 2002, Leach et al., 2002, Thurston et al., 2000). Endothelial cells lining the lumen of these vessels assemble barriers that control the passage of circulating blood constituents into the interstitium (Patterson and Lum, 2001). This suggests endothelial barriers are likely specialized to confer segment-specific phenotypes. The pulmonary microcirculation receives the entire blood volume as a requirement for saturating blood with oxygen. It possesses a relatively large surface area equal to the capillary surface area of the rest of the body (∼ 70 m2), which facilitates this process. Fluid homeostasis in this vascular bed and the adjoining alveolar airspace is therefore critical for perfusion of oxygen into the local circulation and supply of oxygen to all tissues and organs in the body (Crandall et al., 1983). It is clear that the microanatomy of the blood–air barrier measuring only 0.1 μm thick along most of its border is adapted for this unique function, and part of this specialization is most likely the intercellular junctions that maintain barrier integrity.

Indeed, there is a preponderance of functional data indicating lung microvascular endothelial cells possess tight permeability barriers (Chetham et al., 1999, Kelly et al., 1998, Moore et al., 1998, Parker and Yoshikawa, 2002). Protein and fluid conductance per unit surface area is significantly lower in the lung's microcirculation than in the pulmonary artery (Parker and Yoshikawa, 2002). Studies in isolated rat lung preparations and monolayers of cultured lung endothelial cells indicate a more restrictive permeability barrier in pulmonary microvascular endothelial cells (PMVECs) than in pulmonary artery endothelial cells (PAECs) (Chetham et al., 1999, Kelly et al., 1998, Moore et al., 1998). However to date the molecular basis for the unique barrier phenotype in PMVECs has not been elucidated.

Protein and fluid flux across endothelial barriers occurs through paracellular channels between apposed endothelial cells or by a transcellular route involving vesicular transport (Lum and Malik, 1994, Stevens et al., 2000). Several multi-protein complexes play an important role in regulating paracellular transport (Lum and Malik, 1994, Stevens et al., 2000). Adherens and tight junctions promote cell–cell adhesion, integrin receptors mediate cell adhesion to intracellular matrix proteins and cytoskeletal structures exert an intracellular outward tension (Dudek and Garcia, 2001, Gumbiner, 1996, Lum and Malik, 1994, Schnittler, 1998, Stevens et al., 2000). The adherens junction contains vascular endothelial cadherin (VE-cadherin), which is constitutively expressed in all endothelial cells (Schnittler, 1998). There is hemorrhagic pulmonary edema and death likely due to respiratory distress in mice injected with monoclonal VE-cadherin antibody indicating a dominant role for VE-cadherin and multi-protein complexes containing VE-cadherin in lung permeability (Corada et al., 1999). It will be important to identify the profile of this multi-protein complex in PMVECs.

In this study, we used biophysical assays to demonstrate unique intercellular interactions in PMVECs and PAECs. Microarray analysis demonstrated PMVECs and PAECs possessed characteristic gene expression profiles for several adhesion molecules. ALCAM, N-cadherin and VE-cadherin were enriched at cell junctions in PMVECs but were either sparsely distributed or lacking in the junctions in PAECs. ALCAM was linked to β-catenin and Dlg confirming its localization in the adherens junction. These findings highlight unique specialization of the adherens junction as a potential mechanism for tightly controlling vascular permeability at the blood–air barrier.

Section snippets

Antibodies

Generation of primary anti-rabbit ALCAM antibody has previously been described (Matsumoto et al., 1997). Primary non-conjugated monoclonal antibodies used were anti-ALCAM clone ND4 (a gift from Dr. Sviridov), -ALCAM clone MOG/07 (Novacastra, Newcastle, UK), -VE-cadherin clones ab7047 (Abcam Limited, Cambridge, UK) and F-8 (Santa-Cruz Biotech, Santa Cruz, CA), -beta catenin, -alpha-catenin, -p120 catenin (BD Bioscience Pharmigen, San Diego, CA), -ZO-1 and -N-cadherin (Zymed Laboratories Inc. San

Lung microvascular endothelial cells assemble a more restrictive barrier than their macrovascular counterparts

PMVECs and PAECs were seeded in electrical cell impedance system (ECIS) chambers containing 10 evaporated gold electrodes, and cultured for 4–6 days. After confirmation by phase contrast microscopy that the culture was confluent and that each electrode was covered with a monolayer of endothelial cells, the chamber was inserted unto ECIS blocks and TER recorded at 37 °C, 5% CO2 in a humidified chamber. TER was steady for over 20 h in confluent cultures indicating attainment of stable endothelial

Discussion

This study was devised on the principle that endothelial barriers are highly specialized to confer segment-specific permeability phenotypes in different vascular beds. This idea is particularly important in the lung microvascular bed, which contains relatively flat endothelial cells anatomically structured to facilitate efficient alveolar-gas exchange, and likely possess specialized adhesive mechanisms to maintain barrier integrity. To unravel the unique attributes of this barrier we compared

Acknowledgments

This work was supported by research grants HL077769 (SFOA) and HL66299 (TS).

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