The present results demonstrate that the 5-HTT is a major determinant of pulmonary vascular remodeling during exposure to chronic hypoxia. Thus, in mice lacking the 5-HTT gene and exposed to hypoxia for two or five weeks, the number and wall thickness of muscular pulmonary vessels were decreased as compared with wild-type controls. Concomitantly, RVSP was lower and the RV was less hypertrophied in hypoxic 5-HTT–/– mutants than in hypoxic wild-type mice. In contrast, the increase in RVSP elicited by an acute hypoxic challenge was larger in 5-HTT–/– than in 5-HTT+/+ normoxic mice. Therefore, the attenuation of PH development and vascular remodeling in the 5-HTT–deficient mice cannot be explained by decreased pulmonary vasoreactivity to hypoxia. These data strongly support a key role for 5-HTT in vascular SMC proliferation and in the consequent development of PH in response to hypoxia.
Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling. One mechanism that may account for this effect is a direct action of hypoxia on the expression of specific genes involved in SMC proliferation. A recent study has shown that mice partially deficient in hypoxia-inducible factor-1 (HIF-1), an essential mediator of transcriptional responses to decreased dioxygen availability, have decreased development of hypoxic PH with reduced muscularization of distal pulmonary vessels (2
). This is evidence that specific genes expressed under the control of HIF-1 during exposure to hypoxia are involved in pulmonary vascular SMC proliferation. Among vasoactive molecules or growth factors that have been implicated in PH, inducible nitric oxide synthase (13
), heme oxygenase 1 (14
), VEGF (15
), VEGF receptor 1 (16
), and endothelin 1 (ET-1) (17
) are known to be expressed by hypoxia-inducible genes that contain functionally important HIF-1 binding sites. However, these molecules exhibit little or no growth-promoting effects on pulmonary SMCs, and none of them demonstrate selective effects on the pulmonary circulation.
We reported recently that the mitogenic action of 5-HT on rat-cultured pulmonary vascular SMCs was enhanced by hypoxia (6
). Other studies found that the mitogenic action of 5-HT was dependent upon 5-HTT activity in pulmonary artery SMCs from several species but not in systemic vessel SMCs (5
). Hypoxia induces 5-HTT expression in cultured SMCs through a transcriptional mechanism and simultaneously increases the mitogenic activity of 5-HT (6
). Exposure to hypoxia also increases 5-HTT expression in the rat lung, notably in the media of remodeled pulmonary vessels. The presence of two hypoxia-sensitive elements in the promoter region of the 5-HTT gene strongly suggests that 5-HTT may be an effector molecule for pulmonary vascular remodeling in response to hypoxia (6
). However, 5-HTT activity also depends upon the levels of 5-HT. In previous studies, we found that continuous intravenous infusion of 5-HT worsened PH in rats exposed to chronic hypoxia (19
). This effect was prevented by administration of a 5-HT transport inhibitor despite further increases in plasma 5-HT levels (20
). In aggregate, these observations suggest that 5-HTT activity may be involved in the response to exogenous 5-HT. However, they do not provide information on the role of 5-HTT activity in hypoxia-induced PH in the absence of pharmacological modulation of 5-HT.
We used 5-HTT
knockout mice, which have been well characterized in terms of locomotor activity and behavior (8
). Because 5-HTT is encoded by a single gene expressed in several cell types such as neurons, platelets, and pulmonary vascular endothelial cells and SMCs, 5-HTT deficiency can be expected to produce marked alterations not only in central serotoninergic neurotransmission but also in the metabolism and actions of indoleamine at the periphery. Under normal conditions, 5-HT is produced mainly by enterochromaffin cells in the gut, where it plays a role in mechano- and chemotransduction. A large part of the 5-HT released from enterochromaffin cells reaches the portal circulation, where it is avidly taken up by platelets and partly metabolized by the liver. The remaining free 5-HT in blood is cleared by the lung, a process that involves indoleamine uptake by pulmonary vascular endothelial and SMCs. In 5-HTT–/–
mice, high-affinity [3
H]5-HT uptake was absent in platelets in our study and in the brain in an earlier study (8
), confirming the complete absence of 5-HTT gene activity in these mutants. 5-HT blood concentrations were also dramatically reduced in the mutants as compared with wild-type mice, an expected finding since platelet 5-HT is known to contribute at least 90% of whole blood 5-HT. In contrast, blood 5-HIAA levels did not differ significantly between 5-HTT–/–
mutants and wild-type mice, suggesting an absence of marked adaptive changes in 5-HT production and/or degradation in the knockout animals.
The main finding from our study is that mice deficient in 5-HTT developed less PH than their littermate controls when exposed to hypoxia of various durations. Not only was pulmonary artery pressure lower, but pulmonary vessel muscularization and thickening were also less marked in 5-HTT–/–
mice than in wild-type controls, suggesting that impaired pulmonary vascular SMC proliferation owing directly to the deficiency in 5-HTT–mediated 5-HT mitogenic activity was responsible for the attenuation of PH. In theory, protection from pulmonary vascular remodeling and PH could have resulted from other mechanisms. One is decreased pulmonary vasoreactivity to hypoxia. However, we found that the pulmonary pressor response to acute hypoxia as evaluated based on the RVSP increase was enhanced rather than blunted in 5-HTT–/–
mice. The explanation of this finding remains speculative. One likely hypothesis is that the platelet uptake deficiency left more indoleamine available for binding to 5-HT receptors on pulmonary SMCs in 5-HTT–/–
mice. In an earlier study, we found that 5-HT infusion in rats potentiated the in vivo acute pulmonary pressure response to hypoxia (19
). Moreover, treatment with dexfenfluramine, which inhibits platelet 5-HT uptake and, additionally promotes 5-HT release from platelets, also potentiates in vivo acute hypoxic pulmonary vasoconstriction (20
). It is therefore reasonable to assume that the deficiency in platelet 5-HT uptake in the 5-HTT–/–
mice increases hypoxic pulmonary vasoreactivity through the same mechanism. Another possibility is that impaired 5-HT release by platelets or secondary platelet dysfunction may account for the attenuated pulmonary vascular remodeling in 5-HTT–/–
mice. In this regard, a comparison of our results in 5-HTT–/–
mice with those previously reported in the fawn-hooded rat is of interest. Fawn-hooded rats have a genetic deficiency in 5-HT platelet storage and a bleeding tendency owing to a defect in platelet aggregation (21
), but have a normal 5-HTT amino acid sequence (22
). Although fawn-hooded rats share with 5-HTT–/–
mice an increase in pulmonary vasoreactivity to acute hypoxia, responses to chronic hypoxia are diametrically opposed: hypoxia-induced PH is facilitated in fawn-hooded rats, whereas it is attenuated in 5-HTT–/–
mice. This militates against the possibility that attenuated remodeling in 5-HTT–/–
mice was related to platelet dysfunction or to impaired 5-HT release. The increased vascular remodeling in fawn-hooded rats is believed to involve increased lung vessel exposure to 5-HT as a result of the inability of platelets to store 5-HT. Together with the fact that 5-HT infusion worsens hypoxic PH in rats, these findings support a key role for 5-HT in hypoxic PH, provided that 5-HTT expression and/or activity is present in pulmonary vascular SMCs. The observation of attenuated remodeling in our 5-HTT–deficient mice emphasizes the importance of 5-HTT expression by pulmonary SMCs for the development of hypoxic PH.
At present, the mechanisms by which 5-HT may exert its mitogenic effect after being transported inside SMCs remain speculative. Lee et al. observed that 5-HT–induced DNA synthesis was associated with tyrosine phosphorylation of GTPase-activating proteins and that both events were blocked by inhibitors of 5-HT transport or tyrosine kinase (23
). Therefore, although 5-HT–induced mitogenesis of SMCs requires cellular internalization through 5-HTT rather than binding to a membrane receptor, tyrosine phosphorylation of GTPase-activating proteins appears to be a downstream intermediate in the signaling pathway. Recently, involvement of superoxide anion formation in association with 5-HT transport has also been suggested as a possible contributor to the mitogenic effects of 5-HT (24
). Since reactive oxygen species are also considered to be potential mediators of vascular remodeling, we cannot exclude that protection against PH in 5-HTT–deficient mice was indirectly related to decreased formation of superoxide anions.
Interestingly, heart weight was lower in 5-HTT–/–
mutants than in wild-type normoxic mice, although systemic arterial pressure did not differ between the two groups. Clinical studies have established that 5-HT promotes cardiac disease by causing fibroplasia, particularly in the valvular endocardium in the right side of the heart (25
). In addition, patients treated with the 5-HT releaser fenfluramine in combination with phentermine have been reported to develop right and left valvular heart disease (26
). In line with these observations, cardiac alterations may develop in 5-HTT–/–
mice as a consequence of either an excessive amount of 5-HT reaching the heart or a deficiency in 5-HTT. Further studies are needed to explore the nature and the mechanisms of cardiac alterations in 5-HTT–deficient mice.
The importance of 5-HT in pulmonary vascular remodeling is generating renewed interest, in particular because several appetite suppressants that act by inhibiting 5-HT transport have been reported to increase the risk of PH in humans (7
). However, further investigations are needed to determine whether a link exists between the mechanism of appetite suppressant–induced human PH and the present results demonstrating the importance of 5-HTT expression by pulmonary SMCs for the development of hypoxic PH in rodents. Recently, we found marked increases in 5-HTT binding and 5-HTT activity in platelets from patients with primary and secondary forms of PH (27
). Therefore, one reasonable hypothesis is that human PH may be associated with 5-HTT overexpression in platelets and/or pulmonary artery SMCs and that appetite suppressants may act on this process. Further studies exploring 5-HTT expression in the pulmonary circulation of patients with primary or secondary PH are needed to extend the present findings to nonhypoxic forms of human PH.