(Chest. 2005;128:547S-552S.)
© 2005
American College of Chest Physicians
Phenotypic Characterization of Pulmonary Arteries in Normal and Diseased Lung*
Michael Kasper, PhD
* From the Institute of Anatomy, Medical Faculty "Carl Gustav Carus," Technical University of Dresden, Dresden, Germany.
Correspondence to: Michael Kasper, PhD, Institute of Anatomy, Medical Faculty, Fetscherstr. 74, D-01307 Dresden, Germany; e-mail: michael.kasper{at}mailbox.tu-dresden.de
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Abstract
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Vascular endothelium is a continuous cell layer lining the cardiovascular system and serves as an interface between blood and the vascular wall tissue. Although the basic morphology of endothelial cells is similar in blood vessels of different organs and tissues, there is a great heterogeneity in endothelial cell types based on structural, metabolic, and developmental differences within each organ, particularly in the pulmonary vasculature. Current data about the usage of different markers for the immunohistochemical detection of endothelial cells in lung tissue are summarized, and functional aspects of caveolin expression after lung injury and in pulmonary hypertension are discussed.
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Structural Basis of Pulmonary Endothelial Heterogeneity
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The lung has a dual blood supply: the pulmonary or minor circulation system and the bronchial vasa privata. Pulmonary arteries return deoxygenated blood to the lungs, whereas bronchial arteries supply oxygenated blood to maintain the pulmonary tissues. Various communications exist between all arteries and veins of the pulmonary and bronchial systems (reviewed in1). Therefore, a high diversity of blood vessels has to be expected and can be distinguished in different anatomic compartments of lungs, which contain arteriolar, capillary, and venular endothelial cells in the pulmonary as well in the bronchial circulation. The alveolar capillaries are terminal networks in the pulmonary circulation. They are also termed microvascular endothelial cells to distinguish them from all the other macrovascular endothelial cells. Furthermore, there is some evidence about other separate capillary beds surrounding the pulmonary arteries.2
In addition to the arterial and venous blood vessels, there exist lymphatic blood vessels in the different compartments of the lung, which are also covered by endothelial cells.3 The understanding of the complex pattern of pulmonary vasculature is further complicated by anatomic deviations in the pulmonary and bronchial architecture of different species used for experimental animal models of pulmonary disease (for details see45).
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Immunohistochemistry of Pulmonary Blood Vessels
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One important limitation for the immunohistochemical application of specific endothelial markers is the molecular heterogeneity of the endothelium in different organs and tissues and, in our case, in different parts of the pulmonary tree. More important is the modulation of structural and metabolic components of endothelial cells during differentiation, growth, and remodeling in normal and under pathologic conditions in disease or under environmental stress and in other types of injury (reviewed in Aird6).
Table 1
lists some selected suitable markers of lung endothelial cells currently used for monitoring of blood vessel endothelium in vitro and in vivo, and Figure 1
shows an example of a representative immunostaining of pulmonary blood vessels in serial sections of rat lungs to illustrate the endothelial antigen heterogeneity. To the best of our knowledge, no reliable endothelial marker exists that can distinguish precisely between microvascular and macrovascular endothelial cells of the pulmonary tree. Some endothelial antigens such as vascular cell adhesion molecule-1, E-selectin, and P-selectin are inducible adhesion molecules, which under normal conditions show a restricted distribution in different types of endothelia. Another inducible protein is the vascular adhesion protein-1, which can be localized in the majority of macrovascular, but not in microvascular endothelial cells.7 Up to now, markers are not known that solely detect microvascular endothelial cells. Furthermore, under certain metabolic circumstances, endothelial cells are able to express antigens that do not correspond to any known anatomic category of blood vessel, for example, the observation of mosaic-like distribution of von Willebrand factor and/or thrombomodulin in human lung microvascular endothelia.8
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Table 1. Selection of Endothelial Antigens Suitable for the Immunohistochemical Characterization of Blood Vessel Endothelia in Mouse, Rat, and Human Lung
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Figure 1. Paraffin consecutive serial sections of normal rat lung interlobular area. Immunostaining for podocalyxin (top left, A), von Wilebrand factor (top right, B), aquaporin-1 (bottom left, C), and podoplanin (bottom right, D). Podocalyxin is detectable in macrovascular and microvascular endothelial cells (top left, A). von Willebrand factor is not present in the lymphatic endothelium (top right, B; asterisk), which is podoplanin positive (bottom right, D). Note the additional immunoreactivity for aquaporin-5 in smooth-muscle cells, and in cells of a peripheral nerve (arrowheads) [original x 300].
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Another successful approach to stain endothelial cells is the lectin histochemistry. Wheat germ agglutinin, Ulex europaeus agglutinin, Griffonia simplicifolia agglutinin, and Lycopersicon esculentum agglutinin are preferably used for their visualization.9 Species-, age-, sex- and differentiation-dependent differences in lectin binding, however, are critical factors and limit their usage.
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Caveolin and Endothelial Dysfunction
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Caveolin-1 and caveolin-2, the structural proteins of caveolae, seem to be among the best markers of endothelial cells in different blood vessel entities. Caveolae are small plasma membrane invaginations that play a role as biological platforms, eg, microdomains or specific lipid rafts, involved in many cell functions, including cholesterin transport, cellular signaling, as well as proliferation and apoptosis.10 Caveolin-1, -2, and -3 are important constituents of caveolae. Most abundant caveolins of the lung are caveolin-1 and caveolin-2 in all pulmonary endothelial cell types and in alveolar epithelial type I cells. Interestingly, two isoforms of caveolin-1 are existing in the lung, termed 
and ß, which are expressed in a mutually exclusive manner: alveolar epithelial cells express predominantly caveolin-1ß, whereas capillary endothelial cells express caveolin-1.11
In vitro data about the role of caveolins in the endothelium suggest that caveolin-1 regulates nitric oxide and Ca++ signaling. Caveolin-1 also plays a central role in caveolae-mediated transcytosis (reviewed in Minshall et al12). Only sparse data can be found about a modulation of caveolin expression in the lung in vivo. The current knowledge on endothelial caveolin expression in pulmonary disease is low. We13 demonstrated previously that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly down-regulated after irradiation-induced lung injury. In contrast, caveolin expression increased in endothelial cells. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist. These early suggestions were substantiated by recent data of Kogo et al,11 showing that two caveolin-1 and ß isoforms are generated from distinct messenger RNAs in vivo, which may have unique physiologic functions. It has to be proven whether the different caveolin isoforms are responsible for a different resistance of epithelial and endothelial cells after lung injury. A further interesting new finding has been provided by Kathuria et al,14 who investigated the promotor region of caveolin-1 gene and showed an ETS cis-element as important region that accounts for differential caveolin-1 expression in pulmonary epithelial and endothelial cells. Caveolin-1 has further been studied in epithelial and endothelial cells in an experimental model of interstitial edema.15 In edema, caveolin-1 protein distribution increased in the detergent resistant fraction of endothelial plasma membrane as compared to the rest of the plasma membrane.
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Lessons From Knockout Animals
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In different animal models of pulmonary fibrosis (see above) and in various knockouts related to specific lung antigens (advanced glycation endproduct receptor, aquaporin-5, surfactant protein C), we noted a dramatic loss of caveolin expression in the alveolar epithelium but an increase in expression of caveolins in the pulmonary capillary endothelium (Fig 2
;131617). Consistently, all lungs of these knockouts exhibited a fibrosis-like phenotype with enhanced collagen type I expression in thickened alveolar walls and inflammatory reactions.17 That notably not all knockouts of proteins related to alveolar type 1 cells are leading to a fibrotic phenotype, can be seen in the intracellular adhesion molecule (ICAM)-1 knockout, which is even more resistant to pulmonary fibrosis.18
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Figure 2. Immunperoxidase detection of caveolin in normal mouse lung (top left, A), bleomycin- treated mouse lung (top right, B), in surfactant protein-C knockout (bottom left, C) and in glycation endproduct receptor knockout (bottom right, D) mouse. Note the loss of epithelial and the pronounced staining of endothelial caveolin in top right, B, to bottom right, D, as compared with the normal caveolin expression pattern of normal lung tissue (top left, A) [original x 300].
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Mice with a null mutation in the Cav-1 gene highlight the importance of caveolins in the lung. The Cav-1 knockout animals lacking caveolae are viable, fertile, and do not acquire lung tumors. The major pathology is in lung, manifest as thickened alveolar septae containing abundant collagen type I, abnormal cell proliferation, resembling a fibrosis-like phenotype19 and to a certain extent features of pulmonary hypertension.2021
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Is the Caveolin-1 Knockout a Model of Pulmonary Hypertension?
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Plexiform lesions in the lungs from patients with primary pulmonary hypertension as well as the complex vascular lesions of a rat model of severe pulmonary hypertension exhibited a strongly reduced endothelial caveolin-1 expression.22 The results are indicative of a role for Cav-1 in aberrant blood vessel formation. According to immunohistochemical analysis, the proliferating cells in plexiform lesions represent endothelial cells and modified smooth-muscle cells or pericytes (for review see Tudor and Voelkel23).
The situation in caveolin-1-/- mouse is different: pulmonary hypertension in caveolin-1 knockouts could be caused by an intrinsic effect in the pulmonary blood vessels due to the lack of caveolin-1. Pulmonary hypertension could also be secondary to fibrosis-like alterations or caused by cardiac dysfunction.21 In the caveolin-1 knockout, the undefined cells of alveolar septa express the vascular endothelial growth factor receptor flk-1, but not any of the mature endothelial markers such as ICAM-1 or podocalyxin (unpublished data, Fig 3
). They do not proliferative as revealed by double immunostaining with Ki-67 (not shown). Contrary, proliferating endothelial cells of plexiform lesions in humans are podocalyxin positive.
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Figure 3. Paraffin sections of caveolin-1 knockout mouse. The fibrosis-like changes appear as thickened alveolar walls with increased deposition of collagen type I (top, A). Note the podocalyxin-positive capillary endothelial cells (center, B: knockout; bottom, C: wild-type) and the high number of cells, which are immature endothelial or other cells in center, B (original x 300).
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Cardiac dysfunctions in caveolin-1 knockouts may be a secondary phenomenon. The cardiac contractility could be influenced by the loss of caveolin-1 from atrial cardiomyocytes and from specialized cells of the conducting system, which constitutively coexpress caveolin-1 and caveolin-3 in mouse and rat heart (unpublished data; Fig 4
).
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Figure 4. Paraffin sections of rat heart; immunostaining for caveolin-1. Note the immunostaining of blood vessels endothelia in the ventricle myocardium (top, A) and the additional immunoreactivity of specialized cardiomyocytes of the conducting system (top, A, arrows) and of the atrial cardiomyocytes (bottom, B, asterisks) [original x 300].
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Conclusion
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Developmental, structural, and functional diversity of endothelial phenotypes is one reason for the difficulty to distinguish them with immunohistochemical or lectinhistochemical methods. Under normal conditions, microvascular and macrovascular endothelial cells differ both between organs and within a given organ. To find endothelial components or products with site-specific function indicative for different pathologic predispositions and to generate appropriate antibodies against these antigens are important goals for the future.
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Acknowledgements
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The author thanks Drs. Michael Keane (Los Angeles, CA), Angelika Bierhaus (Heidelberg, Germany), and Susan Wert (Cincinnati, OH) for providing of the tissue samples (mouse model of bleomycin-induced fibrosis, glycation endproduct receptor -/- mouse, and surfactant protein-C -/-, respectively). Dr. Marlylin G. Farquhar (La Jolla, CA) is acknowledged for the kind gift of the rabbit antibody against podocalyxin and Mrs. S. Bramke for skillful technical assistance.
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Footnotes
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Abbreviation: ICAM = intracellular adhesion molecule
Dr. Kasper is supported by the Deutsche Forschungsgemeinschaft (Transregio/SFB 13; 2004).
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Abstract
Structural Basis of Pulmonary...
