Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis
Respiratory Research volume 3, Article number: 9 (2001)
Epidemiological studies have established that fenfluramine, D-fenfluramine, and aminorex, but not other appetite suppressants, increase the risk of primary pulmonary hypertension (PH). One current hypothesis suggests that fenfluramine-like medications may act through interactions with the serotonin (5-hydroxytryptamine [5-HT]) transporter (5-HTT) located on pulmonary artery smooth muscle cells and responsible for the mitogenic action of 5-HT. Anorexigens may contribute to PH by boosting 5-HT levels in the bloodstream, directly stimulating smooth muscle cell growth, or altering 5-HTT expression. We suggest that individuals with a high basal level of 5-HTT expression related to the 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 PH.
Appetite suppressant use is now considered an important risk factor for the development of primary PH. An association between the anorexigen aminorex and PH was first reported in the 1960s, and aminorex was withdrawn from the market in 1972 . In the 1980s, fenfluramine use was linked to primary PH and, subsequently, a 30-fold increase in the risk of PH was found in patients who had received these appetite suppressants for longer than 3 months compared with the general population [2,3].
Understanding the molecular mechanism of appetite-suppressant-induced PH has become a major goal for current and future studies. Because amphetamine-like drugs have potential applications in the treatment of obesity, drug dependence, and other psychiatric disorders, a legitimate concern is that new members of this class introduced in the future might cause outbreaks of PH similar to those seen with aminorex and fenfluramine. To ward off this danger, we must identify the drug-related effects likely to generate PH and/or the patient characteristics associated with susceptibility to these effects.
Better knowledge of the pathobiology of primary PH can be expected to flow from elucidation of the mechanisms underlying appetite-suppressant-induced PH. These drugs promote the development of vascular lesions confined to the small muscular arteries and arterioles in the lung, suggesting that one of their molecular targets may be selectively present in pulmonary vessels or may have specific functions in pulmonary vessels compared with systemic vessels. Aminorex, fenfluramine, D-fenfluramine, and phentermine belong to a vast class of amphetamine-like drugs that interact with monoamine systems in the brain. Among appetite suppressants, however, only fenfluramine, D-fenfluramine, and aminorex increase the risk of primary PH. The fact that these three drugs not only inhibit neuronal serotonin (5-HT) reuptake, but also trigger indoleamine release has sparked renewed interest for the 'serotonin hypothesis' of PH.
In our laboratory, we recently tested the hypothesis that the 5-HT transporter (5-HTT) in the lung might be a key determinant of pulmonary vessel remodeling because of its action on pulmonary artery smooth muscle cell (SMC) growth . The 5-HTT transporter is abundantly expressed in the lung, where it is predominantly located on SMCs . The recent observation that aminorex and fenfluramine derivatives interact with 5-HTT in a specific manner has provided further support to the hypothesis that this transporter may be a critical target for appetite suppressants and perhaps for other insults initiating the process of PH .
5-HTT as a key determinant of pulmonary vascular remodeling
A pathological feature shared by secondary and primary PH is increased thickness of the distal pulmonary artery walls, related chiefly to SMC hyperplasia . The 5-HTT in pulmonary vascular SMCs has many attributes suggesting that it may be a key determinant of this process. In addition to contributing to the uptake and subsequent inactivation of 5-HT passing through the lung, 5-HTT mediates the proliferation of pulmonary vascular SMCs through its ability to internalize indoleamine [4,8,9]. The level of 5-HTT expression appears to be much greater in human lung than in human brain, suggesting that altered 5-HTT expression may have direct consequences on pulmonary artery-SMC function . Direct evidence that 5-HTT plays a key role in pulmonary vascular remodeling was recently obtained by showing that mice with targeted 5-HTT gene disruption develop less severe hypoxic PH than wild-type controls , and that selective 5-HTT inhibitors attenuate hypoxic PH. Conversely, increased 5-HTT expression is associated with increased severity of hypoxic PH . Although a heterogeneous population of 5-HT2A and 5-HT1B receptors coexist in pulmonary arteries, 5-HT receptor antagonists do not seem to efficiently protect against development of hypoxic PH (unpublished data). Taken together, these observations suggest a close correlation between 5-HTT expression and/or activity and the extent of pulmonary vascular remodeling during exposure to hypoxia.
5-HTT expression and activity are also increased in platelets and lungs from patients with primary and secondary forms of PH  and in pulmonary artery SMCs from patients with primary PH, as compared with SMCs from controls. Furthermore, compared with SMCs from controls, SMCs from PH patients are more susceptible to the growth promoting effects of 5-HT and serum (which contains high levels of 5-HT) , whereas there is no difference for other growth factors such as platelet-derived growth factor, transforming growth factor-β, fibroblast growth factor-a, and epidermal growth factor. In the presence of 5-HTT inhibitors, the growth stimulating effects of serum and 5-HT are markedly reduced, and the difference between growth of SMCs from patients and controls is abolished. It follows that 5-HTT overexpression and/or activity in pulmonary artery SMCs from patients with PH is responsible for the increased mitogenic responses to 5-HT and serum .
Mechanisms by which appetite suppressants may promote primary PH: is 5-HTT a critical target for drugs linked to primary PH?
5-HT turnover alteration
The anorexigens aminorex, fenfluramine, and dexfenfluramine are potent 5-HT uptake inhibitors. This inhibition occurs not only in neurons of the serotoninergic category, but also in platelets, pulmonary endothelial cells and SMCs, which share the same 5-HTT encoded by a single gene . These facts initially led to the hypothesis that fenfluramine-like medications may elevate circulating 5-HT levels [15,16], which in turn may increase pulmonary artery pressure and pulmonary artery SMC growth, thereby producing primary PH in susceptible individuals. This 'serotonin hypothesis' was consistent with reports of increased plasma 5-HT levels under several conditions leading to PH [17,18]. Recent findings, however, run counter to the serotonin hypothesis: chronic treatment with phentermine and fenfluramine in combination decreases plasma 5-HT levels in humans . Another argument against the serotonin hypothesis is that 5-HTT is also the target of widely used antidepressants, such as selective 5-HT reuptake inhibitors and conventional tricyclic antidepressants, which do not promote the development of primary PH. Conversely, selective 5-HTT inhibitors attenuate the development of experimental PH. Moreover, preliminary data from a case-control study performed in Europe has suggested that the psychoanaleptic drugs may lower the odds ratio for primary PH .
The 5-HT concentrations in blood and plasma may not, however, be indicative of 5-HT concentrations in local microenvironments surrounding pulmonary endothelial cells or SMCs. In addition to their 5-HTT inhibiting properties, aminorex and fenfluramine-like drugs are potent triggers of indoleamine release and, consequently, increase the amount of extracellular 5-HT . Selective 5-HTT inhibitors also increased 5-HT levels in extracellular fluid, but to a much smaller degree than fenfluramine-like drugs . This leaves open the possibility that these drugs may promote 5-HT turnover alterations, thereby increasing the availability of free 5-HT near the pulmonary artery wall. Since 5-HT is also released from pulmonary neuroendocrine cells and neuroepithelial bodies distributed throughout the airways, and possibly from pulmonary artery SMCs , this makes 5-HT more likely to have a role on pulmonary compared with systemic vessels.
Interaction with 5-HTT
Recent studies investigated the possibility that fenflu-ramine and other anorexigens might increase the risk of primary PH by interacting directly with 5-HTT. Interestingly, drugs known or suspected to increase the risk of primary PH (namely, aminorex, fenfluramine, and chlorphentermine) were found to be 5-HTT substrates, whereas drugs not associated with an increased risk of primary PH were less potent in this regard . It has been speculated that medications that are 5-HTT substrates may be translocated into pulmonary cells where, depending on the degree of drug retention, intrinsic drug toxicity, and individual patient susceptibility, they may cause effects similar to or greater than those of 5-HT . According to this hypothesis, 5-HTT substrates other than 5-HT may also be mitogenic. Support for this hypothesis has come from a recent study showing that fenfluramine is mitogenic for rat lung SMCs and for lung fibroblasts .
Potassium-channel inhibition has also been suggested as a potential mechanism of anorexigen-induced cell toxicity . Although this effect requires drug concentrations 10 times greater than those expected with therapeutic drug dosages, it remains plausible that direct vasoconstriction related to potassium-channel blockade or increased intra-cellular Ca2+ levels in SMCs may contribute to the toxic effects of appetite suppressants after internalization and accumulation of these drugs by the SMCs. The fact that 5-HT uptake inhibitors and substrates may bind to different domains on 5-HTT suggest that determining the exact mechanism of action of each drug may help to predict the risk of adverse effects .
Stimulation of 5-HTT expression
Another mechanism by which anorexigens may promote pulmonary vascular remodeling is stimulation of 5-HTT expression. Previous studies have shown that hormones and pharmacological agents  can modulate 5-HTT levels and activity in serotoninergic neurons. Dexfenfluramine given in high doses has been shown to produce long-lasting decreases in both concentration and uptake of 5-HT in forebrain regions, as well as in 5-HTT mRNA levels within the dorsal raphe nucleus . We found that the levels of 5-HTT transcript in lung tissue from rats given chronic dexfenfluramine treatment for 4 weeks remained unchanged compared with those in animals treated with the vehicle alone. However, discontinuation of chronic dexfenfluramine treatment in rats was followed by increased lung 5-HTT expression, which promoted the development of hypoxic PH . 5-HTT overexpression such as that induced by withdrawing dexfenfluramine-like drugs may therefore represent a complementary mechanism promoting 5-HTT-dependent hyperplasia of pulmonary SMCs.
Genetic susceptibility to anorexigen-induced PH
A genetic predisposition has been postulated to explain why primary PH develops in only a minority of appetite-suppressant users. In keeping with this possibility, feeding aminorex or dexfenfluramine to experimental animals fails to elicit PH. Interestingly, it has been established that 5-HTT expression is genetically controlled: a polymorphism in the promoter region of the human 5-HTT gene alters transcriptional activity. This polymorphism consists of two common alleles, a 44 base pair insertion (the L allele) or deletion (the S allele) . The L allele drives a twofold to threefold higher level of 5-HTT gene transcription than the S allele. Preliminary results suggest that the L/L genotype is present in 60–70% of the patients with PH as compared with only 20–30% of a control population of Caucasian subjects . The L/L genotype may therefore confer genetic susceptibility to PH in humans, particularly when it is combined with other factors such as hypoxia, HIV infection, portal hypertension, or other conditions. Individuals with a high basal level of 5-HT uptake related to presence of the long 5-HTT gene promoter variant may be particularly susceptible to one or more of the aforementioned potential mechanisms of appetite-suppressant-related primary PH. Studies examining the potential association between appetite-suppressant-related PH and 5-HTT gene polymorphism are warranted.
In recent studies, mutations in the coding sequence of the BMPR2 gene were shown to occur in more than 50% of patients with familial primary PH and in at least 25% of patients with sporadic primary PH [26,27]. The functional impact of BMPR2 mutations into the pathogenesis of pulmonary vascular remodeling is presently under investigation. An attractive hypothesis is that dysfunction of the BMPR2 protein may result in impaired control of cellular proliferation or gene transcription. Whether abnormal signaling through mutated BMPR2 may contribute to appetite-suppressant-related PH also remains to be determined.
The observation that 5-HTT polymorphism may confer susceptibility to primary PH suggests a mechanism by which appetite suppressants may increase the risk of primary PH in humans. Fenfluramine-like medications may contribute to PH by elevating circulating 5-HT levels, they may act as 5-HTT substrates to produce the same effect as 5-HT or they may alter 5-HTT expression. Individuals with a high basal serotonin uptake that is related to the presence of the long 5-HTT gene promoter variant might be particularly susceptible to one or more of these potential mechanisms of appetite-suppressant-related primary PH.
smooth muscle cell.
Kay JM, Smith P, Heath D: Aminorex and the pulmonary circulation. Thorax. 1971, 26: 262-273.
Abenhaim L, Moride Y, Brenot F, Rich S, Benichou J, Kurz X, Higenbottam T, Oakley C, Wouters E, Aubier M, Simonneau G, Begaud B: Appetite-suppressant drugs and the risk of primary pulmonary hypertension. N Engl J Med. 1996, 335: 609-616. 10.1056/NEJM199608293350901.
Fishman AP: Aminorex to fen/phen: an epidemic foretold. Circulation. 1999, 99: 156-161.
Eddahibi S, Fabre V, Boni C, Martres MP, Raffestin B, Hamon M, Adnot S: Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res. 1999, 84: 329-336.
Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T, Chang AS, Blakely RD: Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning expression, and chormosomal localization. Proc Natl Acad Sci USA. 1993, 90: 2542-2546.
Rothman RB, Ayestas MA, Dersch CM, Baumann MH: Aminorex, fenfluramine, and chlorphentermine are serotonin transporter substrates. Implications for primary pulmonary hypertension. Circulation. 1999, 100: 869-875.
Rubin LJ: Primary pulmonary hypertension. N Engl J Med. 1997, 336: 111-117. 10.1056/NEJM199701093360207.
Lee SL, Wang WW, Moore BJ, Fanburg BL: Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cell in culture. Circ Res. 1991, 68: 1362-1368.
Lee S, Wang W, Fanburg BL: Dexfenfluramine as a mitogen signal via the formation of superoxide anion. FASEB J. 2001, 15: 1324-1325.
Eddahibi S, Hanoun N, Lanfumey L, Lesch K, Raffestin B, Hamon M, Adnot S: Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest. 2000, 105: 1555-1562.
Eddahibi S, Adnot S, Frisdal E, Levame M, Hamon M, Raffestin B: Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats. J Pharmacol Exp Ther. 2001, 297: 148-154.
Eddahibi S, Chouaid C, Sediame S, Housset B, Hamon M, Adnots S: Serotonin uptake and citalopram binding in platelets from patients with chronic pulmonary hypertension [abstract]. Am J Respir Crit Care Med. 1999, 159: A165-
Eddahibi S, Humbert M, Fadel E, Raffestin B, Darmon M, Capron F, Simonneau G, Dartevelle P, Hamon M, Adnot S: Hyperplasia of pulmonary artery smooth muscle cells causally related to overexpression of the serotonin transporter in primary pulmonary hypertension. J Clin Invest. 2001, 108: 1141-1150. 10.1172/JCI200112805.
Lesch K-P, Gross UB, Strauss K, Wolozin BL, Murphy DL, Riederer P: Organization of the human serotonin transporter gene. J Neural Transm. 1994, 95: 157-162.
Celada P, Martin F, Artigas F: Effects of chronic treatment with dexfenfluramine on serotonin in rat blood, brain and lung tissue. Life Sci. 1994, 55: 1237-1243. 10.1016/0024-3205(94)00663-6.
Raleigh MJ, Brammer GL, Ritvo ER, Geller E, McGuire MT, Yuwiler A: Effects of chronic fenfluramine on blood serotonin, cerebrospinal fluid metabolites and behaviour in monkeys. Psychpharmacology. 1986, 90: 503-508.
MacLean M, Herve P, Eddahibi S, Adnot S: 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br J Pharmacol. 2000, 131: 161-168.
Sato K, Webb S, Tucker A, Rabinovitch M, O'Brien RF, McMurtry IF, Stelzner TJ: Factors influencing the idiopathic development of pulmonary hypertension in the Fawn Hooded rat. Am Rev Respir Dis. 1992, 145: 793-797.
Rothman RB, Redmon B, Raatz SK, Kwong CA, Swanson JE, Bantle JP: Chronic treatment with phentermine combined with fenfluramine lowers plasma serotonin. Am J Cardiol. 2000, 85: 913-916. 10.1016/S0002-9149(99)00896-6.
Louis WJ: Primary pulmonary hypertension and anorectic drugs. N Engl J Med. 1999, 340: 482-483. 10.1056/NEJM199902113400614.
Raiteri M, Bonanno G, Vallebuona F: In vitro and in vivo effects of D-fenfluramine: no apparent relation between 5-hydroxytryptamine release and hypophagia. J Pharmacol Exp Ther. 1995, 273: 643-649.
Miyata M, Ito M, Sasajima T, Ohira H, Kasukawa R: Effect of a serotonin receptor antagonist on interleukin-6-induced pulmonary hypertension in rats. Chest. 2001, 119: 554-561. 10.1378/chest.119.2.554.
Blakely RD, Ramamoorthy S, Qian Y, Schroeter S, Bradley CC: Regulation of antidepressant-sensitive serotonin transporters. In Neurotransmitters Transporters: Structure, Function and Regulation. Edited by Reith MEA. NJ: Humana Press Inc;. 1996, 29-72.
Semple-Rowland SL, Mahatme A, Rowland NE: Effects of dexfenfluramine or 5,7-dihydroxytryptamine on tryptophan hydroxylase and serotonin transporter mRNAS in rat dorsal raphe. Mol Brain Res. 1996, 41: 121-127. 10.1016/0169-328X(96)00076-9.
Lesch K-P, Bengel D, Heils A, Sabol SZ, Greeberg BD, Müller CR, Hamer DH, Murphy DL: Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science. 1996, 274: 1527-1531. 10.1126/science.274.5292.1527.
Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, Knowles JA: Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet. 2000, 67: 737-744. 10.1086/303059.
Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA, Loyd JE, Nichols WC, Trembath RC: Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat Genet. 2000, 26: 81-84. 10.1038/79226.
Most of the work performed on 5-HTT in our laboratory has been supported by INSERM and the Fondation de France.
About this article
Cite this article
Eddahibi, S., Adnot, S. Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis. Respir Res 3, 9 (2001). https://doi.org/10.1186/rr181