© 2006 American Thoracic Society doi: 10.1164/rccm.2512006
From Functional to Genetic Studies of a Candidate Gene for Pulmonary HypertensionAny Point?INSERM U651, Hôpital H. Mondor AP-HP, Créteil, France Attention has focused recently on the role of the serotonin transporter (5-HTT) expressed by pulmonary artery smooth muscle cells (PASMCs) and involved in PASMC hyperplasia in various forms of pulmonary arterial hypertension (PAH). A key observation is that PASMCs from lungs of patients with idiopathic PAH (IPAH) exhibit an exaggerated in vitro growth response to serotonin and to serum (which contains high levels of serotonin) as a result of overexpression of 5-HTT, which mediates the mitogenic action of serotonin (1–3). That 5-HTT plays a key role in pulmonary vascular remodeling is now supported by many studies (4–6). In particular, it was recently shown that transgenic animals overexpressing 5-HTT in smooth muscle (at a level close to that seen in PAH) spontaneously develop pulmonary vascular remodeling and PAH (7). Conversely, mice with targeted 5-HTT gene disruption are protected against hypoxic PAH (8), and selective 5-HTT inhibitors reverse or prevent experimental PAH (7, 9). Whether identification of this new pathway can help to understand the susceptibility of individual patients to develop PAH or to experience disease progression is generating interest, as it is well established that 5-HTT expression is genetically controlled. A polymorphism in the promoter region of the human 5-HTT gene alters the level of transcription (10). The polymorphism consists of two common alleles, a 44-bp insertion or deletion, designated the L and S alleles, respectively. In PASMCs with the L/L genotype, 5-HTT expression is 1.5-fold higher than that in LS or SS cells under basal conditions, and this difference is even more marked when 5-HTT expression is increased by external stimuli (1, 11). Consequently, PASMCs with the LL genotype grow faster than LS or SS cells in response to serotonin or serum (1, 2). In previous studies of 89 patients with IPAH (1) and in a subset of 25 lung-transplant recipients (2), homozygosity for the (L) allele was more prevalent in patients than in control subjects. In recent studies, 5-HTT gene polymorphism was shown to strongly influence the severity of PAH in hypoxemic patients with chronic obstructive pulmonary disease (COPD) (11). Thus, it was suggested that the 5-HTT gene polymorphism may either convey susceptibility to PAH or act as an important modifier of the PAH phenotype. In this issue of the Journal (pp. 793–797 and 798–802), Willers and colleagues (12) and Machado and colleagues (13) reexamined this hypothesis by investigating larger populations of patients with IPAH, associated forms of PAH (APAH), or familial PAH (FPAH). Special emphasis was on patients with FPAH, since 80% of patients with a mutation in the bone morphogenic receptor II (BMPRII) gene do not develop the disease. Thus, one of the tested hypotheses was whether the LL genotype promotes IPAH in the general population or promotes FPAH in individuals who carry a BMPRII mutation. Both studies were negative in most respects: they showed no significant differences in the distribution of the LL, LS, or SS genotypes in patients with IPAH, APAH, FPAH, or in unaffected BMPRII mutation carriers. Several aspects deserve discussion in the light of these reports. There is an apparent discrepancy between the present studies and previous work in patients with IPAH. As suggested, the differences may be due to the smaller number of patients in earlier studies, which found the LL genotype in 55 to 65% of patients versus 27% of control subjects. Another possible explanation may involve the specificity of previously studied cohorts, which included a relatively high percentage (28%) of lung-transplant recipients. Because patients referred to lung transplantation are selected on the basis of more rapid disease progression or poor responsiveness to conventional treatment, this may have introduced a bias in the cohort. Support for this possibility comes from our unpublished observation that among 50 patients with PAH who underwent lung transplantation between 1998 and 2005 at the Marie Lannelongue Hospital (Le Plessis Robinson, France), 57% had the LL genotype. Moreover, we previously reported a high frequency of the LL genotype among lung-transplant recipients with APAH, although the frequency of this genotype was not increased in the overall population of patients with APAH (2). Thus, one possible explanation for the differences between these studies may be related to differences in the disease progression profiles of the study populations. Comparing the populations is difficult because, in the study by Willers and colleagues, none of the patients with IPAH underwent lung transplantation. In the article by Machado and coworkers, this information is not included, nor is there any information on survival. Although survival did not differ between LL, LS, and SS patients in the study by Willers and colleagues, this was examined only in a subgroup of 78 patients. Taken together, these studies indicate that the LL genotype may not deserve to be considered a susceptibility factor for the development of IPAH. The possibility that it may affect disease progression remains to be evaluated in a larger population in which transplantation and death are considered censored events. The two studies published in this issue of the Journal appear to have high power for evaluating FPAH, given the large number of included patients. No preponderance of the LL genotype was found in FPAH, which is consistent with observations made in our laboratory. The correlation between the LL genotype and the early age at diagnosis found in the study of Willers and coworkers may be related to chance alone or may reflect a strong interaction between the 5-HTT and the BMPRII pathways. Preliminary results suggest that the BMPRII agonists BMP4 and BMP6 may inhibit 5-HTT expression (14). Mice overexpressing a dominant negative form of BMPRII (15) also express high levels of 5-HTT (personal communication from D. Rodman). An attractive hypothesis is that loss of BMPRII and increased serotonin transport may interact directly to synergistically produce PAH. Studies exploring the molecular pathways that connect the BMPRII mutant genotype and the 5-HTT pathway are therefore warranted. 5-HTT expression, which is markedly increased in PAH, is governed by many factors, including hypoxia, inflammatory cytokines, drugs such as appetite suppressants, BMPRII activation, and other stimuli. None of these factors can explain the three- to fourfold increase in 5-HTT expression found in IPAH or APAH, either in lung tissue or in cultured cells separated from their abnormal environment. In contrast, in hypoxemic patients with COPD, the strong correlation between 5-HTT gene polymorphism and the level of pulmonary artery pressure is associated with a greater stimulatory effect of hypoxia and cytokines on 5-HTT expression in LL cells than in LS or SS cells. In IPAH, APAH, and FPAH, the influence of the 5-HTT gene polymorphism may be far smaller, and the mechanisms responsible for 5-HTT overexpression remain to be elucidated. FOOTNOTES Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. REFERENCES
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