6532

SLC6A4

5-HTT|5-HTTLPR|5HTT|HTT|OCD1|SERT|SERT1|hSERT;solute carrier family 6 (neurotransmitter transporter, serotonin), member 4;SLC6A4;solute carrier family 6 (neurotransmitter transporter, serotonin), member 4

5-hydroxytryptamine transporter|5HT transporter|Na+/Cl- dependent serotonin transporter|sodium-dependent serotonin transporter

17q11.2

protein-coding

Count: Slc6a4; 15567

hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling and pulmonary hypertension development. One mechanism that may account for these effects is the direct action of hypoxia on the expression of specific genes involved in vascular smooth muscle cell (SMC) proliferation. Previous studies demonstrated that the serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) mediates the mitogenic activity of 5-HT in pulmonary vascular SMCs and is overexpressed during hypoxia. Thus, 5-HT-related mitogenic activity is increased during hypoxia. Here, we report that mice deficient for 5-HTT (5-HTT(-/-)) developed less hypoxic pulmonary hypertension and vascular remodeling than paired 5-HTT(+/+) controls. When maintained under normoxia, 5-HTT(-/-)-mutant mice had normal hemodynamic parameters, low blood 5-HT levels, deficient platelet 5-HT uptake, and unchanged blood levels of 5-hydroxyindoleacetic acid, a metabolite of 5-HT. After exposure to 10% O(2) for 2 or 5 weeks, the number and medial wall thickness of muscular pulmonary vessels were reduced in hypoxic 5-HTT(-/-) mice as compared with wild-type paired controls. Concomitantly, right ventricular systolic pressure was lower and right ventricle hypertrophy less marked in the mutant mice. This occurred despite potentiation of acute hypoxic pulmonary vasoconstriction in the 5-HTT(-/-) mice. These data further support a key role of 5-HTT in hypoxia-induced pulmonary vascular SMC proliferation and pulmonary hypertension.

BACKGROUND: The serotonin transporter (5-HTT) is involved in the pulmonary artery smooth muscle hyperplasia that leads to pulmonary hypertension (PH). Because hypoxia and 5-HTT gene polymorphism control 5-HTT expression, we examined 5-HTT gene polymorphism and PH in hypoxemic patients with advanced chronic obstructive pulmonary disease (COPD). METHODS AND RESULTS: In 103 patients with COPD recruited in France (n=67) and the UK (n=36), we determined 5-HTT gene polymorphism and pulmonary artery pressure (PAP) measured during right heart catheterization (France) or Doppler echocardiography (UK). Ninety-eight subjects from the 2 countries served as control subjects. The distribution of 5-HTT gene polymorphism did not differ between patients and control subjects. In patients carrying the LL genotype, which is associated with higher levels of 5-HTT expression in pulmonary artery smooth muscle cells than the LS and SS genotypes, PH was more severe than in LS or SS patients. Mean PAP values in patients from France with the LL, LS, and SS genotypes were 34+/-3, 23+/-1, and 22+/-2 mm Hg (mean+/-SEM), respectively (P<0.01). Corresponding systolic PAP values in the UK were 40+/-3, 28+/-3, and 24+/-3 mm Hg, respectively (P<0.01). Compared with control subjects, platelet 5-HTT protein was increased in COPD patients in proportion to the hypoxemia level, and strong 5-HTT immunostaining was observed in remodeled pulmonary arteries from COPD patients. CONCLUSIONS: 5-HTT gene polymorphism appears to determine the severity of PH in hypoxemic patients with COPD. Because PH is an important prognostic factor in this disease, recognition of patients at risk for PH should be helpful in managing COPD.

Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is a hallmark pathological feature of pulmonary hypertension (PH). Serotonin (5-HT) is involved in the hyperplasia through its interactions with specific receptors and internalization by a specific plasma membrane transporter. We investigated the expression and role of the 5-HT transporter (5-HTT) and 5-HT1B, 5-HT2A, and 5-HT2B receptors in lungs and isolated PA-SMCs from patients with primary PH (n=14), pulmonary veno-occlusive disease (n=4), or secondary PH (SPH, n=8) and nonpulmonary hypertensive control subjects. Whereas strong immunostaining for the three receptor types and 5-HTT was seen in remodeled pulmonary vessels from patients in all PH categories, only 5-HTT expression was increased in lungs and cultured PA-SMCs from patients versus controls. The increased growth response of PA-SMCs from patients with primary PH, pulmonary veno-occlusive disease, or SPH to 5-HT or serum was entirely attributable to 5-HTT overexpression, because 5-HTT inhibitors but not 5-HT receptor antagonists abolished 5-HT mitogenic activity and reduced the serum-induced growth response to similar levels in patients as in controls. The L-allelic variant of the 5-HTT gene promoter, which is associated with 5-HTT overexpression, was present homozygously in 14 of 25 (56%) lung transplantation patients with SPH but in only 27% of controls. Polymorphism of the 5-HTT gene promoter was only partly responsible for the increased 5-HTT expression in PH, because PA-SMCs from patients exhibited higher 5-HTT levels than same-genotype cells from controls and no additional promoter sequence alterations were found. We conclude that 5-HTT overexpression is a common pathogenic mechanism in various forms of PH.

pulmonary hypertension is a potentially lethal condition, which affects adults and children alike. Genetic factors are implicated in the causation of primary pulmonary hypertension. We investigate the role of polymorphism in the 5HTT gene in the etiology of pulmonary hypertension in children aged 1-18.8 years. We have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease.

RATIONALE: The bone morphogenetic receptor type II gene is the major genetic determinant for the inherited form of pulmonary arterial hypertension. However, deleterious mutations of this gene are not observed in the majority of subjects who develop the condition spontaneously and familial disease displays age- and sex-dependent penetrance, indicating the requirement for additional environmental and/or genetic modifiers for disease development. METHODS: We investigated polymorphic variation of the serotonin transporter gene, a biological candidate for predisposition to this vascular disorder. RESULTS: No significant evidence of association between alleles of the serotonin transporter gene and pulmonary hypertension was detected, nor did we observe a relationship with age of onset in familial and idiopathic disease. CONCLUSIONS: Variation of the serotonin transporter gene appears unlikely to confer significant susceptibility to pulmonary arterial hypertension. This study emphasizes the need for adequately powered cohorts for association analyses to identify not only genetic determinants of disease susceptibility but also inherited modifiers for disease development.

One intrinsic abnormality of pulmonary artery smooth muscle cells (PA-SMCs) in human idiopathic pulmonary hypertension (iPH) is an exaggerated proliferative response to internalized serotonin (5-HT) caused by increased expression of the 5-HT transporter (5-HTT). To investigate whether 5-HTT overexpression in PA-SMCs is sufficient to produce PH, we generated transgenic mice overexpressing 5-HTT under the control of the SM22 promoter. Studies in SM22-LacZ(+) mice showed that the transgene was expressed predominantly in SMCs of pulmonary and systemic vessels. Compared with wild-type mice, SM22-5-HTT(+) mice exhibited a 3- to 4-fold increase in lung 5-HTT mRNA and protein, together with increased lung 5-HT uptake activity, but no changes in platelet 5-HTT activity or blood 5-HT levels. At 8 weeks of age, SM22-5-HTT(+) mice exhibited PH, with marked increases in right ventricular systolic pressure (RVSP), right ventricle/left ventricle+septum ratio, and muscularization of distal pulmonary vessels, but no changes in systemic arterial pressure. PH worsened with age. Except a marked decrease in Kv channels, no changes in the lung expression of mediators of pulmonary vascular remodeling were observed in SM22-5-HTT(+) mice. Compared with wild-type mice, SM22-5-HTT(+) mice showed depressed hypoxic pulmonary vasoconstriction contrasting with greater severity of hypoxia- or monocrotaline-induced PH. These results show that increased 5-HTT expression in PA-SMCs, to a level close to that found in human iPH, lead to PH in mice. They further support a central role for 5-HTT in the pathogenesis of PH, making 5-HTT a potential therapeutic target.

BACKGROUND: pulmonary hypertension is common in patients with heart failure (HF); however, for a given degree of left ventricular dysfunction, the range in pulmonary artery pressures (PAPs) is large. Polymorphisms of the serotonin transporter (5-HTT) gene have been implicated in contributing to smooth muscle dysfunction and remodeling of the pulmonary vasculature. This study examined the influence of a repeat length polymorphism in the promoter region of the 5-HTT gene on PAP between patients with HF and healthy control participants. METHODS: A total of 90 patients with HF (age, 55 +/- 14 years; left ventricular ejection fraction, 28% +/- 10%; New York Heart Association, 2.0 +/- 0.9) and 90 age- and sex-matched controls (CTL) (age, 51 +/- 15 years; left ventricular ejection fraction, 63% +/- 7%) were recruited. Patients with HF and CTL participants were divided into 3 genotype groups: homozygotes for the short variant (SS), homozygotes for the long variant (LL), and heterozygotes (LS). For both HF and CTL, there are 20 patients in the SS group, 41 in the LS, and 29 in the LL. Pulmonary artery pressure was calculated from the tricuspid regurgitant velocity. RESULTS: Age and sex did not differ between the HF and CTL groups. Pulmonary artery pressure was higher in HF patients compared with CTL (38.9 +/- 12.5 vs 27.7 +/- 8.2 mm Hg, respectively; P < .01). There was a significant interaction between the HF and CTL groups for PAP by genotype (P < .03), with a significant genotype effect in HF (SS = 34.2 +/- 8.6 vs LL = 43.2 +/- 15.2 mm Hg; P < .05) but not in CTL. The HF group also demonstrated elevated left atrial diameter compared with CTL (48.0 +/- 8.7 vs 32.8 +/- 4.8 mm, respectively; P < .01), although no significant genotype difference was seen within either group. CONCLUSION: These results suggest that the LL variant of the 5-HTT is associated with elevated PAP in patients with HF.

BACKGROUND: pulmonary hypertension (PH) is prognosti- cally important in chronic obstructive pulmonary disease (COPD). Since PH only weakly correlates with hypoxemia, other factors must play a role. OBJECTIVE: To investigate whether polymorphisms of the serotonin transporter (5HTT), serotonin-2a receptor (5HTR2a) and endothelial nitric oxide synthetase (eNOS) are related to PH in COPD. METHODS: In 59 COPD patients who underwent right heart catheterization, 6-min walking distance, NYHA functional class, pulmonary function tests, blood gases and 5HTT, 5HTR2a and eNOS (4ab and T298C) polymorphisms were determined. RESULTS: Forty-nine COPD patients in NYHA functional class III-IV were included. Ten were excluded due to comorbid causes of PH (mainly chronic thromboembolic). PH (mPAP > or =25 mm Hg) was present in 55% and usually mild, but out of proportion (mPAP > or =40 mm Hg) in 12%. Patients with PH had significantly higher frequencies of the 5HTT-L-allele (52%) compared to individuals without PH (36%), and LL homozygote patients had more severe PH. In patients with out-of-proportion PH, the L-allelic frequency was even 75%. We found no association of 5HTR2a and eNOS polymorphism with PH in COPD. CONCLUSIONS: In this COPD cohort we confirm that PH is frequent and usually mild, but out of proportion in a subgroup. We found a significant association of the L-allelic variant of 5HTT with PH overall and especially in out-of-proportion PH. These findings may point towards a role of the serotonin system in COPD-PH and warrant further studies.CI - Copyright 2009 S. Karger AG, Basel.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) share important pathogenic and clinical features. BMPR2 mutations are important in the pathogenesis of IPAH, but little is known about the genetic background in CTEPH. Objective: To search for mutations and polymorphisms in genes involved in the BMPR2, serotonin and nitric oxide pathways possibly associated with pulmonary and cardiac disorders in IPAH and CTEPH. METHODS: In a cohort of Swiss patients with IPAH (n = 16) and CTEPH (n = 16), and in 24 controls with left heart disease without PH, polymorphisms in the BMPR2, 5-HHT, 5-HTR-2A and eNOS genes were analyzed and correlated with various clinical, functional and hemodynamic parameters. RESULTS: We found a BMPR2 missense mutation in a patient with coronary artery disease (CAD) without PH but no BMPR2 mutations in our collective with late-onset sporadic PH. In patients with polymorphic variants of the BMPR2 gene, the number of blood platelets and oxygen saturation were increased. The c.600A-->C synonymous variant was associated with worse exercise capacity and decreased quality of life in PH. We found no significant differences for any measured parameter according to the eNOS, 5-HTR2A and the 5-HTT polymorphisms, although there was a higher allelic frequency of the 5-HTT long variant in IPAH than in CTEPH and controls. CONCLUSION: Our first report of a BMPR2 mutation in a patient with CAD without PH is interesting and warrants further investigation. Our study may reflect the clinical status and genetic background in a typical PH cohort as seen in a single tertiary care referral center.CI - Copyright 2009 S. Karger AG, Basel.

Polymorphisms of serotonin transporter (5-HTT) gene were hypothesized to be associated with ventricular septal defects (VSD) related ultrasonographic pulmonary arterial hypertension (PAH) susceptibility. To test this hypothesis, we performed a case-case study in Chinese population and genotyped two polymorphisms (5-HTTLPR and 5-HTTVNTR). The LL homozygote of 5-HTTLPR and 10/10 homozygote of 5-HTTVNTR were both associated with a significantly increased risk of VSD-related ultrasonographic PAH. Haplotype analysis showed haplotype L-10 were also associated with a significant increased risk of ultrasonic PAH. These findings suggest that 5-HTT polymorphisms may play a role in the etiology of VSD-related possible PAH.

OBJECTIVE: Serotonin is a key contributing factor in pulmonary arterial hypertension (PAH) by inducing pulmonary arterial smooth muscle cell (PA-SMC) proliferation. This relates specifically to the internalization process in PA-SMC of the serotonin transporter (SLC6A4 or 5-HTT). A long (L)/short (S) (44 base pair insertion) functional polymorphism within the promoter of the transporter SLC6A4 gene has been reported to be associated with familial and idiopathic PAH. Our objective was to determine whether polymorphisms of SLC6A4 confer susceptibility to SSc and its vascular phenotype. METHODS: Three Tag single-nucleotide polymorphisms (SNP) (rs2066713, rs1042173, rs6354) chosen using Hapmap and linkage disequilibrium data were genotyped in a total cohort of 667 SSc patients (56 with PAH, 207 with digital ulcerations) and 447 controls. All individuals were of French Caucasian origin. L/S polymorphism genotyping was determined by polymerase chain reaction in a random subgroup of 364 SSc patients (34 with PAH, 138 with digital ulcerations) and 218 controls. RESULTS: Three polymorphisms (L/S, rs2066713, rs1042173) were in Hardy-Weinberg equilibrium in the control population, but rs6354 deviated. Allelic and genotypic frequencies for these 3 polymorphisms were similar in SSc patients and controls. Subphenotype analyses of subsets with PAH and digital ulceration did not detect any difference between SSc patients compared to controls. CONCLUSION: These results from a large cohort of European Caucasian SSc patients do not support the implication of SLC6A4 in the pathogenesis of SSc and its vascular subphenotypes. However, serotonin pathways remain good candidates to contribute to the vasculopathy of SSc.

AIMS: Idiopathic and familial forms of pulmonary arterial hypertension (PAH) predominantly affect females through an unknown mechanism. Activity of the serotonin transporter (SERT) may modulate the development of PAH, and mice overexpressing SERT (SERT+ mice) develop PAH and severe hypoxia-induced PAH. In the central nervous system, oestrogens influence activity of the serotonin system. Therefore, we examined the influence of gender on the development of PAH in SERT+ mice and how this is modulated by female hormones. METHODS AND RESULTS: PAH was assessed via measurement of right ventricular systolic pressure (RVSP), pulmonary vascular remodelling (PVR), and right ventricular hypertrophy. Male SERT+ mice did not develop PAH. Female SERT+ mice demonstrated increased RVSP and PVR and this was abolished by ovariectomy. Following exposure to hypoxia, SERT+ mice exhibited severe PAH and this was also attenuated by ovariectomy. Chronic administration of 17beta oestradiol re-established the PAH phenotype in ovariectomized, normoxic, and hypoxic SERT+ mice. 17beta oestradiol also up-regulated tryptophan hydroxylase-1 (TPH1), 5-hydroytryptamine(1B) (5-HT(1B)) receptor, and SERT expression in human pulmonary arterial smooth muscle cells (hPASMCs). 17beta oestradiol stimulated hPASMC proliferation and this was inhibited by both the TPH inhibitor para-chlorophenylalanine and the 5-HT(1B) receptor antagonist SB224289. CONCLUSION: 17beta oestradiol is critical to the development of PAH and severe hypoxia-induced PAH in female SERT+ mice. In hPASMCs, 17beta oestradiol-induced proliferation is dependant on de novo serotonin synthesis and stimulation of the 5-HT(1B) receptor. These interactions between the serotonin system and 17beta oestradiol may contribute to the increased risk of PAH associated with female gender.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a poorly understood complex disorder, which results in progressive remodeling of the pulmonary artery that ultimately leads to right ventricular failure. A two-hit hypothesis has been implicated in pathogenesis of IPAH, according to which the vascular abnormalities characteristic of PAH are triggered by the accumulation of genetic and/or environmental insults in an already existing genetic background. The multifactor dimensionality reduction (MDR) analysis is a statistical method used to identify gene-gene interaction or epistasis and gene-environment interactions that are associated with a particular disease. The MDR method collapses high-dimensional genetic data into a single dimension, thus permitting interactions to be detected in relatively small sample sizes. AIM: To identify and characterize polymorphisms/genes that increases the susceptibility to IPAH using MDR analysis. MATERIALS AND METHODS: A total of 77 IPAH patients and 100 controls were genotyped for eight polymorphisms of five genes (5HTT, EDN1, NOS3, ALK-1, and PPAR-gamma2). MDR method was adopted to determine gene-gene interactions that increase the risk of IPAH. RESULTS: With MDR method, the single-locus model of 5HTT (L/S) polymorphism and the combination of 5HTT(L/S), EDN1(K198N), and NOS3(G894T) polymorphisms in the three-locus model were attributed to be the best models for predicting susceptibility to IPAH, with a P value of 0.05. CONCLUSION: MDR method can be useful in understanding the role of epistatic and gene-environmental interactions in pathogenesis of IPAH.

Activation of the small GTPase Ras homolog gene family member A (RhoA) and Rho-associated kinase (ROCK) are important in the pathogenesis of pulmonary arterial hypertension (PAH). Selective serotonin reuptake inhibitors inhibit activation of RhoA and ROCK in vitro, and ameliorate PAH and pulmonary arterial remodeling in vivo. However, little is known about whether the RhoA-ROCK signalling pathway is involved in the treatment of PAH with fluoxetine in vivo. The aim of the present study was to investigate the involvement of the RhoA-ROCK signalling pathway in the protective effect of the selective serotonin reuptake inhibitor fluoxetine against monocrotaline (MCT)-induced pulmonary arterial remodeling. MCT was applied to establish PAH in male Wistar rats. Fluoxetine was administered by gastric gavage once a day for 21 d. The results showed that MCT induced pulmonary arterial remodeling, raised the serotonylation and membrane translocation of RhoA in the lungs, and increased serotonin transporter (5-HTT), RhoA, and ROCK2 expression, and extracellular signal-regulated kinase (ERK) and Akt phosphorylation in the pulmonary arteries and the lungs. Fluoxetine markedly inhibited these MCT-induced changes. The findings suggest that fluoxetine inhibits MCT-induced pulmonary arterial remodeling in rats by inhibition of the RhoA-ROCK and Akt signalling pathways.

BACKGROUND: Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. METHODS AND RESULTS: We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT-like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice. CONCLUSIONS: Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

RATIONALE: The bone morphogenetic receptor type II gene is the major genetic determinant for the inherited form of pulmonary arterial hypertension. However, deleterious mutations of this gene are not observed in the majority of subjects who develop the condition spontaneously and familial disease displays age- and sex-dependent penetrance, indicating the requirement for additional environmental and/or genetic modifiers for disease development. METHODS: We investigated polymorphic variation of the serotonin transporter gene, a biological candidate for predisposition to this vascular disorder. RESULTS: No significant evidence of association between alleles of the serotonin transporter gene and pulmonary hypertension was detected, nor did we observe a relationship with age of onset in familial and idiopathic disease. CONCLUSIONS: Variation of the serotonin transporter gene appears unlikely to confer significant susceptibility to pulmonary arterial hypertension. This study emphasizes the need for adequately powered cohorts for association analyses to identify not only genetic determinants of disease susceptibility but also inherited modifiers for disease development.

BACKGROUND: pulmonary hypertension is common in patients with heart failure (HF); however, for a given degree of left ventricular dysfunction, the range in pulmonary artery pressures (PAPs) is large. Polymorphisms of the serotonin transporter (5-HTT) gene have been implicated in contributing to smooth muscle dysfunction and remodeling of the pulmonary vasculature. This study examined the influence of a repeat length polymorphism in the promoter region of the 5-HTT gene on PAP between patients with HF and healthy control participants. METHODS: A total of 90 patients with HF (age, 55 +/- 14 years; left ventricular ejection fraction, 28% +/- 10%; New York Heart Association, 2.0 +/- 0.9) and 90 age- and sex-matched controls (CTL) (age, 51 +/- 15 years; left ventricular ejection fraction, 63% +/- 7%) were recruited. Patients with HF and CTL participants were divided into 3 genotype groups: homozygotes for the short variant (SS), homozygotes for the long variant (LL), and heterozygotes (LS). For both HF and CTL, there are 20 patients in the SS group, 41 in the LS, and 29 in the LL. Pulmonary artery pressure was calculated from the tricuspid regurgitant velocity. RESULTS: Age and sex did not differ between the HF and CTL groups. Pulmonary artery pressure was higher in HF patients compared with CTL (38.9 +/- 12.5 vs 27.7 +/- 8.2 mm Hg, respectively; P < .01). There was a significant interaction between the HF and CTL groups for PAP by genotype (P < .03), with a significant genotype effect in HF (SS = 34.2 +/- 8.6 vs LL = 43.2 +/- 15.2 mm Hg; P < .05) but not in CTL. The HF group also demonstrated elevated left atrial diameter compared with CTL (48.0 +/- 8.7 vs 32.8 +/- 4.8 mm, respectively; P < .01), although no significant genotype difference was seen within either group. CONCLUSION: These results suggest that the LL variant of the 5-HTT is associated with elevated PAP in patients with HF.

BACKGROUND: While modulation of the serotonin transporter (5HTT) has shown to be a risk factor for pulmonary arterial hypertension for almost 40 years, there is a lack of in vivo data about the broad molecular effects of pulmonary inhibition of 5HTT. Previous studies have suggested effects on inflammation, proliferation, and vasoconstriction. The goal of this study was to determine which of these were supported by alterations in gene expression in serotonin transporter knockout mice. METHODS: Eight week old normoxic mice with a 5-HTT knock-out (5HTT-/-) and their heterozygote(5HTT+/-) or wild-type(5HTT+/+) littermates had right ventricular systolic pressure(RVSP) assessed, lungs collected for RNA, pooled, and used in duplicate in Affymetrix array analysis. Representative genes were confirmed by quantitative RT-PCR and western blot. RESULTS: RVSP was normal in all groups. Only 124 genes were reliably changed between 5HTT-/- and 5HTT+/+ mice. More than half of these were either involved in inflammatory response or muscle function and organization; in addition, some matrix, heme oxygenase, developmental, and energy metabolism genes showed altered expression. Quantitative RT-PCR for examples from each major group confirmed changes seen by array, with an intermediate level in 5HTT +/- mice. CONCLUSION: These results for the first time show the in vivo effects of 5HTT knockout in lungs, and show that many of the downstream mechanisms suggested by cell culture and ex vivo experiments are also operational in vivo. This suggests that the effect of 5HTT on pulmonary vascular function arises from its impact on several systems, including vasoreactivity, proliferation, and immune function.

BACKGROUND: pulmonary hypertension (PH) is prognosti- cally important in chronic obstructive pulmonary disease (COPD). Since PH only weakly correlates with hypoxemia, other factors must play a role. OBJECTIVE: To investigate whether polymorphisms of the serotonin transporter (5HTT), serotonin-2a receptor (5HTR2a) and endothelial nitric oxide synthetase (eNOS) are related to PH in COPD. METHODS: In 59 COPD patients who underwent right heart catheterization, 6-min walking distance, NYHA functional class, pulmonary function tests, blood gases and 5HTT, 5HTR2a and eNOS (4ab and T298C) polymorphisms were determined. RESULTS: Forty-nine COPD patients in NYHA functional class III-IV were included. Ten were excluded due to comorbid causes of PH (mainly chronic thromboembolic). PH (mPAP > or =25 mm Hg) was present in 55% and usually mild, but out of proportion (mPAP > or =40 mm Hg) in 12%. Patients with PH had significantly higher frequencies of the 5HTT-L-allele (52%) compared to individuals without PH (36%), and LL homozygote patients had more severe PH. In patients with out-of-proportion PH, the L-allelic frequency was even 75%. We found no association of 5HTR2a and eNOS polymorphism with PH in COPD. CONCLUSIONS: In this COPD cohort we confirm that PH is frequent and usually mild, but out of proportion in a subgroup. We found a significant association of the L-allelic variant of 5HTT with PH overall and especially in out-of-proportion PH. These findings may point towards a role of the serotonin system in COPD-PH and warrant further studies.CI - Copyright 2009 S. Karger AG, Basel.

Voltage-gated potassium (Kv)1.5 is decreased in pulmonary arteries (PAs) of patients with idiopathic pulmonary arterial hypertension (IPAH) and in experimental models including mice with SM22alpha-targeted overexpression of the serotonin transporter (5-HTT). The mechanisms underlying these abnormalities, however, remain unknown. Dichloroacetate (DCA) inhibits chronic hypoxia- or monocrotaline-induced PAH by inhibiting nuclear factor of activated T-cells (NFAT)c2 and increasing Kv1.5. Therefore, we hypothesized that DCA could regress established PAH in SM22-5-HTT+ mice. We evaluated pulmonary hemodynamics, vascular remodeling, NFATc2, and Kv1.5 protein in 20-wk-old SM22-5-HTT+ or wild-type mice treated for 1, 7, and 21 d with DCA, cyclosporine-A (NFAT inhibitor), or vehicle. DCA partially reversed PAH in SM22-5-HTT+ mice by decreasing proliferation and increasing apoptosis in muscularized PAs. Furthermore, serotonin (10(-8)-10(-6) M) dose-dependently increased PA-smooth muscle cell (PA-SMC) proliferation in culture (EC(50)=0.97 x 10(-7) M) and DCA (5 x 10(-4) M) vs. PBS markedly reduced the growth of PA-SMC from IPAH and control patients treated with the highest dose of serotonin by 50 and 30%, respectively. Finally, although serotonin induces NFATc2 activation in PA-SMCs, inhibition of NFATc2 alone with cyclosporine-A was not sufficient for reversing PAH in this model. Our results support the possibility that DCA may be an interesting agent for investigation in patients with PAH.

Polymorphisms of serotonin transporter (5-HTT) gene were hypothesized to be associated with ventricular septal defects (VSD) related ultrasonographic pulmonary arterial hypertension (PAH) susceptibility. To test this hypothesis, we performed a case-case study in Chinese population and genotyped two polymorphisms (5-HTTLPR and 5-HTTVNTR). The LL homozygote of 5-HTTLPR and 10/10 homozygote of 5-HTTVNTR were both associated with a significantly increased risk of VSD-related ultrasonographic PAH. Haplotype analysis showed haplotype L-10 were also associated with a significant increased risk of ultrasonic PAH. These findings suggest that 5-HTT polymorphisms may play a role in the etiology of VSD-related possible PAH.

AIMS: Idiopathic and familial forms of pulmonary arterial hypertension (PAH) predominantly affect females through an unknown mechanism. Activity of the serotonin transporter (SERT) may modulate the development of PAH, and mice overexpressing SERT (SERT+ mice) develop PAH and severe hypoxia-induced PAH. In the central nervous system, oestrogens influence activity of the serotonin system. Therefore, we examined the influence of gender on the development of PAH in SERT+ mice and how this is modulated by female hormones. METHODS AND RESULTS: PAH was assessed via measurement of right ventricular systolic pressure (RVSP), pulmonary vascular remodelling (PVR), and right ventricular hypertrophy. Male SERT+ mice did not develop PAH. Female SERT+ mice demonstrated increased RVSP and PVR and this was abolished by ovariectomy. Following exposure to hypoxia, SERT+ mice exhibited severe PAH and this was also attenuated by ovariectomy. Chronic administration of 17beta oestradiol re-established the PAH phenotype in ovariectomized, normoxic, and hypoxic SERT+ mice. 17beta oestradiol also up-regulated tryptophan hydroxylase-1 (TPH1), 5-hydroytryptamine(1B) (5-HT(1B)) receptor, and SERT expression in human pulmonary arterial smooth muscle cells (hPASMCs). 17beta oestradiol stimulated hPASMC proliferation and this was inhibited by both the TPH inhibitor para-chlorophenylalanine and the 5-HT(1B) receptor antagonist SB224289. CONCLUSION: 17beta oestradiol is critical to the development of PAH and severe hypoxia-induced PAH in female SERT+ mice. In hPASMCs, 17beta oestradiol-induced proliferation is dependant on de novo serotonin synthesis and stimulation of the 5-HT(1B) receptor. These interactions between the serotonin system and 17beta oestradiol may contribute to the increased risk of PAH associated with female gender.

The coding sequence of the serotonin transporter gene was compared in two strains of rat-the Wistar and the fawn-hooded rat (FHR). The FHR has an inherited platelet storage-pool deficiency and a widespread impairment of serotonin storage. It is also susceptible to systemic and pulmonary hypertension. The FHR provides a model to study the genetics in human systemic and pulmonary hypertension. We measured platelet function in these two strains by measuring incorporation of radiolabeled serotonin into a platelet suspension and found significant differences in serotonin uptake and release. The coding sequence for the serotonin transporter in the FHR has yet to be reported. No differences were found in the predicted amino acid sequence between these two strains of rat, either in the platelet or the lung samples or when compared with the published sequence of the brown rat. We conclude that differences in the primary structure of the serotonin transporter gene do not account for the altered serotonin storage in the FHR strain.