Elsevier

Vascular Pharmacology

Volume 52, Issues 5–6, May–June 2010, Pages 175-181
Vascular Pharmacology

Harvesting, identification and barrier function of human lung microvascular endothelial cells

https://doi.org/10.1016/j.vph.2009.12.009Get rights and content

Abstract

Endothelial barrier dysfunction is an important contributor to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Even though approaches that target the prevention and repair of endothelial barrier dysfunction are clearly needed, our understanding of the molecular regulation of pulmonary microvascular endothelial permeability remains incomplete. Cultured pulmonary microvascular endothelial cells represent an attractive paradigm for the study of barrier function. Here, we describe a method for the harvest, identification and culture of human lung microvascular endothelial cells (HLMVEC). HLMVEC thus obtained, grow as a monolayer, exhibit contact inhibition and have the typical cobblestone appearance. They express endothelial proteins, such as von Willebrand factor and endothelial nitric oxide synthase and take up an acetylated LDL. Furthermore, HLMVEC respond predictably and with superior sensitivity to the barrier disruptive effects of Gram positive and Gram negative bacterial products, thrombin, vascular endothelial growth factor and microtubule disrupting agents. These HLMVEC present an in-house-derived alternative to commercially available human cells for the study of mechanisms contributing to ALI and ARDS.

Graphical abstract

Human lung microvascular endothelial cells (HLMVEC) in culture. Phase contrast micrograph (left) and HLMVEC stained with antibody against the von Willebrant factor.

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Introduction

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a continuum of progressive respiratory failure in the absence of left heart failure. ARDS patients represent a subset of ALI patients, distinguished by a greater severity. In ALI/ARDS, the integrity of the capillary barrier is compromised, leading to increased vascular permeability and alveolar flooding. Gram negative sepsis (indirect injury) is by far the most common cause of ALI (Hudson et al., 1995). Sepsis represents the systemic inflammatory response to infection, (Jacobi, 2002). Lungs are among the most frequently affected organs in severe sepsis leading to ALI and ARDS (Martin et al., 2003). The incidence of sepsis has increased by 8.7% from 1979 to 2000 (Martin et al., 2003) and mortality ranges from 30 to 50% (Rangel-Frausto et al., 1995, Angus et al., 2000, Annane et al., 2000). Clinical trials targeting inflammatory mediators have shown no survival benefit (Fisher et al., 1994, McCloskey et al., 1994, Abraham et al., 1998, Dhainaut et al., 1998, Fink, 1998, Abraham et al., 2001) and other strategies have failed to reduce morbidity associated with severe sepsis except for the survival benefit with the use of recombinant activated protein C (Bernard et al., 2001).

Even though approaches that target the prevention and repair of endothelial barrier dysfunction are clearly needed, our understanding of the molecular regulation of pulmonary microvascular endothelial permeability remains incomplete. Cultured pulmonary microvascular endothelial cells represent an attractive paradigm for the study of barrier function. However animal-derived endothelial cells do not necessarily reflect the complex biology of human endothelial cells. Moreover, human endothelial cells, while available commercially, are expensive and, frequently of inconsistent quality. Here we present a method for the harvesting, identification and culture of human lung microvascular endothelial cells. We further provide data to suggest that these cells exhibit a strong baseline barrier function and respond predictably to common edemagenic agents.

Section snippets

Materials

Tosyl activated Dynabeads and Prolong Gold were from Invitrogen; eNOS antibody was from Becton Dickinson; vWF antibody was from Sigma; goat anti-mouse IgG and Cy3 goat anti-rabbit IgG were from Jackson Laboratories. Fetal bovine serum (FBS) was from Hyclone. All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Eight-well arrays were from Applied Biophysics (Albany, NY).

Harvest, culture and identification of human lung microvascular endothelial cells (HLMVEC)

Peripheral lung specimens from patients that were undergoing lobectomy or pneumonectomy at the Medical

Identification of human lung microvascular endothelial cells

The harvesting procedures outlined in the Materials and methods consistently produced endothelial cells free of other cellular contamination. We call these cells microvascular because we utilized the sub-pleural lung segments with no visible large vessels, as well as subjected the chopped tissue to two successive 100 μm filtrations. Furthermore, these cells are endothelial cells for several reasons. As shown in Fig. 1, left upper panel, they are of small size, form a contact-inhibited monolayer,

Discussion

The maintenance of a barrier between blood and tissue is a very important function of vascular endothelium. This is especially true for pulmonary microvascular endothelium, because failure to maintain a healthy barrier may result in impaired gas exchange, reduced blood oxygenation, acid-base disturbances, acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Over the past three decades, cultured endothelial cells have been used frequently as a model for the study of

Acknowledgements

We thank Dr. Chakraborty for providing us with purified listeriolysin and pneumolysin. This work was supported by the American Heart Association, Southeast Affiliate and the National Institutes of Health, HL070214. This work was also supported by a Programmatic Development award (to SMB, JDC, DF, RL, and ADV) from the Cardiovascular Discovery Institute of the Medical College of Georgia.

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