(Chest. 2005;128:558S-564S.)
© 2005
American College of Chest Physicians
Molecular and Cellular Determinants of Lung Endothelial Cell Heterogeneity*
Troy Stevens, PhD
* From the Center for Lung Biology and Department of Pharmacology, University of South Alabama College of Medicine, Mobile AL.
Correspondence to: Troy Stevens, PhD, Professor, Department of Pharmacology, Director, Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL 36688; e-mail: tstevens{at}jaguar1.usouthal.edu
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Abstract
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Idiopathic pulmonary arterial hypertension is a progressive and potentially fatal disease with a limited number of therapeutic options. Two key lesions underlie the pathophysiology of this disease. The principal lesion is found in large- and intermediate-sized blood vessels and is characterized by medial and adventitial hypertrophy/hyperplasia, with distal extension of smooth-muscle layers into normally nonmuscularized vessels. The second lesion, found prominently in severe forms of pulmonary hypertension, originates in small precapillary vessel segments, commonly at blood vessel bifurcations. This "plexiform lesion" is a lumen-obliterative lesion comprised, at least in part, of cells that share endothelial cell attributes, but that have lost the "law of the monolayer." Indeed, the endothelial contribution to the (mal-)adaptive response in pulmonary hypertension is becoming increasingly apparent, with evidence that endothelium plays an important role in promoting the vasoconstriction and hyperproliferation of medial and adventitial cell layers in large- and intermediate-vessel sizes, and lumen obliteration in the plexiform lesion. Recent evidence indicates endothelial cells along the pulmonary artery and precapillary segments are phenotypically distinct and may fulfill different roles in these site-specific lesions. Thus, the present review summarizes our current understanding of pulmonary endothelial cell heterogeneity and discusses the potential role(s) of endothelial cell heterogeneity in the pathogenesis of pulmonary hypertension.
Key Words: epigenetic • permeability • phenotype • pulmonary hypertension • lectins
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Introduction
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Pulmonary arterial hypertension is a vascular disorder that is defined by a sustained increase in BP > 25 mm Hg at rest, or > 30 mm Hg with exercise, in the presence of a low or normal pulmonary capillary wedge pressure (< 15 mm Hg) [for review see 12345]. The principal histologic findings of this disorder include medial (smooth-muscle cells) and adventitial (fibroblast) hypertrophy/hyperplasia, with smooth-muscle cell extension into distal blood vessels that are normally nonmuscularized (Fig 1
). Endothelial abnormalities have been described in these large and intermediate, precapillary blood vessels.6 Their morphology becomes characteristic of an "activated" phenotype with some evidence for loss of barrier function, consequently exposing underlying cells to circulating mitogens.7 The complex of mediators released by endothelium changes from one that promotes vasodilation and quiesces smooth-muscle cell growth, to one that promotes vasoconstriction and increases smooth-muscle cell growth.6 Thus, there is general agreement that pulmonary artery endothelial cells fulfill an important role in the adaptive response to pulmonary hypertension.
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Figure 1. The principal lesion in idiopathic pulmonary arterial hypertension involves intermediate- and large-sized pulmonary arteries/arterioles, with medial and adventitial hypertrophy/hyperplasia. Shown is a histologic section of an intermediate-sized pulmonary arteriole from a patient with pulmonary hypertension. The endothelium is resolved by staining (DAB kit; Vector Laboratories; Burlingame, CA) for activated leukocyte cell adhesion molecule (arrowhead). Nuclei are blue. The abnormally large medial wall is shown (asterisks).
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In severe pulmonary hypertension, an obliterative, angioproliferative lesion is found most prominently in distal blood vessels.8910 This lesion begins in vessels that are approximately 25 µm in diameter, commonly at vessel bifurcations, and can extend proximally into larger precapillary arterioles (Fig 2
). Cells in these lesions appear to have an endothelial origin. However, rather than growing in a typical monolayer that defines a patent vessel, they exhibit a proproliferative and apoptosis-resistant phenotype in which the "law of the monolayer" is lost.11 Attempts to reverse this fixed lesion have been unsuccessful so far, although these efforts have just begun in earnest.
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Figure 2. Lumen-occluding plexiform lesions are seen in severe pulmonary hypertension. While these lesions may extend into large blood vessels, serial sectioning reveals the initiating locus is typically a small, precapillary arteriole in the range of 25 µm. Shown are histologic sections of plexigenic lesions from two patients. Endothelial cells exhibit reduced staining for activated leukocyte cell adhesion molecule (arrowhead) [DAB kit; Vector Laboratories]. Right: A site in the vessel wall where endothelial cells appear to have lost their intimate contact and have grown into the lumen (arrow).
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A growing awareness of the principal role endothelial cells play in the development of pulmonary hypertension has reinvigorated interest in fundamental endothelial cell biology, particularly with respect to the stimuli that control their phenotype and function. New data has revealed that endothelial cell behavior varies along vascular segments, in part due to the unique environment of any given segment at any point in time. It is also evident that endothelial cells in extra-alveolar and alveolar segments, respectively, arise from different cell origins during development and, further, that these unique origins imprint a memory that influences cell behavior (for review see Stevens et al1213). Even under similar environmental conditions, extra-alveolar and alveolar endothelial cells possess a distinct phenotype. Thus, understanding the endothelial cell contribution to pulmonary hypertension requires an integrated view of how environmental influences uniquely interact with either arterial or microvascular endothelium to elicit a maladaptive response.
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Heterogeneity of Lung Endothelium
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Heterogeneity in endothelial cell function is evident along all segments of the lung vascular tree, including the arterial and small precapillary segments that are germane to the pathophysiology of pulmonary hypertension. This heterogeneity is evident in functional studies and in the variable site-specific protein expression patterns (for review see Gebb and Stevens12 and Garlanda and Dejana14). As one example, pulmonary arterial endothelium expresses a greater amount of endothelial nitric oxide synthase (eNOS), and produces more nitric oxide (NO), than capillary endothelium, presumably reflecting the importance of NO in maintenance of low pulmonary vascular tone. Loss of eNOS expression increases both pulmonary vascular reactivity and the susceptibility to developing pulmonary hypertension, whereas eNOS overexpression prevents hypoxic pulmonary hypertension.15 One phenotypic adaptation to the endothelial cell environment in patients with pulmonary hypertension may16 or may not17 be a decrease in eNOS protein. However, even in the event they express enough eNOS protein, they lack bioactive NO because increased arginase activity limits substrate that is available to the enzyme.17 This limitation in NO bioavailability may increase pulmonary artery pressure, although it is not clear whether the NO limitation is a cause of pulmonary hypertension, or reflects the activated state of endothelium in this disease, especially since there appears to be a global alteration is endothelial production of vasoactive autocoids. Decreased expression of prostacyclin synthase,18 accompanied by decreased prostacyclin and increased thromboxane production,19 and increased endothelin-120 release—all environmentally induced pulmonary artery endothelial cell adaptations—contribute to the enhanced vasoconstriction that is apparent in pulmonary hypertension.
While decreased NO and prostacyclin, and increased thromboxane and endothelin-1, establish a "vasoconstrictor milieu," they also promote proliferation of subsets of smooth muscle and fibroblasts that reside in the vessel media and adventitia, respectively. This proproliferative stimulus is not likely to be solely due to these autocoids, however, but also to rely on increased access of growth factors from the circulation to the vessel wall.7 Thus, pulmonary artery endothelial cell barrier function is an important determinant of the vessel wall remodeling that occurs in pulmonary hypertension.
Pulmonary artery and microvascular endothelial cells display different barrier properties that reflect the unique molecular anatomy of their junctional proteins.12212223 In the intact circulation, constitutive fluid filtration is 28-fold and 56-fold greater in pulmonary arterial and venous segments, respectively, than in pulmonary microvascular segments.23 Capillary endothelial cells may express more vascular endothelial cadherin than their macrovascular counterparts,14 and some evidence suggests capillary cells also possess epithelial cadherin,2425 an adhesion protein typically found in alveolar epithelium. Messenger RNA profiling and Western blot analysis revealed capillary endothelial cells express more neural cadherin and activated leukocyte cell adhesion molecule message and protein than do pulmonary artery endothelial cells,12 suggesting these junctional proteins may fulfill an important role in the cell-cell recognition and junctional stability of capillary segments.
Pulmonary hypertension is associated with increased fluid flux across endothelium in pulmonary arterial segments, without parenchymal or alveolar edema. These findings are consistent with the idea that endothelial cells in arterial and capillary segments respond differently to circulating inflammatory agonists. There is experimental support for this idea. Oxidant mediated lung injury—including hydrogen peroxide and ischemia-reperfusion—preferentially targets postcapillary segments, whereas mechanical perturbation principally increases capillary endothelial permeability (for review see Gebb and Stevens12). Inflammatory agonists that activate Gq proteins initiate cytosolic calcium transitions in endothelial cells through store-operated calcium entry channels. The activation of store operated calcium entry increases endothelial cell permeability in arterial and venule segments, while typically sparing capillary endothelial cell segments.21 Based on this available literature, we can understand how the blood chemical environment in pulmonary hypertension may increase pulmonary artery endothelial cell permeability—allowing access of circulating growth factors to the underlying vessel wall—without also increasing capillary endothelial permeability that would promote interstitial and alveolar edema.
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Extra-alveolar and Alveolar Endothelium: a Defining Border
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These findings illustrate the remarkable endothelial cell heterogeneity that exists all throughout the pulmonary vascular circuit. A part of this heterogeneity is due to the unique tissue and blood microenvironments that exist in different lung compartments. Pulmonary artery endothelial cells, for example, are exposed to mixed venous blood gases and overlay a thick basement membrane that links the endothelium to diverse cell populations in the media. In contrast, capillary endothelial cells are exposed to arterial blood gases and overlay a thin protein matrix tightly associated with type I pneumocytes. Clearly, these and other environmental stimuli influence cell behavior to meet metabolic tissue requirements.
However, different cell environments are not sufficient to explain how, or why, segment-specific behavior is so specialized, and cannot explain how endothelial cells isolated from different segments retain their unique functions in vitro, when cell environments are similar. It is becoming increasingly appreciated that macrovascular and microvascular endothelial cells arise from different cell origins during lung development, and that they retain a memory of their vascular origin because they are imprinted to do so.12 These phenotypically different endothelial cells establish a discernible border in the pulmonary circulation that can be resolved at the extra-alveolar/alveolar border, even in the fully differentiated, adult lung.
The extra-alveolar/alveolar endothelial cell border has been resolved in vivo using plant lectins.26 In the rat lung, Helix pomatia recognizes pulmonary artery endothelium, whereas Glycine max and Griffonia simplicifolia recognize microvascular endothelial cells. Simultaneous delivery of tetramethylrhodamine isothiocyanate-conjugated H pomatia and fluorescein isothiocyanate (FITC)-labeled G simplicifolia resolves two adjacent endothelial cells with different lectin binding patterns; all endothelium in larger vessels bind H pomatia and all microvascular endothelial cells bind G simplicifolia, with no evidence of overlap (Fig 3
, A). In vitro studies support these results. Cells in culture retain their lectin-binding patterns, even when they are co-cultured in the same experiment (Fig 3, B). Moreover, co-cultured cells tend to segregate and establish a uniquely discernible border between pulmonary artery and microvascular endothelial cells (Fig 3, C, and D). The mechanism of this unique cell recognition pattern is not understood.
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Figure 3. Pulmonary arterial endothelial cells (PAECs) are distinguishable from pulmonary microvascular endothelial cells (PMVECs) both in vivo and in vitro. A: Dual labeling of pulmonary artery endothelium using tetramethylrhodamine isothiocyanate-conjugated H pomatia and microvascular endothelium using FITC-labeled G simplicifolia reveal a site in the intact circulation where different phenotypes interact, at approximately 25 µm in diameter. All upstream pulmonary artery endothelial cells interact with H pomatia, and all downstream capillary segments interact with G simplicifolia (not shown). B: This distinct lectin binding pattern is retained in cultured pulmonary artery and microvascular endothelial cells. Shown is a co-culture experiment where only microvascular endothelial cells interact with FITC-labeled G simplicifolia. C to D: Cell-cell recognition is different among pulmonary artery endothelial cells and microvascular endothelial cells. Cells from different animals were co-cultured on a gridded coverslip and allowed to grow together. Alike cells form a typical cell-cell border that is indistinguishable. However, the pulmonary artery endothelial cell and microvascular endothelial cell border is notable using light (C) and scanning electron microscopy (D). E: Plexiform lesions are comprised of cells that interact with G simplicifolia, suggesting a microvascular endothelial phenotype. Human lung specimens from patients with severe pulmonary hypertension were used to analyze plexiform lesions for their ability to interact with lectins. This 70-µm lesion interacts selectively with G simplicifolia and not H pomatia (not shown), suggesting cells with a microvascular endothelial cell phenotype are present in the lesion.
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The border between extra-alveolar and alveolar endothelial cells occurs in vessels that are approximately 25 µm in diameter, an important vascular site in pulmonary hypertension. This vessel size is typically poorly muscularized but gains increasing myocyte association as smooth-muscle layers extend distally in pulmonary hypertension. This is also the approximate vessel size where plexiform lesions first appear in severe pulmonary hypertension.8 Extensive work has indicated the plexiform lesion arises from some type of endothelial cell.27 It is not clear whether tissue or circulating endothelial cells account for the lumen-occluding lesion. Perhaps more importantly, it is not clear whether cells in the plexiform lesion exhibit a "macrovascular" or "microvascular" phenotype. We addressed this issue using G simplicifolia lectin. Human lesions in small-vessel segments possessed cell bundles that interacted with G simplicifolia, consistent with a microvascular endothelial cell phenotype (Fig 3, E).
The microvascular endothelial cell phenotype has very unique growth properties that may importantly underscore its contribution to pulmonary hypertension (Fig 4
). These cells grow much faster than do pulmonary artery endothelial cells,26 and global expression profiling experiments revealed the up-regulation of a number of proproliferative molecules in these cells, including vascular endothelial cell growth factor. Serum restriction growth suppresses these cells. However, unlike pulmonary artery endothelial cells, serum stimulation during the lag phase of cell growth is sufficient to support subsequent cell proliferation in the 0.1% serum. It will be critical to establish a more detailed understanding of how these cell populations govern their proliferative potential, to better appreciate how microvascular cells (or pulmonary artery endothelial cells) contribute to the plexiform lesion.
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Figure 4. Pulmonary microvascular endothelial cells grow faster than do pulmonary artery endothelial cells. Top left, A: Serum (10%)-stimulated growth curves were constructed in pulmonary microvascular endothelial cells and pulmonary artery endothelial cells, illustrating the greater growth capacity of pulmonary microvascular endothelial cells. Top right, B: Serum restriction (0.1%) abolished growth in both cells types. Bottom, C: Cell incubation with 10% serum for 48 h, followed by 0.1% serum, was sufficient to reveal the progrowth program in pulmonary microvascular endothelial cells. See Figure 3 legend for expansion of abbreviations.
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Implications of Endothelial Heterogeneity in the Pathophysiology of Pulmonary Hypertension
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Idiopathic pulmonary arterial hypertension is a precapillary phenomenon, although it is not exactly clear why. However, the emerging evidence of developmentally early, segment-specific endothelial cell specification provides some insight into how a disease process may be segment specific. Genetic studies have incriminated bone morphogenetic receptor protein II signaling as a cause of pulmonary hypertension in a subset of patients, while dysfunctional transforming growth factor-ß and activin receptor-like kinase I signaling has been implicated as a cause of hereditary hemorrhagic telangiectasis and pulmonary hypertension (for review see Budhiraja et al6). Activin receptor-like kinase I, in particular, is interesting because its expression is arterial specific during development.2829 Such cell specification undoubtedly plays a key role in the highly site-specific nature in which this disease presents and, perhaps, provides insight into the less asked question of why the vascular adaptation does not continue into capillary segments.
While speculative, extra-alveolar and alveolar endothelial cells may fulfill different roles in the pathogenesis of pulmonary hypertension. The principal precapillary lesion in resistance vessels may involve an extra-alveolar endothelial cell dysfunction that promotes vasoconstriction and proliferation of cells in the underlying media and adventitia, whereas the plexiform lesion may involve uncontrolled growth of microvascular endothelial cells that ultimately occlude the lumen. If correct, then as the field of pharmacogenetics becomes entrenched in clinical medicine, the drug targets, and sensitivity to pharmacotherapy, will exhibit/possess vascular segment-specific efficacy. Better resolving the site-specific nature of the lesions that are pertinent to pulmonary hypertension will therefore greatly improve our understanding of how to treat this devastating disorder.
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Acknowledgements
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The author thanks Drs. Brian Fouty, Kane Schaphorst, and Mark Gillespie for their thoughtful discussions.
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Footnotes
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Abbreviations: eNOS = endothelial nitric oxide synthase; FITC = fluorescein isothiocyanate; NO = nitric oxide
The work discussed in this review was supported, in part, by National Institutes of Health grants HL66299 and HL60024.
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Abstract
Introduction
