Increased serotonin serum levels have been proposed to play a key role in pulmonary arterial hypertension (PAH) by regulating vessel tone and vascular smooth muscle cell proliferation. An intact serotonin system, which critically depends on a normal function of the serotonin transporter (SERT), is required for the development of experimental pulmonary hypertension in rodents exposed to hypoxia or monocrotaline. While these animal models resemble human PAH only with respect to vascular media remodeling, we hypothesized that SERT is likewise required for the presence of lumen-obliterating intima remodeling, a hallmark of human PAH reproduced in the Sugen hypoxia (SuHx) rat model of severe angioproliferative pulmonary hypertension. Therefore, SERT wild-type (WT) and knockout (KO) rats were exposed to the SuHx protocol. SERT KO rats, while completely lacking SERT, were hemodynamically indistinguishable from WT rats. After exposure to SuHx, similar degrees of severe angioproliferative pulmonary hypertension and right ventricular hypertrophy developed in WT and KO rats (right ventricular systolic pressure 60 vs. 55 mmHg, intima thickness 38 vs. 30%, respectively). In conclusion, despite its implicated importance in PAH, SERT does not play an essential role in the pathogenesis of severe angioobliterative pulmonary hypertension in rats exposed to SuHx.
- experimental pulmonary hypertension
- serotonin transporter
pulmonary arterial hypertension (pah) is characterized by progressive remodeling of the pulmonary vessels, increased vascular resistance, and, eventually, fatal dysfunction of the right ventricle (RV) (56). Since the outbreaks of PAH caused by the administration of the appetite suppressants fenfluramine (Ponderal) and Aminorex (Menocil), which are serotonin transporter (SERT) substrates and indirect serotonergic agonists, the serotonin pathway has been attributed a key role in the pathogenesis of PAH (34, 59, 65). Disturbances in the serotonin pathway in PAH patients include increased expression of SERT (31, 34, 54) and tryptophan hydroxase 1 (Tph1) (the protein responsible for serotonin synthesis), increased extracellular serotonin levels (12, 22), and increased expression of the serotonin receptor 5-HT1B (31, 32). Serotonin causes pulmonary vasoconstriction and pulmonary artery smooth muscle cell proliferation (32). The many clinical associations between PAH and the serotonin system have been investigated in preclinical studies of rats exposed to monocrotaline and hypoxia. These animal models, however, only resemble PAH with respect to remodeling of the medial layer of the small pulmonary vessels (Table 1) (61). Therefore, they provide no insight into the role of the serotonin pathway in intima proliferation, which is another critical characteristic of PAH (18–20, 52). In vivo studies of models which display angioproliferation would address the question whether the serotonin pathway is essential in the pathogenesis of endothelial cell disease in PAH. We hypothesized that (increased) presence of SERT is required in the pathogenesis of severe angioproliferative pulmonary hypertension in the Sugen hypoxia (SuHx) rat model of pulmonary hypertension. The SuHx model is based on the combined exposure of rats to the vascular endothelial growth factor (VEGF)-inhibitor Sugen and chronic hypoxia (11, 44, 62). The SuHx rat model develops angioproliferative remodeling of the intima with the formation of plexiform-like angio-obstructive lesions and leads to RV dysfunction (1, 5, 15, 62, 63). Here, we used Slc6a41Hubr+ SERT wildtype (WT) and Slc6a41Hubr− SERT knockout (KO) rats to study the impact of a functional impairment of the serotonin system on the development of angioproliferative pulmonary vascular remodeling induced by Sugen plus hypoxia (27). Our study reveals that SERT KO does not prohibit the development of progressive intima remodeling and pulmonary hypertension in SuHx rats. While an overactive serotonin system may be sufficient to induce pulmonary vascular muscularization, our findings suggest that intact serotonin signaling is not required for the development of angioobliterative pulmonary arterial disease.
The SERT KO rat.
The full SERT KO rat was generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis, by which method the DNA of spermatogonial stem cells was subjected to a point mutation and, by subsequent mating, a full knockout was bred (57, 58). By sequencing, it was found that a codon in the third exon was transferred from encoding cysteine to a stop codon (27). This stop codon served as a full knock out of the SERT assembly in the SERT KO rat. The lack of SERT and the absence of serotonin uptake in tissues were confirmed by the absence of SERT expression in tissues, the absence of d-fenfluramine-induced hypothermia, the absence of citalopram binding in the brain, and by the increased bleeding times due to the absence of serotonin in blood platelets, which are fully dependent on SERT for the uptake of serotonin (25, 27, 40). Matondo et al. (40) measured that the serotonin level in the blood of the SERT KO rat is about 1–6% of that in the WT. Homberg et al. (27) phenotyped the SERT KO rat for neuroscientific use. In the brain, because of the lack of SERT, intracellular serotonin level was decreased and extracellular serotonin was increased. No changes in Tph2 expression were found. Changes in compensatory systems for the serotonin system, such as dopamine and noradrenaline, were not observed, which led to the conclusion that in respect to neurobiology, the phenotypic alterations were limited to serotonin alone (27). The SERT KO rat has impaired object memory (47). The systolic blood pressure was not altered in SERT KO rats (25). The female SERT KO rat has more abdominal fat compared with the male (26).
MATERIALS AND METHODS
Twelve male Slc6a41Hubr WT rats (SERT WT) and twelve Slc6a41Hubr KO male rats (SERT KO) (Radboudumc, Nijmegen, The Netherlands) were allocated to two different groups: naïve control and SuHx-exposed animals. SuHx-mediated pulmonary hypertension was induced according to the protocol published previously (4, 11, 62). Sugen (SU5416, Tocris Bioscience, Bristol, UK) was administered to rats weighing <200 g as a single subcutaneous injection (25 mg/kg) (11, 28). SuHx rats were housed from the day of injection and during the next 3 wk in 10% oxygen (BioSpherix, Lacona, New York) maintained by a nitrogen generator (Avilo, Dirksland, The Netherlands). SuHx rats were subsequently re-exposed to normoxia for 3 wk (11). The study was approved by the local animal welfare committee (study number VU-Fys 12–15).
Echocardiography and hemodynamics.
On the day of necropsy, all animals underwent echocardiographic assessments to measure RV wall thickness (RVWT), RV end diastolic diameter (RVEDD), tricuspid annular plane systolic excursion (TAPSE), stroke volume (SV), heart rate (HR), cardiac output (CO), pulmonary artery acceleration time (PAAT), and cycle length (cl) (Philips Sonos 7500 with a S12 phased array transducer, Andover, MA), as published previously (11, 21). On the day of necropsy, RV systolic pressure (RVSP), mean pulmonary artery pressure (mPAP), end systolic elastance (EES), and arterial elastance (EA) (Millar Instruments, Houston, TX) were measured, as published previously (10a). Total pulmonary resistance (TPR) was calculated as mPAP/CO and arterial ventricular coupling was derived as EES/EA.
Histological and morphometric analyses.
After necropsy, the hematocrit was measured and heart and lungs were weighed before processing for histology. Following examination of stained histological sections, small pulmonary arteries were divided into three classes, based on external diameters: <30, 30–60, and 60–100 μm (11, 24). Media and intima wall thickness and percent of obliteration were measured and recorded according to previously published protocols (11, 45, 46, 62), in which closed vessels are >80% obliterated and open vessels are unaffected. Lungs were homogenized and supernatants were extracted. According to the supplier's manual, a Western blot was performed to quantify cell proliferation [PCNA (FL-261), sc-7907, 1:1,000 (Santa Cruz Biotechnology, Dallas, TX); second antibody HRP anti-goat, 1:5,000 (DAKO, Glostrup, Denmark)]. Novex enhanced chemiluminescence (ECL) (Invitrogen, Carlsbad, CA) was used for protein detection. Optical densities were measured and standardized with β-actin (A3854, 1:20,000, Sigma Aldrich, St. Louis, MO).
Serotonin, SERT and serotonin receptor measurements.
Blood was centrifuged for 5 min at 1,500 g to obtain plasma. Serotonin was determined in plasma and in lung tissue by using a serotonin ELISA kit (Abcam, Cambridge, UK) according to the instruction manual. ELISA measurements of plasma serotonin were confirmed by HPLC with electrochemical detection. Reagents, calibrators and internal standard (N-methylserotonin) were obtained from Chromsystems (Gräfelfing, Germany). The interassay variation was 5% at a level of 0.82 μmol/l. The lower limit of quantification was 0.06 μmol/l. 5-Hydroxyindoleacetic acid (5HIAA) level was measured by liquid chromatography–tandem mass spectrometry (Waters Xevo TQ-MS, Milford, MA). The expression of SERT in lungs was determined by Western blot [ST(C-20) sc-1458 (Santa Cruz Biotechnology); second antibody HRP anti-goat, 1:5,000 (DAKO)]. Novex ECL (Invitrogen) was used for protein detection. Optical densities were measured and standardized with β-actin (A3854, 1:20,000, Sigma Aldrich). Presence of SERT in pulmonary vasculature was determined by immunofluorescence [ST(C-20), sc-1458 (Santa Cruz Biotechnology); second antibody Alexa Fluor 488 anti-goat, 1:100 (ThermoFisher Scientific, Waltham, MA); monoclonal antiactin antismooth muscle, Cy3 antibody (Sigma Aldrich); and Slowfade Gold Antifade Mountant with DAPI (ThermoFisher Scientific)].
The expression of Tph1, responsible for serotonin synthesis, and the receptors 5-HT1B and 5-HT2A were determined by quantitative PCR (qPCR) (GoTaq qPCR Master Mix, Promega Benelux, Leiden, The Netherlands). Primers were designed by using Primer3 online software (http://frodo.wi.mit.edu). qPCR reactions were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) by using the SYBR Green fluorescence quantification system. By using reference genes Ywhaz and Hprt, the cDNA content of the samples was normalized.
After confirmation of a normal distribution, parametric variables were compared between groups by using appropriate two-way ANOVA with Bonferroni post hoc tests. Data is presented as means ± SE (Graphpad, La Jolla, CA).
Clinical observation of the animals throughout the study protocol did not reveal obvious differences between WT and KO rats. Body weight was about 5–10% lower in all KO animals during the entire study. SERT expression was not present in lung tissue nor was SERT present in the pulmonary vasculature (Fig. 1, A and B), which confirms, in line with the conclusions by Smits et al. and Homberg et al. (27, 57), the full knock out of this strain. By using HPLC or ELISA, serotonin could not be detected in the plasma of SERT KO rats, while ELISA did not show presence of serotonin in lung tissue (Fig. 2, A–C). 5HIAA levels were similar in KO and WT rats and were not altered by the SuHx protocol (Fig. 1D). WT rats, but not KO rats, showed a trend of increased Tph1 expression after SuHx exposure (Fig. 1E). Receptor densities of 5-HTR1B and 5-HTR2A in the lung were not altered (Fig. 1, F and G).
Echocardiography and hemodynamics.
Right heart remodeling after SuHx exposure was similar in WT and KO rats. All the animals showed an increase in RVWT and RVEDD and a trend toward a decrease in TAPSE (Fig. 3, A–C). PAAT/cl was decreased after SuHx exposure, but there were no differences between rat strains (Fig. 3D). The CO was significantly lower in the SERT KO when exposed to SuHx, which was caused by a significantly lower HR (Fig. 4, A and B). SV was not different between WT and KO rats (Fig. 4C). SuHx exposure resulted in a significant increases in RVSP, measured by right heart catheterization, compared with naïve control conditions (Fig. 5A), but the RVSP was not different between SERT WT and SERT KO rats. TPR was increased in SuHx rats, WT, and KO rats alike (Fig. 5B).
Pressure volume relationships.
Hematocrit and fulton index.
Hematocrit and Fulton index [RV/(LV+S)] were increased in SuHx rats, but not different between SERT WT and KO rats (Fig. 7).
Pulmonary vascular remodeling.
Vascular remodeling after SuHx exposure was similar in SERT WT and KO rats, with comparable degrees of medial wall and intima thickening and percent of obliteration in all vessel classes (Fig. 8, A–C). A representative overview (Fig. 8D) and examples are shown of changes in ~40-μm vessels in SuHx rats (Fig. 8C). Western blot of proliferation marker PCNA showed no differences between WT and KO in naïve control and SuHx (Fig. 9).
The main result of this study is that the absence SERT did not prevent the development of severe angioproliferative pulmonary hypertension in rats exposed to Sugen and hypoxia. SERT expression was not present in the lungs, indicating that a severely impaired serotonin system does not preclude the development of intima remodeling and vascular obliterations. A comparable increase in RVSP was found in WT and SERT KO SuHx rats, and this was reflected by similar changes in lung histology and functional cardiac measurements. A decrease in intima proliferation in SERT KO rats could have been postulated because serotonin is a known angiogenic factor (9, 49), but did not occur. In contrast to the SuHx study in Tph1 KO mice (9), the KO of SERT in the rat could not prevent the induction of vascular lesions and elevation of RVSP by SuHx. As a powerful regulator of vessel tone, “a serum-tonis regulator” (53), the effect of serotonin is directed toward the medial layer by vasoconstriction and its stimulation of smooth muscle cell proliferation (32). It has been reported that in the mouse, the increase in RVSP upon exposure to hypoxia was partially prevented by knocking out SERT (Table 1) (13, 35). The relative importance of intact serotonin signaling may even be greater in the monocrotaline model (MCT) of pulmonary hypertension, as MCT rats showed a profound upregulation of SERT, and medial hyperplasia was fully inhibited in MCT rats treated with inhibitors of SERT and also with inhibitors of other components of the serotonin pathway (Table 1) (13, 17, 30). In the SuHx model, intima remodeling plays a more dominant role compared with medial remodeling (11), which concentrates the desired effect on the intima solely. Here we show that SuHx induced intima remodeling and angio-obliteration is not impaired in SERT KO rats, which have a complete functional absence of SERT and undetectable levels of serotonin in plasma and lung tissue.
In the rat strain used in our study, SERT is lacking because of an impaired translation (27, 57, 58). Because SERT is essential for transcellular transport of serotonin, platelets from SERT KO rats are serotonin free (25). Here we confirm previous findings in mice that KO of SERT is also associated with undetectable levels of serotonin in the plasma (13). This finding seems unrelated to altered synthesis, metabolism, or degradation of serotonin because Tph1 expression and 5HIAA levels were unchanged, which is in line with the SERT KO mouse, published by Eddahibi et al. (13). By using HPLC or ELISA, serotonin could not be detected in the plasma of SERT KO rats, while ELISA and immunofluorescence did not show presence of serotonin nor SERT in lung tissue, respectively. Because of the protocol used, the plasma was probably not completely platelet free and the presence of ruptured platelets might have caused the increased level of serotonin in plasma of the WT rats. Because the platelets in the SERT KO rat were unable to take up serotonin, as was published previously (25), the serotonin level in plasma of KO rats was below the detectable level of 0.06 μmol/l. This result was already shown for this SERT KO rat, as in whole blood measurements the concentration of serotonin was about 1–6% of that compared with the SERT WT rat and was also reported earlier in SERT KO mice (13, 40). Because 5HIAA levels were not altered in KO rats, true platelet-free serum concentrations of serotonin were probably normal in KO rats. Whereas undetectable plasma serotonin in SERT KO rodents remains unexplained, it does indicate a severely impaired serotonin system. The effect of SERT KO on circulating serotonin is opposite to what is observed after treatment with selective serotonin reuptake inhibitors (SSRIs): by inhibiting SERT function in platelets, SSRIs induce increased levels of circulatory serotonin (34). It is possible that in our experimental setup, very low concentrations of serotonin, although undetectable by HPLC, were still present and exerted vascular effects. 5-HT1B expression was not altered in SERT KO rats, but it is possible that an increased sensitivity to receptor stimulation by serotonin could compensate for a loss of SERT. To fully address this question, future experiments using Tph1 inhibitors could be performed.
Our finding that the lack of SERT did not modify the hemodynamics or structural remodeling in the SuHx model, indicates that the pulmonary vascular changes in this rat model of severe PAH do not require a fully intact serotonin system. We have shown previously that changes in the medial layer in the SuHx model are rather modest (11). As such, the SuHx model mirrors findings in the end-stage PAH of major changes in the intima and not in the media of small pulmonary vessels (11, 60). In our study, intima remodeling was present to the same degree in SuHx WT and SuHx KO rats, which implies that in this model an intact serotonin pathway does not play an essential role in the development of hyperproliferative angioobliterative intimal lesions. We cannot exclude the possibility that this situation only applies to male rats, as we did not include female rats in our study. Recent publications have pointed to an increased sensitivity to alterations in the serotonin pathway in female compared with male mice (36, 66, 67). Female smooth muscle cells have higher concentrations of serotonin and show higher proliferation in response to serotonin administration than male smooth muscle cells (37). Estrogens are known to increase the protein expression of 5-HT1B receptor in smooth muscle cells (3) and therefore it has been suggested that serotonin based treatments are only, or at least more, effective in female patients (36, 37). As indicated above, the major changes in the SuHx model take place in the vascular intima, and sex differences in endothelial cell responses to serotonin signaling have not been explored. Although one could speculate that angioproliferative remodeling in the SuHx model is driven by serotonin-independent mechanisms in male rats only, there is no evidence to support this hypothesis.
Serotonin has been attributed a positive inotropic action (50), and in chronic heart failure the density of 5-HT2A and 5-HT4 receptors is increased (33, 51). However, it is unclear whether the serotonin system directly affects cardiac function in pulmonary hypertension. The available data is inconclusive, because all studies in this context have been performed using interventions that primarily affect the pulmonary circulation. Experiments of pulmonary arterial banding would be better suitable to address cardiac-specific effects of the serotonin system in a situation of pressure overload. We observed a lower CO in SERT KO SuHx rats, while all other parameters of cardiac function were not different between SERT WT and KO rats. The effect of SERT KO on CO was driven by a decrease in HR. Serotonin is known to regulate the autonomic regulation of HR (48), and although no differences in HR were observed in the naïve SERT KO rat, bradycardia was observed after exposure to the SuHx protocol. A mechanism to explain these findings is currently lacking. Paradoxically, the lack of SERT in the brain locally leads to increased concentrations of extracellular serotonin, which is produced by Tph2 (27). Apparently this feedback mechanism is lacking outside the brain, as we found serotonin to be completely absent from the blood plasma and lungs.
Despite these new findings in SuHx rats, the fact that the serotonin pathway affects human PAH is well established (34). In PAH patients, increased circulatory serotonin levels (22) were observed and increased SERT expression was measured in the lungs from PAH patients (54). High intracellular serotonin levels trigger vasoconstriction and smooth muscle cell proliferation (42). Increased serotonin signaling has been implicated in the development of PAH after use of serotonin reuptake inhibitors or serotonin receptor agonists (the anorexigens dexfenfluramine and aminorex; antidepressants, for example, fluoxetine; and illicit drugs, for example, amphetamines and cocaine) (6, 8, 14, 64). However, as only a fraction of the population taking these compounds will develop PAH, a second hit, in addition to excessive serotonin signaling, is apparently required. MacLean and Dempsie (34) suggested combined inhibition of SERT and serotonin receptors as a new treatment option for PAH. Our findings of unhindered angioproliferative remodeling even in the absence of SERT and with undetectable levels circulatory serotonin question this therapeutic approach, within the limits of translating from animal models to human disease. In conclusion, a fully intact serotonin system does not play an essential role in the pathobiology of severe angioobliterative pulmonary hypertension in the rat SuHx model.
Grant support was received from PAH patient association Live Life Max and the Dutch Lung Foundation (Longfonds No. 3.3.12.036). We acknowledge the support from the Netherlands CardioVascular Research Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centers, The Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences, as well as the LeDucq foundation (PHAEDRA grant).
No conflicts of interest, financial or otherwise are declared by the author(s).
M.A.D.R. and H.J.B. conception and design of research; M.A.D.R., Y.K., A.M., and H.d.J. performed experiments; M.A.D.R. and H.d.J. analyzed data; M.A.D.R., F.S.d.M., H.d.J., A.V.N., C.d.K., N.F.V., J.H., and H.J.B. interpreted results of experiments; M.A.D.R. prepared figures; M.A.D.R. and H.J.B. drafted manuscript; M.A.D.R., Y.K., A.M., F.S.d.M., A.V.N., C.d.K., N.F.V., J.H., and H.J.B. edited and revised manuscript; M.A.D.R., Y.K., A.M., F.S.d.M., H.d.J., A.V.N., C.d.K., N.F.V., J.H., and H.J.B. approved final version of manuscript.
We acknowledge Sjef van Hulten, Rick van der Doelen, Stefan Janssen, Dora Lopresto, Karin de Haas, (Radboud University medical center, Nijmegen, The Netherlands), Ingrid Schalij, Liza Botros (VU University medical center, Amsterdam, The Netherlands), and Manon de Raaf-Beekhuijzen for their contribution to this study by sharing their knowledge and expertise. The endocrine laboratory department (VU University medical center, Amsterdam) is acknowledged for the HPLC measurements of serotonin.
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