© 2004 American Thoracic Society doi: 10.1164/rccm.2406008
Fishing in the BloodstreamInsights into the Mechanisms of Pulmonary Hypertension?Department of Medicine and the Lung Biology Center University of California, San Francisco San Francisco, California The ability to simultaneously measure expression of essentially all genes in a cell or organism has the potential to accelerate greatly the pace of discovery in biology and medicine. However, application of this technology to the study of the pathogenesis of human disease is still in its infancy. One major challenge for effective use of expression microarrays to study disease is the difficulty in obtaining the most informative cells and/or tissues in sufficient quantity and purity. For a small number of diseases, such as hematologic malignancies, this is not a serious problem, because large numbers of affected cells are readily accessible by phlebotomy and can be easily purified. Similarly, the current treatment of most solid tumors, surgical resection, makes it relatively straightforward to obtain large numbers of tumor cells. Although solid tumors also contain large numbers of nonmalignant cells, the expression profiles of these cells can also be highly informative. It should therefore not be surprising that the most impressive examples of application of microarray technology to the study of human disease have been applications to hematologic malignancies (1–4) and solid tumors (5–8). For nonmalignant diseases of complex organs like the lung, microarrays are proving to be useful for identifying unexpected molecular mechanisms in experimental models. Tractable systems are most obvious for murine models, where homogeneity of genetic background and the ease of genetic manipulation have allowed investigators to target their studies by manipulating genes involved in already well characterized molecular pathways. Examples of the successful use of such approaches include the identification of a role for arginine metabolism in experimental asthma (9) and identification of roles for an epithelial integrin and transforming growth factor ß in modulation of pulmonary emphysema (10). One pleasant surprise from these sorts of experiments has been the realization that the variability between animals in lung gene expression is quite small, probably as a result of genetic homogeneity in inbred strains and the powerful normalizing effects of in vivo homeostasis. One serious problem for interpretation of changes in whole organ (i.e., whole lung) gene expression is the often dramatic change in cellular composition in response to experimental intervention or disease. This issue of cellular heterogeneity is especially problematic for studies of lung tissue obtained from human patients, where cellular composition can vary dramatically in different regions of a single patient's lungs and at different temporal stages of development of any lung disease. In mice, one way to get around this problem is by engineering animals that can only respond to a given stimulus in a single cell type (11). However, such approaches are obviously of little help for clinical studies in patients. The issues of cellular heterogeneity and accessibility of informative cell types remain daunting challenges for scientists interested in applying microarray technology to the study of lung disease in humans. For example, large amounts of lung tissue are rarely obtained from living patients with airway disease. Although open lung biopsy can provide large amounts of RNA from some patients with interstitial lung diseases, cellular heterogeneity remains a substantial problem. Several groups are working on methods to use microdissection to obtain sufficient amounts of high quality RNA from specific lung cells for microarray analysis. However, these approaches remain challenging and the optimal methods have not yet been determined. For investigators studying pulmonary vascular diseases, such as pulmonary arterial hypertension, access to affected tissue is especially problematic. Biopsies are rarely performed in patients with pulmonary hypertension, because the diagnosis can often be convincingly made without obtaining lung tissue, and lung biopsy has a high morbidity and mortality in these patients. Bull and colleagues (pp. 911–919) addressed this problem by performing expression microarrays on mononuclear cells obtained from the peripheral blood of 15 patients with pulmonary hypertension and 6 control subjects (12). Seven of the patients had idiopathic pulmonary hypertension and the remaining eight had pulmonary hypertension secondary to another defined illness. The authors reasoned that patterns of gene expression shared by this diverse group of patients whose common characteristic was markedly elevated pulmonary artery pressures might shed light on core pathways that contribute to the development or maintenance of this condition. The results convincingly demonstrated that there is a distinct subset of genes that are specifically induced or inhibited in the blood cells of patients with pulmonary hypertension. Importantly, this subset of genes was chosen based on results in one group of patients and then in a separate population was able to distinguish normal subjects from patients with pulmonary hypertension. Furthermore, using a "leave one out" strategy, the authors were able to correctly identify each of the 21 subjects based on the array results generated from the other 20. The results confirmed the authors' hypothesis that blood cells do indeed carry a gene expression signature that identifies the presence of pulmonary hypertension. The number of genes whose expression differed in patients with pulmonary hypertension of any cause and normal control subjects was substantially larger than the number whose expression differed between patients with primary and secondary hypertension, so that it was not possible to identify a robust gene expression signature for primary pulmonary hypertension. As the authors point out, this result could be due to the relatively small sample size. Nonetheless, it appears that the major gene expression changes reflected in circulating mononuclear cells are likely those related to the shared structural alterations in the pulmonary vasculature. Because pulmonary hypertension itself can be diagnosed by other methods, it is not likely that pulmonary physicians will be performing expression profiling as part of the diagnostic work-up of patients with suspected pulmonary hypertension anytime soon. As with any microarray experiment, the major utility of the results described by Bull and colleagues is that they provide a platform for generating hypotheses about biological meaning. One possible meaning that the authors favor is that at least some of the genes that are differentially expressed will provide insights into the pathways that led to the development of pulmonary pathology (for example, the endothelial cell growth factor, ECGF-1). The possibility that any of the 106 differentially expressed genes contributes to pathology could be evaluated in more tractable experimental systems (e.g., by gene knockout or transgenic expression in mice). It is also possible that some of the differentially expressed genes (for example, the potent vasodilator, adrenomedullin) will provide insight into homeostatic mechanisms induced to compensate for that pathology. However, an equally likely possibility is that many, or even potentially all, of the differentially expressed genes reflect the responses of circulating blood cells to their passage through a mechanically and biochemically deranged pulmonary vasculature. This issue might be a bit more challenging to address, but could be approached by evaluating expression profiles over time in blood cells from animals in which pulmonary hypertension is induced in a variety of disparate models. Microarray experiments cannot distinguish among these possibilities but, as demonstrated by the elegant work of Bull and colleagues, they can provide a series of testable hypotheses that should keep experimentalists busy for some time. FOOTNOTES Conflict of Interest Statement: D.S. does not have a relationship with a commercial entity that has an interest in the subject of this manuscript. REFERENCES
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