Hypoxiainduced Serotonin Transporter

The serotonin transporter (5-HTT) in pulmonary vascular SMCs has many attributes suggesting that it may be a key determinant of pulmonary SMC proliferation. It belongs to a family of integral membrane proteins responsible for terminating the action of neurotransmitters released from neurons. In addition to 5-HT reuptake into neurons, 5-HTT is responsible for indoleamine uptake by platelets, endothelial cells, and SMCs. These processes result in very low levels of free 5-HT in plasma under physiological conditions. Interest in the potential role for 5-HTT in PH was sparked by the observation that 5-HTT is a target for drugs that have been shown to increase the risk of PH development in humans.

In addition to contributing to the uptake and subsequent inactivation of serotonin passing through the lung, 5-HTT mediates the proliferation of pulmonary vascular SMCs through its ability to internalize indoleamine The requirement of 5-HTT as a mediator of 5-HT mitogenic activity appears specific for SMCs from the pulmonary vessels, since it has not been demonstrated in SMCs from systemic vessels. We have recently reported that the mitogenic action of serotonin on cultured pulmonary vascular SMCs from rats is enhanced by hypoxia, which induces 5-HTT expression through a transcriptional mechanism and simultaneously increases 5-HTT activity, an effect associated with potentiation of the mitogenic effect of 5-HT (4). 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.

In previous studies, we found that continuous intravenous infusion of serotonin worsened PH in rats exposed to chronic hypoxia (7). This effect was prevented by administration of a 5-HT transport inhibitor, despite a further increase in plasma serotonin levels. Moreover, when a 4-week course of the 5-HTT substrate dexfenfluramine (2g/kg body weight per day) was discontinued, 5-HTT expression increased transiently in lung tissue and, concomitantly, hypoxic PH worsened (3, 6).

Further information on the key role for 5-HTT activity in hypoxia-induced PH has been provided by experiments on mice lacking the 5-HTT gene. After exposure to hypoxia for 2-5 weeks, PH and vascular remodeling are less marked in 5-HTT-deficient mice than in littermate controls (5). This protection from pulmonary vascular remodeling and PH cannot be ascribed to decreased pulmonary vasoreactivity to hypoxia since the pulmonary pressor response to acute hypoxia is enhanced rather than blunted in 5-HTT'1' mice. The mechanism underlying the protective effect remains speculative. One likely hypothesis is that the platelet uptake deficiency leaves more indoleamine available for binding to 5-HT receptors on pulmonary vascular SMCs. We found that 5-HT infusion in rats potentiated the in vivo acute pulmonary pressure response to hypoxia (7). Moreover, treatment with dexfenfluramine, which inhibits platelet 5-HT uptake and promotes 5-HT release by platelets, also potentiated acute hypoxic pulmonary vasoconstriction in vivo. It is therefore reasonable to assume that deficient platelet 5-HT uptake in mice increases hypoxic pulmonary vasoreactivity through the same mechanism. Impaired serotonin release by platelets or secondary platelet dysfunction is unlikely to 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 Fawn-Hooded rats is of interest. Fawn-Hooded rats have a genetic deficiency in 5-HT platelet storage and a bleeding tendency due to a defect in platelet aggregation, yet their 5-HTT amino acid sequence is normal. 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 exacerbated in Fawn-Hooded rats and attenuated in 5-HTT"'" mice. Therefore, attenuation of hypoxic PH is not related to platelet 5-HT depletion when 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 key role for 5-HTT expression by pulmonary SMCs in the development of hypoxic PH.

5-HTT can be competitively inhibited by the antidepressants fluoxetine, paroxetine, and citalopram. Consequently, these drugs inhibit the in vitro SMC proliferative response to 5-HT and, to a lesser extent, the growth response to serum. In vivo, we also recently observed that these drugs impaired hypoxia-induced pulmonary vascular remodeling, despite their potentiating effect on acute hypoxic pulmonary vasoconstriction.

Recent studies have emphasized the contribution of 5-HT(B and 5-HT2B receptors to hypoxia-induced PH. 5-HT1B receptors mediate contraction to 5-HT in human pulmonary arteries. 5-HT1B-mediated contraction is enhanced in pulmonary arteries from hypoxic rats. Rats treated with a selective 5-HT1D/1B receptor antagonist or mice deficient in 5-HT1B receptors (5-HT IB"'") have less severe PH and vascular remodeling than do wild-type controls, suggesting that 5-HT,b receptors may play a role in the development of PH via enhanced 5-HT1B-mediated vasoconstriction (12). This effect, however, is modest, possibly because serotonin-induced pulmonary vasoconstriction does not greatly influence pulmonary vascular remodeling. Moreover, in a recent study, we found that treatment with the 5-HT1B receptor antagonist GR127935 abrogated acute hypoxic vasoconstriction in mice, whereas the 5-HT2A receptor antagonist ketanserin had no effect. When given chronically, neither GR nor ketanserin affected the development of chronic PH. When GR was given in association with citalopram or fluoxetine, the pulmonary vasoconstriction enhanced by the antidepressant was reduced to the level observed in the absence of antidepressant, but no further benefit on chronic PH was obtained.

Using the chronic hypoxic mouse model of PH, Launay et al. (14) recently showed that PH was associated with a substantial increase in 5-HT2B receptor expression in pulmonary arteries and that PH did not develop in hypoxic mice with genetic or pharmacological inactivation of 5-HT2B receptors. However, these results are difficult to compare with our findings on the role for 5-HTT, since the severity of hypoxic PH in the control animals differed markedly between the two studies.

Several conclusions can be drawn from our results showing less hypoxic pulmonary vascular remodeling despite potentiation of the acute pressor response to hypoxia. First, precapillary vasoconstriction, which is considered an important contributor to pulmonary arterial muscularization, may not fully explain the pathophysiology of hypoxic PH. Second, SMC proliferation, which is the main component of pulmonary vascular remodeling, may be independent from the severity of hypoxic pulmonary vasoconstriction and closely linked to the 5-HTT pathway.

At present, the mechanisms by which 5-HT may exert its mitogenic effects after being transported inside the SMCs remain speculative. Lee et al. (15) have reported that 5-HT-induced DNA synthesis is associated with tyrosine phosphorylation of GTPase-activating protein and that both events are blocked by inhibitors of 5-HT transport or tyrosine kinase. Therefore, although 5-HT-induced mitogenesis in SMCs requires cellular internalization through 5-HTT rather than binding to a membrane receptor, subsequent tyrosine phosphorylation of GTPase-activating protein appears to be an 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 (16). 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 is indirectly related to decreased formation of superoxide anions.

We recently reported an increased growth response to 5-HT or serum of pulmonary artery-derived SMCs from patients with primary PH, an effect ascribable to increased 5-HTT expression (3, 6). Proliferation in response to 5-HT is dose dependently inhibited by the highly selective 5-HT transport inhibitors fluoxetine and citalopram but not by the 5-HT receptor antagonists ketanserin and GR 127935. These findings are ascribable in part to a variant in the upstream promoter region of the 5-HTT gene. 5-HTT expression is genetically controlled, and a polymorphism in the promoter region of the human 5-HTT gene affects transcriptional activity. The long (L) promoter variant of the 5-HTT gene is associated with increased 5-HTT expression, as compared with the short (S) variant, and causes an increase in 5-HT uptake. Pulmonary artery SMCs from controls with the LL genotype take up more 5-HT than do SS or LS

cells and, accordingly, the growth-stimulating effects of 5-HT are more marked in LL cells than in LS or SS cells. Thus, the capability of pulmonary artery SMCs to proliferate in response to 5-HT is directly linked to the functional polymorphism of the 5-HTT gene promoter. Interestingly, the L variant of the 5-HTT gene polymorphism associated with 5-HTT overexpression appears more common in patients with primary PH than in controls: the L variant was present in homozygous form in 65% of 89 patients with primary PH but in only 27% of normal controls. Although these results demonstrate that the long allele ofthe 5-HTT gene promoter is associated with increased serotonin uptake in pulmonary artery SMCs, they do not fully explain the increased 5-HTT expression in patients with primary PH, since 5-HTT expression is higher in cells from patients than in same-genotype cells from controls. 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. However, these data are consistent with the hypothesis that 5-HTT polymorphism confers susceptibility to primary PH. Whether 5-HTT gene overexpression is associated with other forms of secondary PH deserves further study. A role for 5-HTT in experimental hypoxic PH is now clearly established, and recent data from our laboratory demonstrating that 5-HTT gene polymorphism may determine the severity of PH in patients with chronic obstructive pulmonary disease extend this concept to humans. Agents capable of selectively inhibiting 5-HTT-mediated pulmonary artery SMC proliferation deserve to be investigated as potential treatments for PH.

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