The present results show that PA-SMCs from patients with PPH grow faster than PA-SMCs from controls when stimulated by serotonin or serum and that these effects are due to increased expression of the serotonin transporter, which mediates internalization of indoleamine. Because the L allelic variant of the 5-HTT gene promoter, associated with 5-HTT overexpression, was more common in patients with PPH than in controls, the results suggest that 5-HTT gene polymorphism may confer susceptibility to PPH.
A primary objective of the present study was to assess the role of 5-HTT in mediating the mitogenic and comitogenic effects of 5-HT in human PA-SMCs from patients with PPH versus controls. We found that 5-HT was a potent inducer of human PA-SMC proliferation and that its effect was dose-dependently inhibited by the highly selective 5-HT transport inhibitors fluoxetine and citalopram but not by the 5-HT receptor antagonists ketanserin and GR127935. The comitogenic activity of 5-HT measured in the presence of PDGF was also inhibited by fluoxetine, but this drug had no effect on cell proliferation due to PDGF alone. The growth-stimulating effect of serum was also partly inhibited by 5-HTT blockade with fluoxetine, a finding consistent with the presence in serum of micromolar concentrations of serotonin.
The major finding of this study is that PA-SMCs from patients with PPH proliferated at a faster rate than PA-SMCs from controls when stimulated by serum or serotonin. This increased growth responsiveness of PA-SMCs from patients with PH was selective to serotonin: it did not occur with growth factors such as EGF, TGF-β, FGF, or PDGF. 5-HTT expression was increased in patients with PPH as compared with controls, suggesting that 5-HTT may mediate the greater growth-stimulating effect of serotonin. 5-HTT mRNA and 5-HTT protein levels, as well as 5-HTT uptake activity, were increased two- to threefold in PA-SMCs from patients with PPH as compared with those from controls. Furthermore, the data obtained with the selective 5-HTT inhibitors fluoxetine and citalopram demonstrate clearly that the increased 5-HTT activity in PA-SMCs from patients with PPH was responsible for the increased mitogenic response to serotonin, and consequently to serum.
We found marked increases in 5-HTT mRNA and 5-HTT immunoreactivity in lung specimens from patients with PPH. Immunoreactive 5-HTT predominated in the media of thickened pulmonary arteries and in onion-bulb lesions. In contrast, only a low level of immunostaining was found in endothelial cells and plexiform lesions. Since plexiform lesions are thought to result from proliferation of endothelial cells (19
), this suggests that 5-HTT expression in the lung may occur mainly in PA-SMCs. The observation that 5-HTT immunoreactivity was prominent at sites of vascular remodeling suggests a close relation between PA-SMC proliferation and 5-HTT expression. Importantly, the increased 5-HTT protein expression shown by immunohistochemistry in the SMC layer of pulmonary arteries from patients with PPH was also found in PA-SMCs cultured after repeated cell passages and explant transfers. It has been suggested that the arterial media may contain functionally distinct PA-SMC populations with different migratory and proliferative capabilities, notably in response to PDGF and FGF, as well as different cell levels of contractile and cytoskeletal proteins (20
). In our study, cells were isolated in the absence of a 5-HTT inhibitor, which may have promoted preferential growth of SMCs with increased 5-HTT activity. However, PA-SMCs from both patients and controls exhibited similar growth responses to PDGF, EGF, FGFa, and TGF-β and similar cell levels of α-SM-actin, desmin, and vinculin. These observations are strong evidence against the presence of distinct PA-SMC populations or PA-SMC phenotypes in patients versus controls. Interestingly, the increase in 5-HTT protein expression in patients with PPH was not restricted to the lung but was also found in platelets. An increase in platelet 5-HTT protein indicates increased 5-HTT synthesis by platelet precursor cells and consequently any indirect effects of altered pulmonary hemodynamics on 5-HTT synthesis are unlikely. The observation that cultured PA-SMCs from patients with PPH retain their ability to overexpress 5-HTT in the absence of additional stimuli, together with the increased 5-HTT activity in the platelets of patients with PPH, strongly support a genetic basis for 5-HTT overexpression in PPH.
Compelling evidence has been obtained that 5-HTT expression is genetically controlled and that a polymorphism in the promoter region of the human 5-HTT gene affects transcriptional activity (12
). Studies of human lymphoblasts showed that the long (L) promoter variant was associated with increased 5-HTT expression as compared with the short (S) variant and resulted in increased 5-HTT uptake (8
). In our studies of PA-SMCs from controls, we found that cells with the LL genotype took up more 5-HT than cells with the SS or LS genotype. Accordingly, the growth-stimulating effects of 5-HT or serum were more marked in LL cells than in LS or SS cells, indicating that the capability of PA-SMCs to proliferate in response to serotonin or serum was directly linked to the functional polymorphism of the 5-HTT gene promoter. These results differ to some extent from those reported by Lesch et al. (12
), who found that the S variant appeared to exert a dominant influence, since homozygous SS cells and heterozygous LS cells showed identical 5-HTT expression and 5-HT uptake. In contrast, in our studies conducted in PA-SMCs, 5-HTT activity was higher in LS than in SS cells, suggesting a more subtle effect of 5-HTT gene polymorphism on 5-HTT activity than that inferred from studies of lymphoblasts. We were unable to look for differences in SMC proliferation across 5-HTT gene polymorphisms because of the marked predominance of LL subjects in this group of patients with PPH. Evaluation of the proliferative response of SS cells would be of interest because these cells may show either no response or intermediate response enhancement as compared with cells from controls.
Interestingly, we found that the L variant of the 5-HTT gene polymorphism associated with 5-HTT overexpression was considerably more common in patients with PPH than in controls, implying that this allele may confer susceptibility to PPH. It is unlikely that population heterogeneity could explain our results: in each population, the cases and controls were carefully selected to minimize ethnic heterogeneity; all our study subjects were white. Moreover, the distribution in the control group was similar to that recently reported by Gustavsson et al. in a general population of white individuals (22
). Therefore, 5-HTT gene polymorphism appears to be much more strongly associated with PPH than with mood disorders. 5-HTT is the main target of many antidepressants, and considerable attention has been directed to the possible role for 5-HTT in the pathophysiology of depressive disorders (17
). Linkage studies of 5-HTT gene polymorphisms have shown relations between the S allelic variant of the 5-HTT gene promoter and anxiety symptoms in normal individuals (12
), suicidal behavior (23
), and severe alcoholism (24
). A preliminary report of a case-control study performed in Europe indicated lower levels of psychoanaleptic drug use in PPH patients than in controls (25
) and suggested that the use of psychoanaleptic drugs lowered the odds ratio for developing PPH. An alternative hypothesis is that patients with PPH, who are less likely to carry the S allelic variant than the population at large, may be less prone to anxiety-related traits than subjects without PPH.
Although the present results demonstrate that the long allele of the 5-HTT gene promoter is associated with increased serotonin uptake in PA-SMCs and is strongly associated with PPH, they do not fully explain the increased 5-HTT expression in patients with PPH: 5-HTT expression in PA-SMCs from patients with PPH was higher than in same-genotype cells from controls. Thus, concomitant factors are probably needed to produce 5-HTT overexpression in PPH. Whether this overexpression results from an alteration in the 5-HTT gene itself or from alterations in other factors involved in regulating 5-HTT gene expression remains to be determined. In recent studies, mutations in the coding sequence of the BMPR2 gene were found in more than 50% of patients with familial PPH and in at least 25% of patients with sporadic PPH (2
). The functional impact of BMPR2 mutations on the pathogenesis of pulmonary vascular remodeling is under investigation. An attractive hypothesis is that BMPR2 protein dysfunction may impair the control of cellular proliferation or gene transcription. If this is the case, increased 5-HTT gene expression in some individuals may result from abnormal signaling through the mutated BMPR2 protein. Alternatively, PPH may result from several genetic predisposing factors, and altered genetic control of BMPR2 and 5-HTT may play independent roles in the pathogenesis of the vascular lesion characteristic of PPH.
Our finding that 5-HTT gene expression is a key determinant of SMC proliferation in patients with PPH supports novel therapeutic approaches. Given that selective 5-HTT inhibitors reduce SMC proliferation, they may protect from the vascular lesions associated with PPH. In keeping with this hypothesis, we recently found that citalopram treatment or 5-HTT gene disruption in mice diminished the pulmonary vascular remodeling induced by chronic hypoxia exposure (9
The evidence from this study that 5-HTT polymorphism may confer susceptibility to PPH suggests a mechanism by which appetite suppressants may increase the risk of PPH in humans. Epidemiological studies have established that fenfluramine, dexfenfluramine, and aminorex, but not other appetite suppressants, increase the risk of PPH (5
). A genetic predisposition has been postulated to explain why PPH develops in only a minority of the individuals who use these drugs. Fenfluramine-like medications may elevate circulating 5-HT levels by releasing platelet 5-HT, which in turn may increase pulmonary artery pressure and PA-SMC growth, thereby producing PPH in susceptible individuals (7
). More recently, it was shown that the above-mentioned appetite suppressants acted as 5-HTT substrates and produced the same effect as 5-HT (27
). Another mechanism by which these drugs may promote pulmonary vascular remodeling is stimulation of 5-HTT expression. We found recently that discontinuation of chronic dexfenfluramine treatment in rats was followed by increased lung 5-HTT expression, which promoted the development of hypoxic PH (28
). Individuals with a high basal serotonin uptake related to presence of the long 5-HTT gene promoter variant may be particularly susceptible to one or more of these potential mechanisms of appetite suppressant–related PPH. Interestingly, the distribution of 5-HTT gene polymorphisms was similar in the PPH patients who had and had not received dexfenfluramine treatment. This observation is consistent with the hypothesis that the long 5-HTT gene promoter variant may confer susceptibility to PPH associated with dexfenfluramine use. Confirmation of this hypothesis, however, would require further studies of subjects who took appetite suppressants but did not develop PPH.
Whether 5-HTT gene overexpression is associated with other forms of secondary PH deserves discussion. Most cases of PH occur in association with diseases such as collagen vascular disease, portal hypertension, HIV infection, congenital systemic-to-pulmonary shunt, respiratory disorders, or chronic thromboembolic lung disease (1
). In most of these conditions in adults, as well as in persistent PH in neonates, a genetic predisposition has been suggested. Moreover, a role for 5-HTT in experimental hypoxic PH is now clearly established, and we have also reported increased 5-HTT activity in platelets from patients with chronic hypoxemic lung disease (29
). Thus, the associations linking 5-HTT overexpression to PH and 5-HTT gene polymorphism to susceptibility to PH probably exist in various types of PH in humans.
In conclusion, the present results support a crucial role of 5-HTT activity in the pathogenesis of pulmonary vascular remodeling and suggest that functional polymorphism of the 5-HTT gene promoter may confer susceptibility to PPH. Agents capable of selectively inhibiting 5-HTT–mediated PA-SMC proliferation deserve to be investigated as potential treatments for PH.