Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes
Introduction
Although endothelium lines blood vessels throughout the circulation, it exhibits highly specialized functions in different vascular sites. In the systemic circulation, permeability edema is prominent at post-capillary venules (Thurston et al., 2000). White blood cell recruitment to sites of inflammation occurs at high endothelial venules Cavender, 1990, Colditz, 1985 and, while the blood brain barrier consists of endothelium with tight cell–cell junctions (Gloor et al., 2001) that are highly restrictive, both renal glomerular (Stan et al., 1999) and liver sinusoidal endothelium (Grisham et al., 1975) possess fenestrations that are highly permeable. It is clear that these distinct endothelial cell characteristics are at least partly directed by environmental cues (Stevens et al., 2001).
The embryological origin of endothelial cells may also contribute to their site-specific function (Stevens et al., 2001). Studies in the developing lung suggest two distinct processes form the circulation deMello and Reid, 2000, deMello et al., 1997, Hall et al., 2000, Schachtner et al., 2000, Schwarz et al., 2000, including angiogenesis of large vessels and vasculogenesis of small vessels. deMello et al. (1997) used a casting technique to temporally illustrate vascular tube formation. The earliest formed vascular structures were observed at embryonic day 14 (E14) in the developing mouse lung. These structures progressively branched from large vessels at sharp angles, consistent with angiogenesis, but did not form a contiguous vessel. The parallel growth of blood lakes/islands that were filled with precursor cells of hematopoietic origin was observed by transmission electron microscopy until, at E15, a fusion between angiogenic sprouts and vasculogenic blood islands could be resolved using the vascular casting technique. This issue has also been addressed by assessing the temporal expression pattern of endothelial cell markers in developing lung (Schachtner et al., 2000). Endothelial cells of both large(r) and small vessels express the VEGF receptor Flk-1 during development, which has been interpreted to suggest vessels larger than originally suspected may originate from vasculogenesis. Thus, while this issue is not completely understood, in the simplest form, it appears that endothelial cells in large and small blood vessels are likely to arise from different progenitors.
Functional studies in in vitro models illustrate that lung microvascular endothelial cells possess a more restrictive barrier than their macrovascular counterparts Chetham et al., 1999, Kelly et al., 1998, Moore et al., 1998b, and exhibit unique signaling responses to similar agonists Chetham et al., 1999, Kelly et al., 1998, Moore et al., 1998a, Stevens et al., 1997, Stevens et al., 1999, Stevens et al., 2001. Distinct site-specific vascular responses are observed in the intact lung Chetham et al., 1999, Khimenko and Taylor, 1999, Qiao and Bhattacharya, 1991. The lung's microcirculation is more restrictive to protein and water flux than is the macrocirculation (Parker and Yoshikawa, 2002). In contrast, macrovascular endothelial cells express more eNOS (Stevens, unpublished) and generate more nitric oxide (Al-Mehdi, unpublished) than do microvascular endothelial cells. Large and small pulmonary vessels appear to exhibit unique growth or survival properties. Indeed, the lung's microcirculation exhibits significantly more plasticity than previously appreciated Massaro and Massaro, 1997, Massaro and Massaro, 2000, Massaro and Massaro, 2001, Massaro and Massaro, 2002, Massaro et al., 2000. Emphysema-like lesions are associated with a decrease in alveolar and capillary (e.g., microvascular endothelial cell) density, a portion of which can be rescued by retinoic acid. These findings are generally compatible with evidence that alveolar cells and microvascular endothelial cells uniquely regulate one another's function, partly dependent upon vascular endothelial cell growth factor (VEGF) signaling to orchestrate capillary development along the basement membrane of airway epithelium Acarregui et al., 1999, Dumont et al., 1995, Gebb and Shannon, 2000, Lassus et al., 2001, Shalaby et al., 1997. VEGF stimulates small vessel formation and microvascular endothelial cell survival. The VEGF receptor Flk-1 null mice die because blood islands are disorganized and microvessels do not form (Shalaby et al., 1995). In the fully developed lung inhibition of VEGF signaling reduces alveolar septation (as in emphysema) (Kasahara et al., 2000) and, in combination with hypoxia, generates microvascular (≈100 μm) plexigenic lesions (Taraseviciene-Stewart et al., 2001). Thus, lung endothelial cell origin may be an important determinant of cell phenotype and function. To further determine the unique attributes of lung macro- and microvascular endothelial cells, we undertook studies to examine whether pulmonary artery (PAEC) and microvascular (PMVEC) endothelial cells isolated from the fully differentiated organ exhibit distinct structure and function, even under similar environmental conditions.
Section snippets
Isolation and culture of rat main pulmonary artery endothelial cells (PAECs)
Main pulmonary arteries were isolated as previously described Creighton et al., 2003, Stevens et al., 1999. Briefly, 300–400 g Sprague–Dawley rats were euthanized by an intraperitoneal injection of 50 mg of pentobarbital sodium (Nembutal, Abbott Laboratories, Chicago, IL). The heart and lungs were excised en bloc after sternotomy and the mainstem pulmonary artery and two vessel generations were isolated and removed. The artery was inverted and the intimal lining was carefully scraped using a
Lectin binding to lung macro- and microvascular endothelial cells
Lectin binding has previously been utilized as an effective method of discriminating between macro- and microvascular endothelial cells Abdi et al., 1995, Del Vecchio et al., 1992, Fischer et al., 2000, Gumkowski et al., 1987, Lotan et al., 1994, Magee et al., 1994, Norgard-Sumnicht et al., 1995, Schnitzer et al., 1994. Nine different lectins were therefore screened for binding to the rat pulmonary artery and microvascular endothelial cell surface (Table 1). Of the nine lectins examined, six
Discussion
Our present studies were founded on the hypothesis that PAEC and PMVEC phenotypes are distinct, in part due to their epigenetic origin. If this hypothesis is true, then the cells should retain distinct functions in vitro when their environments are similar. We approached this hypothesis using structure–function analyses, evaluating morphological characteristics of the cells along with functional endpoints.
Lectins are protein agglutinins isolated from various plant and animal sources that have
Acknowledgements
We thank Dr. Ray Hester for his participation in this work. Supported by HL66299 and HL60024 (T. Stevens).
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